Crop Plant Resistance to Biotic and Abiotic Factors ... - Die DPG

DPG Spectrum Phytomedizin 

F. FELDMANN, D. V. ALFORD, C. FURK (EDS.) 

Crop Plant Resistance to Biotic and Abiotic 

Factors: Current Potential and Future Demands 

Proceedings of the 

3rd International Symposium on Plant Protection and Plant Health in Europe 

held at the Julus Kühn-Institut, Berlin-Dahlem, Germany, 14-16 May 2009 

jointly organised by 

the German Phytomedical Society (DPG) and the British Crop Production 

Council (BCPC) 

in co-operation with the 

Faculty of Agriculture and Horticulture (LGF), Humboldt University Berlin, and 

the Julius Kühn-Institut (JKI), Berlin, Germany 

Selbstverlag 

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ISBN: 978-3-941261-05-1 

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© 2009 DPG Selbstverlag 

Messeweg 11-12, 38104 Braunschweig 

Email: geschaeftsstelle@phytomedizin.org 

Internet: www.phytomedizin.org 

Lectorate: Dr. David V. Alford, Mr. Chris Furk, Dr. Falko Feldmann 

Production: Dr. C. Carstensen, InterKulturIntern, Edenkoben 

Design (cover): C. Senftleben, Braunschweig 

Foto (cover): Feldmann (DPG), Wehling (JKI), Heupel (LWK Rheinland) 

Printed in Germany by Lebenshilfe Braunschweig gGmbH 

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PREFACE 

Plant production has to meet considerably mounting demands in the future. Expanding global 

markets and the competition of food and non-food uses require further significant progress in 

productivity levels. In Europe as well as globally, increased production will have to be 

achieved on the same or decreasing area of arable land. If global welfare is to be maintained or 

improved an increased efficiency per unit area is required. At the same time, climatic changes 

may aggravate the conditions of growth in less favourable locations. Thus, the scenario which 

agriculture is facing is further intensified crop rotations with a limited number of high-yielding 

crops for the food or raw materials market, under aggravated climatic conditions. Altogether, 

these developments will result in a significant increase in problems caused by biotic and abiotic 

stresses, which will inevitably limit yield levels. One way out will be improvement of cultivars. 

Breeding programmes are currently set up to meet the new challenges. Recent biotechnological 

progress has opened new avenues for further and faster advances in crop breeding. Cultivars 

with better resistance to biotic and abiotic stress are becoming a real option. However, a 

number of emerging questions had to be answered. What will be the major threats in crop 

production systems over the next few decades? Which traits are needed and which can be 

expected to become available in new cultivars within the next few years? How can the new 

biotechnologies be helpful in producing cultivars harbouring the desired new traits? 

This symposium seeked to gather experts from the fields of crop production, crop protection, 

plant breeding and crop plant biotechnology in order to stimulate answers to these questions. In 

particular, this symposium addressed the following topics: 

Driving forces for modifications of production systems in a changing world. This topic 

gathered knowledge on the main factors influencing crop production systems and seeked to 

project how crop production systems might look like in Europe in the next decade, taking into 

account diversity in product uses, altered markets and a changed climate. 

New challenges for crop protection through changed climate and markets. Based on the 

current status reports on new emerging pests and diseases resulting from altered crop rotations 

and a changed climate were given. The economic impact was estimated for major crops based 

on the relative damage potential of the various stress factors. 

Resistance in crop plants – current status. Status reports were presented highlighting the 

currently available resistance traits in the most important European crops and crop cultivars. 

Resistance in crop plants – current potential and future innovations. Current potential and 

future innovations in crop resistance to biotic and abiotic stress were outlined. The role of 

modern biotechnology vs. conventional breeding technology has been critically reviewed. 

We invited to present oral and poster contributions and received a huge amount of valuable 

papers which are provided in this conference report. 

F Feldmann, DPG, Braunschweig, Germany, D V Alford, BCPC, Cambridge, UK, & C Furk, 

BCPC, York, UK. 

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SYMPOSIUM ORGANISERS 

General management 

Falko Feldmann, DPG, Germany 

Local Organising Committee 

Christoph Reichmuth & Birgit Hering, Julius Kühn-Institut, Berlin, Germany 

Carmen Büttner & Martina Bandte, Humboldt University, Berlin, Germany 

Programme Committee 

Falko Feldmann & Andreas von Tiedemann, DPG, Germany 

David V. Alford & Chris Furk, BCPC, United Kingdom 

International Advisory Committee 

Olaf Christen, University of Halle, Germany 

Charles-Erik Durel, Institut National de la Recherche Agronomique (INRA), France 

Milka Glavendekic, University of Belgrad, Serbia 

Bernd Holtschulte, KWS, Einbeck, Germany 

Ahmed Jahoor, The Royal Veterinary and Agriculture University, Copenhagen, Denmark 

Graham Jellis, HGCA, London, UK 

Spiros Kintzios, Agricultural University Athens, Greece 

Jozef Kotleba, Slovak Crop Protection Association, Bratislava, Slovakia 

Thomas Miedaner, University of Hohenheim, Germany 

Frank Ordon, Julius-Kühn-Institut, Quedlinburg, Germany 

Vladimir Rehak, C. Spolecnost Rostlinolekarska, Praha, Czech Republic 

Chris-Carolin Schön, Center of Life and Food Sciences Weihenstephan, Germany 

Mark Varrelmann, University of Göttingen, Germany 

Peter Wehling, Julius-Kühn-Institut, Groß Lüsewitz/Sanitz, Germany 

Acknowledgement 

DPG wishes to thank the German Research Foundation (DFG) for their support of this 

symposium. 

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PROGRAMME AND CONTENT 

1-1 Crop Plant Resistance to Biotic and Abiotic Factors: Combating the Pressures on Production 

Systems in a Changing World 

Jellis G J 15 

2-1 Atmospheric composition – a threat to crop growth and health? 

Weigel H J, Bender J 22 

2-2 The use of the Water Potential Index and some ecophysiological and morphological parameters as 

reliable indicators of crop adaptation to drought 

Karamanos A J 33 

2-3 Spatial Presentation (GIS) of Winter Apple Tree Phenology in Conditions of the Slovak Republic 

Influenced by Expected Climate Change 

Mezeyová I, Šiška B, Mezey J, Paulen O 47 

2-4 Use of Forest Tree Species Under Climate Change 

Grundmann B M, Roloff A 53 

2-5 Forestry in a Changing Climate − the Necessity of Thinking Decades Ahead 

Profft I, Frischbier N 66 

3-1 Interaction of free air carbon dioxide enrichment (FACE) and controlled summer drought on fungal 

infections of maize 

Oldenburg E, Manderscheid R, Erbs M, Weigel HJ 75 

3-2 Plant tissue colonization by the fungus race 1.2 of Fusarium oxysporum f. sp. melonis in resistant 

melon genotypes 

Chikh-Rouhou H, González-Torres R, Álvarez JM 84 

3-3 Wheat double haploid lines with improved salt tolerance: in vitro selection and RAPD analysis 

Beckuzhina, S, Kochieva E 87 

3-4 Antioxidants in wild and cultivated potato species 

Wegener C B, Jansen G 91 

3-5 Removal of a selectable marker in transgenic potato by PVX-Cre virus vector 

Kopertekh L, Schiemann J 96 

3-6 Effects of temperature on yield parameters of Lupinus angustifolius and Pisum sativum cultivars 

Jansen G 100 

3-7 Antioxidative enzymes in buckwheat (Fagopyrum esculentum Moench) leaves subjected to flooding 

stress 

Majic D 101 

3-8 Analysis of Barley Genotypes with Contrasting Response Towards Salinity Using Complementary 

Molecular and Biochemical Approaches 

Witzel K, Hensel G, Kumlehn J, Hajirezaei M, Rutten T, Melzer M, Börner A, Mock H-P, Kunze G 102 

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3-9 Root Characteristics and N Uptake of Potato Genotypes Grown in vitro in Response to Nitrogen 

Deficiency Stress 

Schum A, Balko C, Debnath M 103 

3-10 Correlation between soil characteristics and in-field variation of soil-borne pathogens 

Jonsson A, Almquist C, Wallenhammar A-C 104 

3-11 First report of three grapevine viruses in Kazakhstan 

Ryabushkina N, Askapuly A, Stanbekova G, Galiakparov N 105 

3-12 Influence of the fungal root endophyte Piriformospora indica on tomato growth and spread of 

Pepino mosaic virus 

Fakhro A, Schwarz D, von Bargen S, Bandte M, Büttner C, Franken P 106 

3-13 First results of mapping and exploitation of new sources of resistance to tan spot (Pyrenophora 

tritici-repentis) in wheat 

Engelmann U, Kopahnke D, Ordon F 107 

3-14 The role of biotic factors in haricot (Phaseolus vulgaris L. Savi) cultivation on the south part of West 

Siberia 

Babenko A, Mikhailova S, Chikin J, Nikolaeva I 108 

3-15 Diabrotica virgifera virgifera (Col.: Chrysomelidae) and its abundance in maize and neighbouring 

non-maize fields of West Romania 

Dinnesen S, Nedelev T, Hummel H E, Grozea I, Carabeţ A, Stef R, Ulrichs Ch 109 

3-16 Monitoring Diabrotica virgifera virgifera (Col.: Chrysomelidae) with different lures and traps 

Dinnesen, S, Humme H E, Grozea I, Carabeţ A, Stef R, Ulrichs C 117 

3-17 Interactions between mycorrhizal fungi and medicinal plants 

Zubek S, Stojakowska A, Kisiel W, Góralska K,Turnau K 124 

3-18 Identification of physical and biochemical agents related to resistance in different sugarcane 

cultivars to stalk borers, Sesamia spp. (Lep.: Noctuidae) 

Abbasipour H, Askarianzadeh A 130 

3-19 Comparison of feeding indexes of Sesamia nonagrioides Lef. (Lep., Noctuidae) 

Minaeimoghadam M, Askarianzadeh A 137 

3-20 Varietal resistance against Jassid, Amrasca biguttula biguttula (Ishida) on Okra under Faisalabad 

ecological conditions 

Mansoor-ul-Hasan, Ashfaq M, Iqbal J, Sagheer M 138 

3-21 Seasonal abundance of the Cotton jassid Amrasca biguttula biguttula on Okra and their Correlation 

with Abiotic Factors 

Iqbal J, Mansoor-ul-Hasan, Sagheer M 146 

3-22 Screening of various genotypes of rice against rice leaf folder, Cnaphalocrocis medinalis Guenee 

Sagheer M, Ashfaq M, Hasan M-ul 154 

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3-23-Field assessment of antibiosis resistance of different wheat cultivars to the Russian Wheat Aphid, 

Diuraphis noxia (Hom.: Aphididae) at stem elongation growth stage 

Kazemi M H, Mashhadi Jafarloo M 155 

3-24 Production of Sunflower Hybrids Based on New Cytoplasmic Male Sterility Sources 

Zeinalzadeh Tabrizi H, Ghaffari M 163 

3-25 Relationship between Antioxidant activity and biochemical components of wheat and sorghum 

genotypes under salinity stress 

Heidari M, Ghanbari A 164 

3-26 Different response of intact siliques and naked seeds of turnipweed (Rapistrum rugosum) to light 

Ohadi S, Rahimian Mashhadi H, Tavakol Afshari R 165 

3-27 Seasonal Abundance of Alfalfa Aphid (Therioaphis trifolli Monell) in Berseem Field 

Mari J M 166 

3-28 Antifungal activity of endophytic fungi isolated from Tylophora indica in India 

Kumar S, Kaushik N, Proksch P 167 

3-29 Management of disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. 

lycopersici using different combinations of Karanj oilseed cake and/ or VA Mycorrhiza, Glomus 

fasciculatum, on tomato 

Jain A, Mohan J, Singh M 175 

3-30 Insect Resistance in Tomato Accessions in Tamilnadu, South India 

Selvanarayanan V 185 

3-31 Current situation of insecticide resistance of major agricultural insect pests in Ghana 

Obeng-Ofori D, Oduro Owusu E 186 

3-32 The new sources of resistance of some cowpea genotypes to the cowpea aphid (Aphis craccivora 

Koch) in Ghana 

Kusi F, Obeng-Ofori D 187 

3-33 Evaluation of the resistance status of twenty varieties of maize to infestation and damage by 

Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) 

Ofuya T, Balogun A O 188 

3-34 In planta Biological Control of Potato Brown Rot Disease in Egypt 

Kabeil S S, Amer M A, Matar S M, El-Masry M H 189 

3-35 Production of Bio-Active protein from some soil bacteria and biological use in controlling Erwinia 

amylovora 

Kabeil S S, Hafez E E, Daba A S, Botros W, El-Saadani M A 190 

3-36 Survival, proliferation of Trichoderma harzianum in Egyptian soil and the role of Trichoderma on 

the plant growth 

Yasser, M 191 

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3-37 Wheat leaf-rust infection response – from apoplast proteomics to transcriptional aspects in nearisogenic 

lines of the ’Thatcher’ cultivar 

Pós V, Hunyadi-Gulyás É, Manninger K, Szikriszt B, Rab E, Kabai M, Medzihradszky K, Lukács N 192 

3-38 Assessment of Organophosphate and Carbamate Pesticide Residues with a Novel Cell Biosensor 

Mavrikou S, Flampouri K, Moschopoulou G, Michaelides A, Kintzios S 194 

3-39 Phytopathogenic and mycotoxigenic characterization of laboratory mutant strains of Fusarium 

verticillioides resistant to triazole fungicides 

Markoglou A N, Vitoratos A G, Doukas E G, Ziogas B N 195 

3-40 New Lignin-Phenolic Compounds for Plant Protection 

Hamolka L, Krut'ko N, Gutkovskaya N 196 

3-41 Dry matter partitioning parameterization in wheat infected by sporulating wheat leaf rust (Puccinia 

triticina) 

Bancal M O, Hansart A, Sache I, Bancal P 197 

3-42 Regulation of Grain N Accumulation in Wheat 

Ben Slimane R, Bancal P, Bancal M-O 198 

3-43 Characterization of novel endophytic Bacillus licheniformis strain CRP-6 from apple seedlings 

displaying multiple plant growth promoting activities 

Chand S, Rishi M, Preeti M, Anjali C, Saurabh K 209 

3-44 Dissection of plant resistance to pest using a genomic approach: Arabidopsis-Two Spotted Spider 

Mite Tetranychus urticae, a novel model for plant-herbivore interactions 

Grbic M, Grbic V 210 

3-45 FieldClimate.Com offering Weather data based Plant Disease Information for Growers and 

Advisors 

Denzer H W 211 

3-46 Sustainable Development – Good Agriculture Practice Eriterion in Crop Production System 

Rajkovic S, Tabakovic – Tosic M 214 

3-47 Role of Phytomedcine and Plant Health Clinic in Plant Health Security 

Srivastava M P 222 

3-48 Potential for Exploiting Host Plant Resistance to Insects for Food Security Under Subsistence 

Farming Conditions in the Semi-Arid Tropics 

Sharma H C 231 

4-1 Mixed Infections of Geminiviruses and Unrelated RNA Viruses or Viroids in Tomato: A Multitude 

of Effects with a Highly Probable Impact on Epidemiology and Agriculture 

Wege C 233 

4-2 Cherry Leaf Roll Virus in birch – an old problem or an emerging virus in Finland? 

Bargen S v, Arndt N, Grubits E, Büttner C, Jalkanen R 242 

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4-3 Importance of Insect-Transmitted Viruses in Cereals and Breeding for Resistance 

Habekuß A, Riedel C, Schliephake E, Ordon F 251 

4-4 Strategy for pathogen-derived resistance in Nicotiana benthamiana to Beet yellows virus and Beet 

necrotic yellow vein virus 

Vinogradova S, Rakitin A, Kamionskaya A, Agranovsky A, Ravin N 262 

4-5 Current Status of Rhizomania Resistance in Sugar Beet - Still Holding or Breaking of Resistance? 

Varrelmann M, Thiel H 263 

4-6 Geminivirology in the Age of Rolling Circle Amplification 

Jeske H, Krenz B, Paprotka T, Wyant P 275 

4-7 Elicitor-Induced Sugar Beet Defence Pathways Against Beet Mosaic Virus (BtMV) infection 

Haggag W M, Mahmoud Y S, Farag W M 276 

5-1 Mechanisms of Fungal Infection 

Deising H B, Horbach R, Ludwig N, Münch S, Schweizer P 290 

5-2 Altered Distribution and Life Cycles of Major Pathogens in Europe 

Evans N, Gladders P, Fitt B D L, & Tiedemann A v 302 

5-3 Genetics of the Plasmodiophora brassicae - Brassica napus interaction 

Diederichsen E, Werner S, Frauen M 308 

5-4 Differential Gene Expression in Wild Sunflower with Resistance to the Necrotrophic Pathogen 

Sclerotinia Sclerotiorum 

Müllenborn C, Krause J-H, Muktiono B, Cerboncin C 309 

5-5 Mapping of genes controlling development and resistance to Verticillium longisporum in Brassica 

alboglabra 

Konietzki S, Socquet-Juglard D, Diederichsen E 319 

5-6 Dynamics of adaptation of powdery mildew to triticale 

Mascher F, Marcello Z, Celeste L 320 

5-7 Screening of Triticum Monococcum and T. Dicoccum to Identify New Sources of Resistance to 

Fusarium Head Blight 

Kopahnke D, Brunsbach G, Miedaner T, Lind V, Rode J, Schliephake E, Orden F 321 

6-1 Invasive Species Following New Crops 

Glavendekić M, Roques A 328 

6-2 The Current Status of Diabrotica virgifera virgifera in Selected European Countries and Emerging 

Options for its Pest Management 

Hummel H E, Bertossa M, Deuker A 338 

6-3 Evaluation of Ear Infestation by Thrips and Wheat Blossom Midges in Winter Wheat Cultivars 

Gaafar N, Cöster H, Volkmar C 349 

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6-4 Varying Glucosinolate Profiles in Arabidopsis Influence Plant Defense Against Generalist and 

Specialist Caterpillars Differently 

Mewis I, Rohr F, Tokuhisa J G, Gershenzon J, Ulrichs C 360 

6-5 Promising Acquired Resistance Against Some Wheat Insects by Jasmonate Application 

El-Wakeil N E, Volkmar C, Sallam A A 366 

7-1 Resistance versus susceptibility in grapevine - Response of different grapevine genotypes to the 

biotrophic pathogen Plasmopara viticola 

Kassemeyer H H, Seibicke T, Boso S 378 

7-2 Detection of wheat resistance to bunts by real-time PCR 

Kochanova M, Prokinova E, Rysanek P 384 

7-3 Genetics of Resistance Against the Vascular Pathogen Verticillium Longisporum in Brassica and 

Arabidopsis Thaliana 

Häffner E, Konietzki S, Socquet-Juglard D, Gerowitt B, Diederichsen E 385 

7-4 Genetical Analysis of Resistance to Powdery Mildew in Triticale 

Herrmann M, Ruge-Wehling B, Hackauf B, Klocke B, Flath K 393 

7-5 Management of Coffee Leaf Rust, Hemileia Vastatrix, in a Changing Climate 

Kairu G M 401 

8-1 Improving Resistance to Late Blight (Phytophthora Infestans) by Using Interspecific Crosses in 

Potato (Solanum Tuberosum Ssp.) 

Hammann T, Truberg B, Thieme R 407 

8-2 A QTL-Study on Quantitative Maturity-Corrected Resistance to late Blight (Phytophthora infestans) 

in Tetraploid Potato (Solanum tuberosum) 

Truberg B, Thieme R, Hammann T, Darsow U 415 

8-3 Study of Wild Solanum Species to Identify Sources of Resistance Against the Green Potato Aphid, 

Myzus Persicae 

Askarianzadeh A, Birch A NE, McKenzie G, Ramsay G, Minaeimoghadam M 419 

8-4 Wild Potato Species of the Series Pinnatisecta - Progress in their Utilisation in Potato Breeding 

Thieme R, Schubert J, Nachtigall M, Hammann T, Truberg B, Heimbach U, Thieme T 428 

8-5 Can natural resistance help to control nematode transmissible tobacco rattle virus in potato? 

Varrelmann M 437 

9-1 delta13C values - an indicator for drought tolerance of different potato genotypes 

Giesemann, A, Balko, C 438 

9-2 Minimizing Ergot Infection in Hybrid Rye by a SMART Breeding Approach 

Hackauf B, Truberg B, Wortmann H, Fromme F J, Wilde P, Menzel J, Korzun V, Stojałowski S 439 

9-3 Maintenance of NAD homeostasis - a promising route towards multiple stress tolerance in plants 

Hannah M, Metzlaff M 451 

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9-4 The Formation of Biochemical Resistance to Biotic and Abiotic Stress in Cereals 

Molodchenkova O O, Adamovskaya V G 452 

9-5 Contribution of chemical treatments to Crop Stress Tolerance 

Brahm L, Gladwin R, Semar M, Gomes-de-Oliveira C 455 

10-1 Breeding Strategies for Wheat Improvement: Creating Semi-Dwarf Phenotypes with Superior 

Fusarium Head Blight Resistance 

Perovic D, Welz G, Förster J, Kopahnke D, Lein V, Löschenberger F, Buerstmayr H, Ordon F 456 

10-2 Wheat double haploid lines with improved salt tolerance: in vitro selection and RAPD analysis 

Beckuzhina S, Kochieva E 462 

10-3 Induction of defense-related genes in downy-mildew infected sunflower plants treated with 

resistance inducer 

Körösi K, Virányi F 465 

10-4 Sources of Resistance and Development of Molecular Markers for Anthracnose Resistance in 

Narrow-Leafed Lupin (Lupinus angustifolius ) 

Ruge-Wehling B, Thiele C, Eickmeyer F, Wehling P 473 

10-5 Combining Stress Shield Chemistry and Parp-Silencing to Improve Productivity in OSR 

Thielert W, Boer B d 480 

11-1 Arbuscular Mycorrhizal Fungi and Their Roles in Relieving Abiotic Stress 

Bothe H 489 

11-2 Mycorrhizal Cotton Seedlings Withstand the Stress of Verticillium dahliae 

Long X, Bu J, Cui W, Yong R 499 

11-3 Should we breed for effective mycorrhiza symbioses? 

Feldmann F, Gillessen M, Hutter I, Schneider C 507 

12-1 Registration of Plant Protection Products in Poland and the Problem of Resistence 

Matyjaszczyk E 523 

12-2 The Adoption of Bt-Maize in Germany: An Econometric Analysis 

Consmüller N, Beckmann V, Petrick M 530 

12-3 Environmental Risk Assessment of Transgenic Plants Resistant to Biotic and Abiotic Factors 

Schiemann J, Devos Y, Lheureux K 542 

12-4 ENDURE Foresight Study: A Tool for Exploring Crop Protection in Europe in 2030 and Its 

Implications for Research 

Latxague E, Barzman M, Bui S, Abrassart C, Ricci P 554 

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Accompanying IUFRO meeting unit 7.02.04: Viruses in forest and urban trees 

IUFRO-1 Double strand RNA patterns indicate plant viruses associated with dieback affected 

Dalbergia sissoo trees in Bangladesh 

Vogel S, Tantau H, Mielke N, Sarker R H, Khan S, Mühlbach H-P 559 

IUFRO-2 Detection and distribution of European Mountain Ash Ringspot-Associated Virus 

(EMARAV) in Finland 

Kallinen A, Lindberg I, Valkonen J 560 

IUFRO-3 European mountain ash ringspot-associated virus (EMARAV) and relationship to other ss(-) 

RNA viruses: Protein characterisation, RNA localisation and quantification 

Schlatermund N, Mielke N, Mühlbach H-P 561 

IUFRO-4 Co-expression of viral proteins P2 and P3 of European mountain ash ringspot-associated 

virus (EMARAV) in plant protoplasts 

Novikova L, Ikogho B, Ludenberg I, Mielke N, Muehlbach H-P 562 

IUFRO-5 Investigation on virus-like particles associated to decline of Quercus suber 

Nóbrega F, Vidal R, Serrano R 563 

IUFRO-6 Cherry leaf roll virus (CLRV) - genome organisation of the RNA1 

Bargen S von, Langer J, Rumbou A, Gentkow J, Büttner C 564 

IUFRO-7 Study on transmission modes of Cherry leaf roll virus: genetic basis of seed transmissibility 

based on the model system CLRV/A. thaliana and investigation of possible vectors 

Rumbou A, Bargen S von, Büttner C 566 

IUFRO-8 Molecular properties of Cherry leaf roll virus 

Langer J, Bargen S von, Büttner C 567 

IUFRO-9 Epidemiological investigations on Cherry leaf roll virus 

Langer J, Bargen S von, Büttner C 568 

IUFRO-10 Analysis of the 3´ non-coding region of Cherry leaf roll virus, a nepovirus of subgroup c 

Czesnick H, Langer J, Bargen S von, Büttner C 569 

IUFRO-11 Cherry leaf roll virus: a threat to Finnish Betula spp. 

Grubits E, Bargen S von, Langer J, Jalkanen R, Büttner C 571 

IUFRO-12 Occurrence of EMARAV and CLRV in tree species native to Finland 

Arndt N 1 , , Bargen S von 1 , Grubits E 1 , Jalkanen R 2 , Büttner C 1 573 

IUFRO-13 Investigations on virus-diseased elm trees (Ulmus laevis L.) in eastern Germany 

Bandte M, Essing M, Büttner C 575 

IUFRO-14 Studies on Quercus robur - a perspective 

Bandte M, Fabich S, Bargen S von , Büttner C 576 

13

14 

PLENARY SESSIONS 1 & 2

Jellis G J: Crop Plant Resistance to Biotic and Abiotic Factors: Combating the Pressures on Production Systems in 

a Changing World. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 

(2009), 15-20; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

1-1 Crop Plant Resistance to Biotic and Abiotic Factors: Combating the 

Pressures on Production Systems in a Changing World 

Jellis G J 

HGCA, Caledonia House, 223 Pentonville Road, London, N1 9HY, UK 

Email: graham.jellis@hgca.com 

ABSTRACT 

Changes in crop production and the impact of new food and environmental 

legislation are having an influence on the significance of pests and diseases which 

attack plants and reduce yields. Climate change will also have an impact on pests 

and pathogens, and may increase exposure to abiotic stresses such as drought and 

heat. With the expected increase in world population outstripping the land available 

for cropping, maximising utilisable yields by breeding for resistance to biotic and 

abiotic stresses is becoming an imperative. It is therefore important that plant 

breeders can identify the most important constraints on production in a particular 

crop and region. 

INTRODUCTION 

In recent years, food concerns in Europe have been largely centred on safety and quality, on 

how food is grown and on the impact of agriculture on the environment. The food shortages of 

two years ago raised the spectre of basic food security after many years when this was not 

regarded as top priority. With predictions that climate change will reduce the land available for 

cropping, maximising yields has increased as a priority but not at the expense of quality and 

safety. This paper explores this tension and examines the role for plant breeding, especially 

breeding for biotic and abiotic stresses, within modern agriculture. 

FOOD DEMAND 

For the last half a century, global grain demand and production have more than tripled for 

wheat, and for maize (corn) the increase is even greater, with a recent rapid acceleration due to 

the demand in the USA for biofuels. However, throughout the world, approximately 800 

million people are malnourished and the demand for food will increase as the population 

increases. It is predicted that by 2050 the world population will increase from approximately 

6.7 billion to over 9 billion and that the current trend for more resource-intensive diets, which 

15

include more dairy and meat products, will continue. It is estimated that current global 

production of wheat must increase annually by about 2% (Singh & Trethowan 2007). At the 

same time, the demand for land from uses other than agriculture is increasing. Biofuels have 

already been mentioned; other demands include housing and industrial buildings, timber and 

forest conservation. (Evans 2009). The recognition that biodiversity and the quality of the 

environment need to be preserved for future generations also leads to competition for land use 

and can also negatively affect crop yields through a reduced use, or total exclusion of, 

inorganic fertilisers and pesticides as in, for example, organic systems. 

A comprehensive study of pest, disease and weed losses in eight crops which occupy half the 

world’s cropped land to date was published by a team of German crop scientists (Orke et al. 

1994). The study found that, overall, pests accounted for preharvest losses of 42% of the 

potential value of output, with 15% attributable to insects and 13% each to weeds and 

pathogens. An additional 10% of the potential value was lost postharvest. Losses in Europe 

alone were lower (for example a loss of 9.7% of production caused by plant diseases) but were 

still very significant, in spite of a substantial use of pesticides. Abiotic stresses such as drought, 

heat and salinity add considerably to these losses, and are likely to increase with climate 

change (see below). In a world demanding more food from a limited amount of land, improved 

resistance to biotic and abiotic stresses is a priority 

CLIMATE CHANGE 

At the same time as the demand for food is intensifying, the climate is changing, with 

inevitable consequences for agriculture and the world’s food supply. The potential 

consequences have been discussed by Rosenzweig & Hillel (1995). They state that 

“vulnerability to climate change is systematically greater in developing countries, which in 

most cases are located in lower, warmer latitudes. In those regions, cereal grain yields are 

projected to decline under climate change scenarios, across the full range of expected warming. 

Agricultural exporters in middle and high latitudes …stand to gain, as their national production 

is predicted to expand, and particularly if grain supplies are restricted and prices rise. Thus, 

countries with the lowest income may be the hardest hit.” 

In Europe, predicted changes in climatic conditions depend on location, with the greatest levels 

of warming predicted for Mediterranean and north-eastern areas, increased precipitation in 

northern areas (particularly in winter) and decreased precipitation in southern areas (Brooker & 

Young 2006). The challenge in many areas of the world will be to produce more food with 

limited supplies of water, and breeding for drought tolerance and water use efficiency are key 

to this (Ober 2008). Globally, drought already results in greater yield loss than any other single 

biotic or abiotic factor (Boyer 1982) and even in the UK drought losses are estimated to be 1-2 

t/ha (Foulkes et al. 2007). However, Semenov (2008) considers that, in England and Wales, 

heat stress around flowering might represent a greater risk to wheat production in England than 

drought, because although the summer is predicted to be drier in the 2050s, winter is predicted 

to be wetter and water might still be available to the growing crop in late spring and summer. 

16

Collier et al. (2008) evaluated the potential impact of future extreme weather events on 

horticultural crops in the UK using a stochastic weather generator linked with UKCIP02 

(Hulme et al. 2002) projections of future climate. This study indicated that episodes of summer 

drought severe enough to interrupt the continuity of supply of salads and other vegetables will 

increase and there will be a requirement for winter cauliflowers with different temperature 

sensitivities from those used currently. Important pests, such as cutworm (Agrotis segetun) and 

diamond-back moth (Plutella xylostella) could become a greater threat: in the case of the 

former, the number surviving to third instar increased with time in the model; in the latter, 

there was an increase in the number of generations. The impact of climate change on other 

pests due to changes in life cycles might be expected throughout Europe and may pose a 

serious threat to production systems. 

Climate change is also expected to impact on pathogens and pathosystems. Turner (2008) 

reported modelling work to predict potential levels of disease under climate change for the 

years 2081-2090. She concluded that wheat brown rust (Puccinia recondita) will become the 

primary target for disease control strategies, because it is favoured by warmer, drier summers. 

Conversely, Septoria leaf blotch (Mycosphaerella graminicola), currently the most important 

disease of wheat in the UK, was predicted to decline. However, Turner acknowledged that the 

model only accounted for effects on the pathogen and climate will also affect the host, making 

risk prediction more challenging. Roche et al. (2008) modelled the potential impact of climate 

change on wheat brown rust for four contrasting French sites, taking into account a range of 

climatic factors and their effect on the pathogen and plant-pathogen interactions. Surprisingly 

they found no clear trend in infection rates, which they concluded was due to opposing effects, 

for example an increase in temperature accelerated the disease cycle but was counteracted by a 

reduction in leaf surface wetness duration. Also, when plant development changes were taken 

into account, although temperature accelerated the disease cycle it also had the same effect on 

the development of the crop, maintaining the status quo. 

The spread of diseases may well also be affected by climate change. Insect vectors of 

pathogens such as the fungi causing Dutch elm disease (Ophiostoma ulmi and O. novo-ulmi) 

are likely to respond to warmer summers by extending their geographic ranges and hence the 

ranges of disease incidence. Another important pathogen of trees, Phytophthora cinnamomi, 

an aggressive introduced fungus which causes root and stem-base diseases of oaks, chestnuts 

and many other tree species, is predicted to become more active across coastal areas of the UK 

and Europe (Lonsdale & Gibbs 2002) 

Agriculture is potentially very sensitive to climate change but there are clearly many 

uncertainties, which create difficulties for plant breeders who are making a long-term 

investment. However, breeding for disease resistance may not only be beneficial in adaptation 

to climate change, it may have a role in limiting greenhouse gas emissions. Berry et al. (2008) 

calculated the reductions in emissions that could be achieved in the UK from disease control: 

with current cultivars and fungicide use, there is the potential to save up to 1.14Mt CO2 eq. per 

annum. This saving could be improved through the use of more effective disease resistance, 

17

providing it is not associated with a yield penalty. A general increase in resistance to Septoria 

leaf blotch of one point on the 1-9 scale used by the HGCA Recommended Lists (Anon. 2009) 

would decrease greenhouse gas emissions by approximately13kg CO2 eq. 

LAND USE 

In an area as diverse as Europe, it is not possible to generalise on changes in land use. 

However, a desk-based study has been carried out on cropping on the chalkland of the East 

Anglian region of the UK, which is nowadays primarily arable (Parry et al. 2006) Over recent 

years there has been a decline in mixed farming and a switch from spring to autumn cropping. 

The study suggested that if market forces determine land use in this area in the future, the 

landscape would become more aggregated, with oilseed rape and wheat dominating. Wheat 

would be farmed on the larger fields on average, with oilseed rape on smaller fields. 

Economies of scale and greater use of contractors would lead to block-cropping, with large 

areas (>200ha) of a single crop. 

The report was principally commissioned to study the impact of land use changes on the 

environment but clearly disease and pest pressure will increase if fewer crop types are grown in 

larger blocks. Since 2006, when the report was published, set-aside has been abolished within 

the EU, reducing the amount of land left fallow. 

EU LEGISLATION 

EU legislation introduced in recent years as a response to concerns about food safety and 

agriculture’s impact on the environment may have a impact on our ability to control diseases 

and pests, especially in those countries which have a considerable reliance on pesticides, such 

as the UK where the mild and wet climate encourages the development of many diseases. 

Pesticide legislation 

Currently the EU is in the final negotiation phase of a new legislative package on pesticides (a 

revision of Directive 91/414/EEC). Among other things, this introduces cut-off criteria based 

on hazards rather than risks. It is still not clear how many pesticides will eventually be 

withdrawn as a result of this legislation. It has been suggested that the list may include 

fungicides such as some of the triazoles (which control powdery mildews, rusts, and many leafspotting 

fungi on a wide range of crops), mancozeb (which controls downy mildews and potato 

blight (Phytophthora infestans) and is widely seen as vital to prevent resistance developing in 

other fungicides) and quinoxyfen (which controls powdery mildews on, for example, cereals 

and grape vines). They will add to the 60% which have already been withdrawn from the 

European market over the last ten years (Anon. 2008a) 

18

Water Framework Directive 

The implementation of the Water Framework Directive is likely to have an impact on a number 

of pesticides. Although herbicides are particularly vulnerable, many insecticides are also at 

risk, and metaldehyde, used for controlling slugs, is already under scrutiny as it has been found 

in water at concentrations above the EU limit (Twining et al. 2009). Metaldehyde is 

particularly important for growers of vegetables, potatoes and oilseed rape. Some crops, such 

as potatoes (Johnston & Pearce 2008), differ significantly in cultivar resistance to attack by 

slugs, whereas others, such as oilseed rape, do not. 

In addition to these products, important pesticides have been lost in recent years due to 

pesticide resistance and there is concern that, with a reduction in the number of active 

chemicals, there will be an increase in selection pressure for resistance. Many organisations 

have been advocating a greater use of integrated pest management systems for a number of 

years and enhanced disease and pest resistance clearly has a significant part to play in this 

(Anon 2008a). 

Mycotoxin legislation 

In 2006 the European Commission introduced regulation No. 881/2006 setting maximum 

levels for certain contaminants in foodstuffs. This included a number of important mycotoxins, 

including aflotoxins in dried fruit and cereals, Ochratoxin A in cereals, dried vine fruit and 

wine, patulin in apples, cider and fruit juices, deoxynivalenol (DON) and zearalenone in 

processed and unprocessed cereals including maize, and fumonisins in maize (Anon. 2006). 

Maximum levels for the Fusarium mycotoxins (DON, zearalenone and fumonisins) in maize 

were lowered the following year, in order to avoid a disruption of the market whilst 

maintaining a high level of public health protection. 

Mycotoxin regulations are having a significant effect on food production. For example, in the 

UK, control of Fusarium head blight has become a much more important issue, because of the 

production of DON. In the field, infection of ears by some Fusarium species can result in the 

production of mycotoxins when weather is warm and wet at flowering but there is little 

correlation between Fusarium-damaged grain and mycotoxin occurrence. In the past two 

seasons, until January 2008, millers relied on a risk assessment developed by the HGCA and 

completed by farmers (Anon. 2008b). However, concentrations of DON above the EU 

accepted limit have been found in batches of grain for milling classified as at low risk which 

has led to compulsory testing for mycotoxin and a review of the risk assessment system. 

Fungicide control of head blight is not easy, as fungicides have to be applied at precisely the 

right time. Improved resistance would be of great benefit as it has been demonstrated that DON 

levels are lower in wheat varieties with higher disease resistance (Edwards 2007). Until 

recently, this disease was not regarded as of high priority for UK plant breeders but this has 

changed because of the legislation. 

19

THE CHALLENGE FOR PLANT BREEDING 

Even with modern techniques which can speed up the breeding process, plant breeders have to 

determine their objectives many years in advance of a cultivar being released. This has never 

been easy but with the pressures on production systems described above and the largely 

unpredictable effect of a number of them, determining the importance of specific traits 10-15 

years ahead of the release of a new cultivar is a real challenge. To add to the difficulty, a whole 

range of traits alongside resistance to biotic and abiotic stresses have to be considered. For 

example, the HGCA Recommended List 2009/10 for winter wheat (Anon. 2009) measures 

treated and untreated yield plus eight grain quality characteristics and five agronomic features 

alongside resistance to seven diseases and one pest. There is also a need to try and ensure that 

the sources of resistance to pathogens employed are durable, to escape from the ‘boom and 

bust’ cycle common with some pathosystems. Many sources of durable resistance are 

controlled by a number of genes which makes breeding much more challenging. 

Molecular biology has developed rapidly in the past two decades and this is benefiting 

resistance breeding programmes, although the impact is not as great as it could be if genetic 

modification was more acceptable in Europe. The production of markers for resistance genes 

and other traits is developing rapidly and will enable much more effective selection to take 

place. In addition to this, there is a greater understanding of the mechanisms of resistance, 

through the cloning and characterization of resistance genes and an understanding of biotic and 

abiotic stress signalling pathways. These scientific developments have the potential to provide 

new strategies for effective breeding for host resistance to stresses in the future. 

REFERENCES 

Anonymous (2006). Commission Regulation (EC) No 1881/2006 setting maximum levels of 

certain contaminants in foodstuffs. Official Journal of the European Union L364, 5-24. 

Anonymous (2008a). ENDURE’s position on the consequences of the European pesticide 

policy. www.endure-network.eu (date of access 11.03.09). 

Anonymous (2008b). Managing Fusarium mycotoxin risk in wheat intended for human food – 

harvest 2008. Topic Sheet No 102. HGCA: London. 

Anonymous (2009). Recommended Lists 2009/10 for cereals and oilseeds. HGCA: London. 

Berry P M; Kindred D R; Pavely N D (2008). Quantifying the effects of fungicides and disease 

resistance on greenhouse gas emissions associated with wheat production. Plant 

Pathology 57, 1000-1008. 

Boyer J S (1982). Plant productivity and environment. Science 218, 443-8. 

Brooker R; Young J (eds) (2006). Climate change and biodiversity in Europe: a review of 

impacts, policy responses, gaps in knowledge and barriers to the exchange of in 

formation between scientists and policy makers. 

http://nora.nerc.ac.uk/3301/1/WC02018_3361_FRP.pdf (date of access 11.03.2009). 

Collier R; Fellows J; Adams S; Semenov M; Thomas B (2008). Vulnerability of horticultural 

crop production to extreme weather events. In: Effects of climate change on plants: 

Implications for agriculture, eds N Halford, H D Jones & D Lawlor, pp. 3-13. 

Association of Applied Biologists: Warwick. 

20

Edwards S (2007). Investigation of Fusarium mycotoxins in UK wheat production. HGCA 

Project Report No. 413. HGCA:London. 

Evans A (2009). The Feeding of the Nine Billion: Global Food Security for the 21 st Century. 

Chatham House: London. 

Field C (2008). Agriculture in a changing environment. (Abstract) Phytopathology 98, 52. 

Foulkes M J; Sylvester-Bradley R; Weightman R; Snape J W (2007). Identifying traits 

associated with improved drought resistance in winter wheat. Field Crops Research 

103, 11-24. 

Hulme M; Jenkins G J; Lu X; Turnpenny J R; Mitchell T D; Jones R G; Lowe J; Murphy J M; 

Hassell D; Boorman P; McDonald R; Hill S (2002). Climate change scenarios for the United 

Kingdom: The UKCIP02 scientific report. University of EastAnglia: Norwich. 

Johnston K A; Pearce R S (2008). Biochemical and bioassay analysis of resistance of potato 

(Solanum tuberosum L.) cultivars to attack by the slug Deroceras reticulatum (Muller). 

Annals of Applied Biology 24, 109-131. 

Lonsdale D; Gibbs J N (2002). Effects of climate change on fungal diseases of trees. In: 

Climate change: impacts on UK forests, ed. M S J Broadmeadow pp. 83–97. Forestry 

Commission Bulletin No 125. Forestry Commission: Edinburgh. 

Ober E S (2008). Breeding for improved drought tolerance and water use efficiency. In: 

Proceedings HGCA R&D conference: Arable cropping in a changing climate 2008, pp. 

28-37. 

Orke E C; Dehne H W; Schonbeck F; Weber A (1994). Crop production and crop protection: 

Estimated losses in major food and cash crops. Elsevier: Amsterdam. 

Parry H; Ramwell C; Bishop J; Cuthbertson A; Boatman N; Gaskell P; Dwyer J; Mills J; 

Ingram J (2006). Quantitative approaches to assessment of farm level changes and 

implications for the environment. (date of access 11.03.2009): 

https://statistics.defra.gov.uk/esg/ace/research/pdf/obs03.pdf. 

Roche R; Bancal M-O; Gagnaire N; Huber L (2008). Potential impact of climate change on 

brown wheat rust: a preliminary study based on biophysical modeling of infection 

events and plant-pathogen interactions. In: Effects of climate change on plants: 

Implications for agriculture, eds N Halford, H D Jones & D Lawlor, pp. 135-142. 

Association of Applied Biologists: Warwick. 

Rosenzweig C; Hillel D (1995). Potential impacts of climate change on agriculture and food 

supply. Consequences 1, 23-32. 

Semenov M A (2008). Extreme impacts of climate change on wheat in England and Wales. In: 

Effects of climate change on plants: Implications for agriculture, eds N Halford, H D 

Jones, D Lawlor, pp. 37-38. Association of Applied Biologists: Warwick. 

Singh R P; Trethowan R (2007). Breeding spring bread wheat for irrigated and rainfed 

production systems of the developing world. In: Breeding major food staples, eds M S 

Kang & P M Priyadarshan, pp 109-140. Blackwell: Ames. 

Turner J A (2008). Tracking changes in the importance and distribution of diseases under 

climate change. Proceedings HGCA R&D conference: Arable cropping in a changing 

climate 2008, pp. 68-77. 

Twining S; Berry P; Cook S; Ellis S; Gladders P; Clarke J; Wynn S (2009). Pesticide 

availability for cereals and oilseeds following revision of Directive 91/414/EEC. HGCA 

Research Review No 70. HGCA: London. 

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Weigel H J, Bender J: Atmospheric composition – a threat to crop growth and health? In: Feldmann F, Alford D 

V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 22-32; ISBN 978-3-941261-05-1; © 

Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

2-1 Atmospheric composition – a threat to crop growth and health? 

Weigel H J, Bender J 

Institute of Biodiversity, Johann Heinrich von Thünen-Institute (vTI), Federal Research 

Institute for Rural Areas, Forestry and Fisheries, Bundesallee 50, 38116 Braunschweig, 

Germany Email: Hans.Weigel@vti.bund.de 

INTRODUCTION 

Anthropogenic activities have significantly changed the composition of the global atmosphere. 

Especially, the concentrations of several trace gases have undergone significant changes during 

the past century and continue to change. Plants are important mediators in the exchange of the 

different gaseous and particulate compounds between the atmosphere and the biosphere (Table 

1). The transport of these compounds from the atmosphere into vegetation is by dry and wet 

deposition of gases, aerosols and sedimenting particles. Many atmospheric constituents can 

influence crop performance, both directly by affecting growth and qualiy or indirectly by 

altering the plant´s ability to cope with other abiotic and biotic stresses. In terms of their impact 

on agricultural ecosystems they can be broadly divided into: 

− compounds which act as macro- or micronutrients (e.g. the gases CO2, SO2, NO, NO2, 

NH3 and particulate NH4, NO3-N, SO4-S, P, Ca, Fe, Mg) and 

− compounds which may cause adverse or toxic effects (e.g. the gaseous pollutants O3, 

SO2, NO2, NH3, HF, PAN, NMHC or VOC, metals like Pb, Cd, Hg) or excess nutrient 

substances (e.g. N, S, Zn, Al) which alter normal pattern of growth and development in 

ecosystems (Dämmgen & Weigel, 1998). 

The latter category is mostly termed as air pollutants. 

Ambient air is always composed of pollutant mixtures, with the concentrations of individual 

pollutants varying in time and with location. For example, a particular air pollutant such as SO2 

or NH3 can be dominant only in the vicinity of its sources, i.e. those pollutants are primarily of 

local importance. In comparison, among secondary pollutants, O3 is of widespread global 

occurrence and can currently be considered to be the most important air pollutant (Fuhrer 

2009). Among the different environmental factors which determine crop growth potential 

recent and predicted further changes of climate (i.e. increased temperature, altered pattern of 

rainfall intensity and frequency) including atmospheric CO2 concentration as well as other 

atmospheric compounds have become and will be of growing importance. Therefore, 

projections of global food security must similarly consider the likely impacts of climate change 

and air pollution. With respect to effects, this paper will focus on responses of crops to O3 and 

22

CO2 as these trace gases are key variables of climatic and atmospheric change for future global 

food production (Long et al. 2005). 

Table 1: Examples of atmospheric compounds involved in element flux between vegetation 

and atmosphere (after Dämmgen & Weigel 1998). 

___________________________________________________________________________ 

Species Potential effects 

• H2O-vapor, CO2, CH4, N2O, NO2, O3 � trapping of infrared radiation, 

contribution to the greenhouse effect 

• NH3, CO, HC � effects on reactivity of the atmosphere 

• CH4, CO2, SO2, H2S,NO2, NO, NH3 , � involved in nutrient cycling, 

NH4-/NO3-N, SO4-S, P, Ca, K, act as macro- and micronutrients 

Fe, Mg (particles) 

• O3, SO2, NO2, HF, H2O2, PAN, � potentially toxic, affecting “normal” 

NMHC/VOC (gases), heavy metals growth and performance of organisms, 

(e.g. Pb,Cd, Hg), surplus nutrients populations and ecosystems 

(bioavailable forms of N, S, Zn, Al) 

Abbreviations: Al: aluminum; Ca: calcium; Cd: cadmium; CH4: methane; CO: carbon monoxide; CO2: carbon 

dioxide; Fe: iron; HC: hydrocarbons; HF: fluoride; Hg: mercury; H2O: water vapor; H2O2: hydrogen peroxide; 

H2S: hydrogen sulfide; K: potassium; Mg: magnesium; N: nitrogen; N2O: nitrous oxide; NH3: ammonia; NH4 + : 

ammonium; NH4NO3: ammoniumnitrate; NO: nitrogen monoxide; NO2: nitrogen dioxide; NO3 - : nitrate; NOX: 

NO + NO2; NMHC: non-methane hydrocarbons; O3: ozone; P: phosphorus; PAN: peroxyacetylnitrate; Pb: lead; 

S: sulfur; SO2: sulfur dioxide; SO4 2- : sulfate; VOC: volatile organic compounds; Zn: zinc. 

ATMOSPHERIC CHANGE: SPATIAL AND TEMPORAL TRENDS 

The concentrations of several of the compounds listed above in many parts of the industrialized 

world have changed significantly during the last century (Dämmgen & Weigel 1998). While 

local emissions of urban or industrial sources still occur, emissions particularly of SO2, and to a 

smaller extent of NOx (NO+NO2), VOC and particulate matter have declined during the past 

decades in Europe and North America. This was due to successful policies to reduce emissions, 

as well as a decline of polluting heavy industries (UNECE 2007). SO2 levels and sulphur bulk 

deposition, for example, are now usually low during the growth periods of crops (

threat for annual crops. For the majority of heavy metals (e.g. Pb, Cd, Ni, Hg, Zn) a similar 

decline of emission and subsequent deposition is observed since the late 1980s in most of 

Europe, although higher metal deposition is still found in some eastern European countries 

(Harmens et al. 2008). 

In contrast to the situation in Europe and North America, air pollutant emissions have been 

increasing over the last two decades in many developing countries, particularly in rapidly 

growing regions of Asia, Africa and Latin America, where rapid industrialization and 

population growth is taking place accompanied by increasing energy demand and road traffic, 

but with poor emission controls (Emberson et al. 2003). China and India are now the leading 

emitters of SO2 in the world (Marshall 2002). Also, the predicted increase in global NOx 

emissions may be attributed largely to the high percentage increases in developing countries, 

such as China (Marshall 2002) 

Tropospheric O3 is a widespread secondary air pollutant found in all industrialized countries 

worldwide and meanwhile also in many of the developed countries in the world where it has 

reached levels in ambient air which are of concern with respect to vegetation damage and 

human health effects (Emberson et al. 2003), and these trends are expected to continue as 

economies continue to expand. While at least in most parts of western Europe there is a clear 

trend of decreasing O3 peak values (“photosmog episodes”), predictive models indicate that 

background O3 concentrations will continue to increase at a rate of 0.5% to 2% per year in the 

Northern Hemisphere during the next several decades. Currently, the background O3 

concentration in the Northern hemisphere is within the range of 23-34 ppb, however, global 

surface O3 concentration is expected to be in the range of 42-84 ppb by 2100 (Vingarzan 

2004). Figure 1 shows the projected global increase in O3 concentration over the next 100 years 

from Prather et al. (2003), based on IPCC global emission scenarios. According to that, the 

locations of the major O3 increases ("hot-spots") in the future are expected to be Asia, southern 

Africa, southern Europe and USA. 

In contrast to the different temporal trends of the “classical” air pollutants like SO2 and NOx 

between industrialized and developing countries, atmospheric CO2 concentration has risen 

steadily all over the globe from a pre-industrial concentration of about 280 ppm to a current 

value of about 385 ppm, and could reach > 550 ppm already by 2050 (IPCC 2007). Due to the 

direct effects of rising CO2 levels on crop photosynthesis, growth and quality, assessments of 

future air pollution effects on plants and crops have to consider this rapid change. 

CROP RESPONSES TO AIR POLLUTION AND CLIMATE CHANGE 

Direct effects on yield and quality 

Gaseous atmospheric compounds are transferred from the atmosphere onto plant canopies by 

diffusion which is governed by micro-meteorological conditions (radiation, temperature, wind 

etc.). The major path of entry into the leaf is through the stomata. The reaction of a plant to a 

given air pollutant depends on the exposure characteristics, plant properties, and external 

24

growth conditions (Bender & Weigel 2003). Short-term exposures to relatively high 

concentrations generally result in acute visible foliar injury. Long-term chronic exposures to 

lower concentrations can cause physiological alterations that may result in chlorosis, premature 

senescence, and in growth and yield reductions. For agriculture, chronic effects of air 

pollutants such as O3 are of particular concern, because they are due to exposures for weeks, 

months, or over the entire lifecycle of the crop. It is well known that increasing O3 levels 

causes a decline in the yield of many crop species, such as wheat, rice, soybean and cotton 

(Ashmore 2005). Such yield losses have been attributed to reduced photosynthetic rate, altered 

carbon allocation, and accelerated leaf senescence (Fiscus et al. 2005; Fuhrer 2009). Mills et 

al. (2007) analysed O3 exposure-response data for 19 agricultural and horticultural crops, 

respectively, and identified wheat, water melon, pulses, cotton, turnip, tomato, onion, soybean 

and lettuce as the most ozone-sensitive crops, while, for instance, barley was classified as O3 

resistant. Holland et al. (2006) estimated crop losses and the associated economic loss in 

Europe for 23 horticultural and agricultural crops for the base year 2000 and found an overall 

loss of 3% of all crop species considered, which would be equivalent to € 6.7 billion economic 

damage. The global impact of O3 on crop yields was recently evaluated by Van Dingenen et al. 

(2009). Their estimates of present day global relative yield losses ranged between 7% and 12 % 

for wheat, between 6% and 16% for soybean, between 3 % and 4% for rice, and between 3% 

and 5% for maize. When translating the production losses into global economic damage for the 

four crops considered, they estimated an economic loss in the range of $14-26 billion. About 

40% of this damage is occuring in China and India. However, the uncertainty on these 

estimates is large.This is primarily due to the O3 exposure metrics used in the estimates, which 

are based on the exposure concentrations in ambient air, either on a regional, national or global 

scale, rather than on the actual uptake of O3 and thus do not account for the dose-specific 

nature of plant responses. In addition, only the direct O3 effects on crop growth are considered, 

i.e. indirect growth effects e.g. mediated by phytosanitary problems are not taken into account 

(see 3.2). Moreover, wide variability in O3-sensitivity among various crop cultivars is common 

(USEPA 2006). 

By contrast, a future rise in atmospheric CO2 levels principally will have a positive effect on 

crop growth and yield, as CO2 directly affects plant physiology and growth by serving as a 

primary substrate for photosynthesis. Generally, elevated CO2 concentrations increase biomass 

and yield substantially in C3 crops by increasing photosynthesis and decreasing 

photorespiration, but with large differences among species in the magnitude of the yield 

stimulation (Kimball et al. 2002). No significant stimulation of yield was found so far in C4 

crops, at least under well watered conditions, because C4 photosynthesis is saturated under 

ambient CO2 (Long et al. 2005). However, in all crops (both C3 and C4) higher CO2 

concentrations reduce stomatal conductance and transpiration and improve water-use 

efficiency, i.e. crops will experience a reduced demand for water. 

25

26 

Fig. 1: Increases in global surface O3 concentration from 2000 to 2100. 

Adapted from Prather et al. (2003) 

In comparison to air pollutant and climate change effects on crop growth and yield, much less 

is known about potential effects on the quality or the nutritive value, respectively, of 

agricultural and horticultural crops. Changes in crop quality due to O3 exposure have been 

studied in a limited number of crops. For example, in wheat, O3 reduced yield but increased 

grain protein concentration (Pleijel et al. 1999; Piikki et al 2008). Moreover, O3 was found to 

have positive effects on the quality of potato tubers by decreasing reducing sugars and 

increasing the vitamin C content (Vorne et al. 2002). In contrast, O3 has been found to reduce 

the oil, protein, and carbohydrate contents of the seeds of rape (Ollerenshaw et al. 1999). 

Recent evidence suggests that O3 can also alter the plant food quality for ruminant animals.

Decreased nutritive quality of forages was found in a number of pasture species (Krupa et al. 

2004; Bender et al. 2006). 

Pollutant-induced visible injury is of particular significance when the quality and the 

marketable value of the crop depend on the appearance of the foliage as it is the case for a 

number of horticultural crops. For example, Kostka-Rick et al. (2002) have shown that 

environmentally-relevant concentrations of O3 can cause visible foliar injury on species like 

lettuce, spinach or onion, which would make these crops unmarketable. 

A frequently observed phenomenon is that plants grown at high CO2 levels exhibit significant 

changes of their chemical composition (Idso & Idso, 2001; Loladze, 2002). A prominent 

example of a CO2 effect is the decrease of the nitrogen (N) concentration in vegetative plant 

parts as well as in seeds and grains and, related to this, the decrease of the protein 

concentrations (Cotrufo et al.1998; Taub et al. 2008; Wieser et al. 2008). Other CO2 

enrichment studies have shown changes in the composition of other macro- and microelements 

(Ca, K, Mg, Fe, Zn) and in concentrations of secondary compounds, vitamins and sugars (Idso 

& Idso, 2001). Overall, these CO2 induced changes may have negative consequences with 

respect to nutritional quality of foods and feeds, the plant-herbivore interaction and the element 

turnover of ecosystems, respectively. 

The examples above indicate that there may be economically important effects of air pollution 

and climate changes on the quality of crops and forage species, although the avilable 

information is still inconsistent. 

Indirect effects 

Atmospheric compounds and air pollutants, respectively, may interact with other biotic and 

abiotic growth or stress factors (e.g. water and nutrient supply; heat and water stress; salinity, 

pesticide application; pests and pathogens; symbiontic relationships) in a complex manner thus 

causing indirect effects on crop performance. For example, while it is well accepted that 

reduced vitality to O3 stress can make plants more susceptible to plant pathogens, general 

predictions of O3 effects on particular plant-pathogen systems are difficult, because the 

available data for specific pests and diseases are often controversial (USEPA 2006; Fuhrer 

2009). Increased susceptibility after O3 exposure has been reported for necrotrophic pathogens, 

while obligate biotrophic infections tend to be diminished by O3 (Manning & von Tiedemann 

1995; USEPA 2006). With regard to insect pathogens, there is a general trend that some pests 

may have a preference for and grow better when feeding on O3 stressed plants, but there are 

also other observations where insect growth was not changed (USEPA 2006). Viral infection 

often provides some protection from O3 injury, however, the type and degree of protection 

depend on the specific host and virus (Manning & von Tiedemann 1995). 

The direct effects of elevated CO2 levels on tissue chemical composition can have an indirect 

effect on plant-herbivore interactions, as host plants growing under enriched CO2 environments 

usually exhibit e.g. decreased tissue N concentration, increased C/N ratio and generally altered 

27

secondary metabolism of C-based secondary and structural compounds. This in turn may affect 

food consumption by herbivores and related population development (Stiling & Cornelissen 

2007). However, there is almost no information about how O3 effects on plant-pathogen 

systems may be modified in a future climate with elevated CO2 (Chakraborty et al. 2000; 

Fuhrer 2009). For example, while host plants growing under enriched CO2 environments 

usually exhibit larger biomass, increased C/N ratios and decreased tissue N concentration, O3 

has the opposite effect (Pleijel et al. 1999; Piikki et al. 2008). Hence, it remains open, how 

food consumption by herbivores and population development is affected under future 

atmospheric conditions characterized by elevated O3 and CO2 concentrations (Stiling & 

Cornelissen, 2007). 

Another important interaction may occur between the effects of air pollutants and soil moisture 

availability. Water supply directly affect stomatal conductance and hence the uptake and 

effects of gaseous air pollutants. For example, it is known that reduced soil moisture limit O3 

uptake by decreasing stomatal conductance, which increase O3 tolerance (Bender & Weigel, 

2003). However, other findings suggest that, in some species, soil moisture stress may reduce 

rather than increase O3 tolerance (Bungener et al. 1999). The complex physiological and 

morphological changes due to water deficit impair plant vitality itself, e.g. by promoting 

senescence processes. Therefore, decreased pollutant uptake may not necessarily be connected 

with decreased pollutant injury. As outlined before, elevated CO2 concentrations often improve 

water use efficiency, i.e. may mitigate drought stress effects (Manderscheid & Weigel 2007), 

which is an important feedback effect in future climate change scenarios. 

Although the available information is clearly insufficient to understand the importance of 

interactions between air pollutants and biotic or abiotic factors, it is suggested that these 

indirect effects could be more important under certain circumstances than the directs effects of 

the gases on plants. 

Interactive effects of atmospheric compounds 

Under field conditions plants are exposed to different environmental factors including more 

than only one atmospheric compound. Based primarily on experimental work it has been 

shown that mixtures of atmospheric compounds and air pollutants, respectively, modify the 

magnitude and nature of the response to individual compounds. Generally, pollutant 

combinations may result in either more-than additive (synergistic) or less-than-additive 

(antagonistic) effects. Based on the prevailing conditions at that time interactions of O3 with 

other air pollutants (e.g. SO2, NO2) have been studied quite frequently in the 1980's (reviewed 

by Fangmeier et al. 2002). Currently, at least for Europe and North America, a simultaneous 

occurence of O3, SO2, NO2 or NH3 at phytotoxic levels is rather unusual and far less frequent 

than sequential or combined sequential/concurrent exposures. From experiments where 

exposure conditions have been more realistic in terms of their likelihood of occurrence in 

ambient air it can be concluded that: (1) antagonistic interactions are tend to be found when 

gases were applied sequentially (e.g. O3/NO2) and/or when e.g. nitrogenous or sulphurous air 

28

pollutants were combined with O3 at relatively low levels, suggesting that plants were able to 

utilize the additional S or N source, and, (2) synergistic interactions are more likely to be found 

when O3 was applied simultaneously with another pollutant at high concentrations (Fangmeier 

& Bender 2002). For the situation in Europe and North America this would imply that both 

SO2 or NO2 seems unlikely to pose an additional risk to the one related to O3. However, the 

effects of pollutant combinations on crop growth and yield should have a much higher 

significance in many developing countries where air pollutants such as SO2, NOx and O3 are 

rapidly increasing. 

With respect to the future there is some evidence that elevated CO2 has the potential to mitigate 

negative effects of O3 (and other gaseous pollutants), mainly due to a CO2-induced reduction in 

stomatal conductance, which reduces O3 uptake. On the other hand, O3 limits positive CO2 

responses in many plants as well (Fiscus et al. 2005). All climate change factors (CO2, 

warming, changes in precipitation etc.) which may affect stomatal conductance and thus the 

flux of gaseous air compounds into leaves will exert a modification on the effects of individual 

pollutants (Bender & Weigel, 2003; Harmens et al. 2007). 

CONCLUSIONS 

Crops, similar to all other types of vegetation, are closely linked to the exchange of matter 

between atmosphere and biosphere. After deposition of atmospheric compounds to canopies, 

crop growth and quality may be affected in various ways. For the situation in most parts of 

Europe and North America exposure to compounds like SO2, NO2/NO, VOC´s and heavy 

metals is reduced and is currently no major threat to crops. However, in many regions of both 

continents continuously increasing background levels of tropospheric O3 remain a problem 

which poses an additional risk to crop growth and health during the growing season. In the 

growing economies of many developing countries the concentrations of atmospheric 

compounds such as SO2, NOx , NH3 and particularly O3 are rapidly increasing. Already now, 

these pollutants can lead to serious reductions of crop growth and yields, a situation which may 

exacerbate in the future. On a global scale the rapid change in atmospheric composition by the 

increase of the atmospheric CO2 concentration accompanied by climate change has two major 

implications. A possible benefit to crop growth by direct stimulation of photosynthesis and by 

mitigation of e.g. gaseous air pollutant and water stress, but concomitantly a threat to crop 

production due to an enhancement of crop quality losses. 

REFERENCES 

Ashmore M R (2005). Assessing the future global impacts of ozone on vegetation. Plant, Cell 

and Environment 28, 949-964. 

Bender J; Weigel H -J (2003). Ozone stress impacts on plant life. In: Ambasht R S; Ambasht N 

K (Eds.), Modern Trends in Applied Terrestrial Ecology. Kluwer Academic/Plenum 

Publishers, New York, pp. 165-182. 

29

Bender J; Muntifering R B; Lin J C; Weigel H J (2006). Growth and nutritive quality of Poa 

pratensis as influenced by ozone and competition. Environmental Pollution 142, 109- 

115. 

Bungener P; Nussbaum S; Grub A; Fuhrer J (1999). Growth response of grassland species to 

ozone in relation to soil moisture conditions and plant strategy. New Phytologist 142, 

283-294. 

Chakraborty S; von Tiedemann A; Teng P S (2000). Climate change: potential impact on plant 

diseases. Environmental Pollution 108, 317-326. 

Cotrufo M F; Ineson P; Scott A (1998). Elevated CO2 reduces the nitrogen concentration of 

plant tissue. Global Change Biology 4, 43-54. 

Dämmgen U; Weigel H J (1998). Trends in atmospheric composition (nutrients and pollutants) 

and their interaction with agroecosystems. In Sustainable agriculture for food, energy 

and industry: strategies towards achievement. El Bassam N; Behl R; Prochnow B eds. 

(James & James), pp. 85-93. 

Davison A W; Cape J N (2003). Atmospheric nitrogen compounds – issues related to 

agricultural sytems. Environment International 29, 181-187. 

Emberson L D; Ashmore M R; Murray F (Eds.) (2003). Air Pollution Impacts on Crops and 

Forests: a Global Assessment. Imperial College Press, London. 

Fangmeier A; Bender J (2002). Air pollutant combinations: significance for future impact 

assessments on vegetation. Phyton 42, 65-71. 

Fangmeier A; Bender J; Weigel H J; Jäger H J (2002). Effects of pollutant mixtures. In: Bell J 

N B & M Treshow (eds.). Air pollution and plant life, pp. 251-272. John Wiley & Sons, 

Chichester. 

Fiscus E L; Booker F L; Burkey K O (2005). Crop responses to ozone: uptake, modes of 

action, carbon assimilation and partitioning. Plant Cell and Environment 28, 997-1011. 

Fuhrer J (2003). Agroecosystem responses to combinations of elevated CO2, ozone, and global 

climate change.Agriculture, Ecosystems and Environment 97, 1-20. 

Fuhrer J (2009). Ozone risk for crops and pastures in present and future climates. 

Naturwissenschaften 96, 173-194. 

Harmens H; Mills G; Emberson L D; Ashmore M R (2007). Implications of climate change for 

the stomatal flux of ozone: A case study for winter wheat. Environmental Pollution 

146, 763-770 

Harmens H; Norris D and the participants of the moss survey (2008). Spatial and temporal 

trends in heavy metal accumulation in mosses in Europe (1990-2005). Programme 

Coordination Centre for the ICP Vegetation, Bangor, UK. 

Holland M; Kinghorn S; Emberson L; Cinderby S; Ashmore M; Mills G; Harmens H (2006). 

Development of a framework for probabilistic assessment of the economic losses 

caused by ozone damage to crops in Europe. CEH Project No. C02309NEW. Report to 

U.K. Department of Environment, Food and Rural affairs. 

Idso S B; Idso K E (2001). Effects of atmospheric CO2 enrichment on plant constituents related 

to animal and human health. Environmental and Experimental Botany 45, 179-199. 

IPCC (Intergovernmental Panel on Climate Change) (2007). The 4 th Assessment Report. 

Working Group I Report: The physical scientific basis. www.ipcc.ch 

Kimball B A; Kobayashi K; Bindi M (2002). Responses aof agricultural crops to free-air CO2enrichment. 

Advances in Agronomy 77, 293-368. 

30

Kostka-Rick R; Bender J; Bergmann E & H J Weigel (2002). Symptoms of ozone-induced 

foliar injury on horticultural crops. In: Klumpp A; Fomin A; Klumpp G & W Ansel. 

Bioindication and air quality in European cities, pp. 191-196. G. Heimbach, Stuttgart. 

Krupa S; Muntifering R; Chappelka A (2004). Effects of ozone on plant nutritive quality 

characteristics for ruminant animals. The Botanica 54, 1-12. 

Loladze I (2002). Rising atmospheric CO2 and human nutrition: toward globally imbalanced 

plant stoichiometry. Trends in Ecology and Evolution 17, 457-461. 

Long S P; Ainsworth E A; Leakey A D B; Morgan P B (2005). Global food insecurity. 

Treatment of major food crops with elevated carbon dioxide or ozone under large-scale 

fully open.air conditions suggests recent models may have overestimated future yields. 

Philosophical Transactions of the Royal Society B 360, 2011-2020. 

Manderscheid R; Weigel H J (2007). Drought stress effects on wheat are mitigated by 

atmospheric CO2 enrichment. Agronomy and Sustainable Development 27, 79-87. 

Manning W J; v Tiedemann A (1995). Climate change: Potential effects of increased 

atmospheric carbon dioxide (CO2), ozone (O3), and ultraviolet-B (UV-B) radiation on 

plant diseases. Environmental Pollution 88, 219-245. 

Marshall F M (2002). Effects of air pollutants in developing countries. In: Bell, J N B & M 

Treshow (eds.). Air pollution and plant life, pp. 407-416. John Wiley & Sons, 

Chichester. 

Mills G; Buse A; Gimeno B; Holland M; Emberson L; Pleijel H (2007). A synthesis of 

AOT40-bases response functions and critical levels of ozone for agricultural and 

horticultural crops. Atmospheric Environment 41, 2630-2643. 

Ollerenshaw J H; Lyons T; Barnes J D (1999). Impacts of ozone on the growth and yield of 

field grown winter oilseed rape. Environmental Pollution 104, 53-59. 

Piikki K; De Temmerman L; Ojanperä K; Danielsson H; Pleijel H (2008), The grain quality of 

spring wheat (Triticum aestivum L.) in relation to elevated ozone uptake and carbon 

dioxide exposure. European Journal of Agronomy 28, 245-254. 

Pleijel H; Mortensen L; Fuhrer J; Ojanpera K; Danielsson H (1999). Grain protein 

accumulation in relation to grain yield of spring wheat (Triticum aestivum L.) grown in 

open-top chambers with different concentrations of ozone, carbon dioxide and water 

availability. Agriculture, Ecosystems and Environment 72, 265-270. 

Prather M; Gauss; M; Berntsen T; Isaksen; I; Sundet J; Bey I; Brasseur G; Dentener F; 

Derwent R; Stevenson D; Grenfell L; Hauglustaine D; Horowitz L; Jacob D; Mickley 

L; Lawrence M; von Kuhlmann R; Muller J; Pitari G; Rogers H; Johnson M; Pyle J; 

Law K; van Weele M; Wild O (2003). Fresh air in the 21 st century. Geophysical 

Research Letters 30, 72-1 – 72-4. 

Stiling P; Cornelissen T (2007). How does elevated carbon dioxide (CO2) affect plantherbivore 

interactions ? A field experiment and meta-analysis of CO2-mediated changes 

on plant chemistry and herbivore performance.Gobal Change Biology 13, 1823-1842. 

Taub D R; Miller B; Allen H (2007). Effects of elevated CO2 on protein concentration of food 

crops: a meta-analysis. Global Change Biology 14, 111 

UNECE (United Nations Economic Commission for Europe) (2007). Review of the 

Gothenburg Protocol. Report of the Task Force on Integrated Assessment Modelling 

and the Centre for Integrated Assessment Modelling. CIAM report 1/2007, Bilthoven, 

Laxenburg. 

31

USEPA (U.S. Environmental Protection Agency) (2006). Air Quality Criteria for Ozone and 

Related Photochemical Oxidants. Report no. EPA/600/R-05/004aF-cF. U.S. 

Environmental Protection Agency, Washington, D.C. 

Van Dingenen R; Dentener F J; Raes F; Krol M C; Emberson L; Cofala J (2009). The global 

impact of ozone on agricultural crop yields under current and future air quality 

legislation. Atmospheric Environment 43, 604-618. 

Vingarzan R (2004). A review of surface ozone background levels and trends. Atmospheric 

Environment 38, 3431-3442. 

Vorne V; Ojanpera K; DeTemmerman L; Bindi M; Högy P; Jones M B; Lawson T; Persson K 

(2002). Effects of elevated carbon dioxide and ozone on tuber quality in the European 

multiple-site experiment 'CHIP-projekt. European Journal of Agronomy 17, 369-381. 

Wieser H; Manderscheid R; Erbs M; Weigel H J (2008). Effects of elevated CO2 

concentrations on the quantitative protein composition of wheat grain. Journal of 

Agricultural Food Chemistry 56, 6531-6535. 

32

Karamanos A J: Spatial Presentation (Gis) of Winter Apple Tree Phenology in Conditions of the Slovak Republic 

influenced by expected Climate Change. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and 

Abiotic Factors (2009), 33-46; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, 

Braunschweig, Germany 

2-2 The use of the Water Potential Index and some ecophysiological and 

morphological parameters as reliable indicators of crop adaptation to 

drought 

Karamanos A J 

Agricultural University of Athens, Faculty of Plant Science, 75, Iera Odos, 11855 Athens, 

Greece 

Email: akaram@aua.gr 

ABSTRACT 

The regression technique of the Water Potential Index suggested by Karamanos & 

Papatheohari (1999) and some widely used ecophysiological and morphological 

parameters related to drought resistance were evaluated for their reliability as 

indicators of adaptation to drought on 20 bread and durum wheat landraces of 

Greek origin grown in the field. Considerable differences in the grain yield 

sensitivity to water stress among the examined landraces were detected. Osmotic 

adjustment, stomatal control of transpiration, leaf rolling, leaf temperature 

depression, leaf senescence, root growth at the surface soil layers were expressed to 

a different extent by the landraces. However, the yield response of individual 

landraces to water stress could not be ascribed to a single or to a small number of 

the above-mentioned traits. 

INTRODUCTION 

Global water shortage is very likely to become a serious problem by the year 2025, especially 

in areas with high population density (Cosgrove & Rijsberman, 2000). Crises are also likely to 

spread in other areas, since water consumption per capita has been increased 45 times in 

comparison to that estimated three centuries ago, due to the irrigation of agricultural lands, the 

industrial development and the increase in population. Climate change may also cause severe 

water deficits in many parts of the globe, such as the Mediterranean basin and extended areas 

in low latitudes (Palutikof 1993). Thus, water stress may well be the major yield limiting factor 

33

in many agricultural lands of the world in the near future. Accordingly, it is important to assess 

the drought resistance of crop genotypes and investigate how crop plants could be productive 

under restricted water availability. 

The identification of plant physiological parameters which could be considered as indices for 

drought resistance has been the subject of many investigations. Although most of these 

parameters have a sound physiological basis and are related to plant water status, their 

association with crop productivity under drought conditions is either weak or absent 

(Karamanos, 1984; Turner, 1986). The effort to extrapolate to natural conditions results 

obtained with plant tissues subjected to artificial water stress in vitro, was proved not to be 

successful owing to the interaction of factors involved in the expression of certain 

characteristics in the field (Sullivan & Ross, 1979). From the practical point of view, any crop 

reactions to drought are of little value if they are not related to their overall impacts on crop 

productivity. For example, the control of transpiration via leaf folding or the closure of stomata 

can be also considered as acting negatively by reducing photosynthesis and dry matter 

accumulation. 

The difficulty to adopt a certain physiological parameter as a reliable index for drought 

resistance led breeders in using the productivity of genotypes over a range of environments as 

an indication for their drought resistance. Accordingly, a number of methods based on the 

regressions of yields against some environmental indices as independent variables were 

developed. Finlay & Wilkinson (1963) and Eberhart & Russel (1966) used the average yield of 

the examined genotypes in a given location as an index expressing the local environment. 

Fischer & Maurer (1978) proposed the ‘drought susceptibility index’ (yield of a genotype 

under drought as a function of the yield without drought), whereas Lin & Binns (1988) used 

the ‘superiority index’ (the mean square of the distance of the yield of a genotype from the 

maximum yield of all genotypes at a given location) as estimates of genotype adaptability over 

a range of environments. 

The regression techniques and the indices mentioned above present a bulk estimation of the 

combined effects of many environmental factors without a possibility to evaluate their separate 

effects. The weakness lies in the lack of a direct quantification of a given environment by 

specific environmental factors (e.g. drought, temperature etc.). In an effort to estimate the 

water stress experienced by crop plants, Idso et al. (1981) suggested a ‘crop water stress index’ 

derived from the increase in average canopy temperature induced by stomatal closure in waterstressed 

crops. 

Because the plant water potential (Ψ) is an adequate expression of plant water balance at any 

time (Karamanos, 2003), it could be a useful and objective indicator of the intensity of water 

stress in genotype evaluation trials. By taking regular measurements of Ψ throughout the 

growing season, we form an integrated view of the water stress history experienced by a plant 

or crop. Thus, the ‘water potential index’ (WPI) is calculated from the seasonal patterns of Ψ 

by a simple method proposed by Karamanos & Papatheohari (1999). According to this method, 

the magnitude of the regression coefficient of the linear regression between WPI and yield or 

34

any other plant growth parameter is expressing the ‘sensitivity’ of the yield or the parameter to 

water shortage. Thus, the evaluation of adaptability to water shortage in genotype trials will be 

based in the comparison of the regression coefficients obtained from the regressions mentioned 

above among genotypes. 

In the present work, an assessment of the drought resistance of 20 bread and durum wheat 

landraces of Greek origin using the WPI-method will be performed. In addition, some 

ecophysiological and morphological traits traditionally used for drought resistance evaluation 

(e.g. osmotic adjustment, cell wall elasticity, stomatal behaviour, leaf cooling, leaf rolling, leaf 

shedding, root characteristics etc.) will also be estimated in an effort to provide information 

concerning the mechanisms available for each genotype to adapt to water stress conditions. The 

final aim is to draw conclusions on the prevailing adaptive mechanisms, in parallel with the 

overall yield behaviour under drought for each landrace. 

MATERIALS AND METHODS 

Experimental layout 

The results were taken from three field experiments carried out during the seasons 2002-2003, 

2003-2004 and 2004-2005 in the experimental field of the Agricultural University of Athens. 

Ten landraces of durum wheat (Triticum turgidum ssp. durum) and ten of bread wheat 

(Triticum aestivum ssp. aestivum) were subjected to different degrees of water stress. The soil 

was a clay loam (35.6% sand, 35.9% silt, and 29.8% clay), slightly alkaline (pH 7.24) with a 

high concentration in total CaCO3 (16%). 

Α split-plot design with three replicates was applied. Landraces (20, in total), were the main 

plots and four levels of water shortage the subplots. The total area of the experimental field 

was 320m 2 . Seeds were kindly supplied by the Gene Bank of the National Agricultural 

Research Foundation (Thessaloniki). Table 1 shows the landraces and climatic characteristics 

of their origin in Greece. 

Increasing levels of water shortage were induced by increasing the distance from the water 

source, namely from the drippers in each irrigation line. This is a slight modification of the 

technique proposed by Hanks et al. (1976). The four levels (W1, W2, W3, and W4, from the 

wettest to the driest) were set along every sowing line (main plots) at 37.5cm-distances from 

the irrigation line. The discharge of each dripper was 6l h -1 . The durations (between 1.5 and 3 

hours) and frequencies of irrigations (between 2 and 6 days) were adjusted according to the 

values of leaf water potential in the W1-treatments and the course of the meteorological 

conditions. Rainout shelters of 200cm maximum and 120cm minimum height were installed 

over the plots 80 days after sowing in the first, 103 days in the second and 106 days in the third 

experiment. The shelters consisted of polyethylene sheets fixed on metallic frames. 

35

36 

Table 1. The bread and durum wheat landraces examined with their origin and a brief 

climatic description 

Landraces Origin Climate 

Hasiko 

Asprostaro 

Skylopetra 

Giulio 138 

Atheras 137 

Atheras 184 

Atheras 186 

Grinias Zante 

Grinias 148 

T. aestivum 

Chania, Crete Temperate Mediterranean, 

hot, wet winters 

Central-Western 

Macedonia 

Ionian Islands 

(Corfu, Zante) 

Continental, cold and wet 

winters 

Mild, wet winters, 

cool summer 

Zoulitsa Arcadia, C. Peloponnese Mountainous, cold, wet 

winter, cool summer 

T. durum 

Romanou 10 Eastern Aegean 

Lemnos 

Kontopouli 

Kontopouli 16 


Moudros 5 

Moudros 11 

Atsiki 6 

Mavrotheri 

(Islands of Lemnos, Chios) 

Temperate, cool winter, 

hot and dry summer, 

windy 

Heraclion Heraclion, Crete Temperate Mediterranean, 

hot and dry summer

Sowing was performed on 17 January 2003 for the first experiment, on 22 December 2003 for 

the second and on 24 November 2004 for the third one in lines 15cm-apart at a uniform 

seeding rate of 16,5g m -2 . Pre-emergence weed control was applied one day after sowing by 

using chlorsulfuron 5% a.i. (Glean) at a rate of 10g ha -1 . Hand-weeding was applied during the 

cultivation period, when necessary. 

Plant water status and drought resistance parameters 

Plants were sampled twice a week at 12.00 hrs, when their leaf water potential (Ψl) reached its 

most negative daily value, in all three seasons. The youngest fully expanded leaf (third from 

the top of the plant) was sampled until ear emergence. From then on, the flag leaf was sampled 

up to maturity. Ψl was determined by the pressure bomb technique (Schollander et al., 1964). 

From the time course of Ψl, the water potential index (WPI) was calculated according to 

Karamanos & Papatheohari (1999). WPI represents the water stress history of plants during 

any period of their growth cycle. The value of the osmotic potential at zero turgor (ψso), as an 

index of osmotic adjustment was determined in the second and third seasons from pressurevolume 

curves according to Tyree & Hammel (1972). 

Stomatal resistance (rst) of the lower (abaxial) epidermis was measured on the second and third 

seasons twice a week at 12.00 hrs (minimum daily value) using a cyclic diffusion porometer 

model AP4 (Delta-T Devices Ltd., Burwell, Cambridge, U.K.). In addition, diurnal 

measurements of rst at approximately two-hour intervals were taken on two occasions in each 

cultivation season (days 120 and 135 after sowing in the second and days 126 and 154 after 

sowing in the third season). Leaf temperature measurements were taken in all seasons twice a 

week at 12.00 hrs, when Ψl was also determined, by using an infrared thermometer (Raytek, 

Model RAYST 2XU). The leaf-air temperature difference, an indication of leaf cooling 

through transpiration, was calculated by referring to the air temperature measured by a 

minimum-maximum thermometer installed above the crop canopy under the shelters. 

The degree of leaf rolling, a response frequently encountered in cereals, was visually estimated 

in the second and third seasons when sampling for Ψl was taking place. Rolling was scored as 

percentage in increasing intensity as follows: 0% (no rolling), 33% (low rolling), 66% (high 

rolling), 100% (maximum rolling). 

The course of leaf senescence was determined in all seasons by counting the number of yellow 

leaves on two marked plants per plot twice a week. The rate of leaf senescence was determined 

as the regression coefficient of the linear regression between the total number of yellow leaves 

against time (days after sowing) (Ritchie & Nesmith, 1991). 

Soil sampling for root system determination was carried-out when plants were fully mature 

(150 days after sowing, in all seasons). Soil cores of 12cm diameter were extracted with a soil 

sampler from a depth of 25cm. The sample was divided into two equal portions of 12.5cmlength 

and then treated with a 0.5% solution of sodium polyphosphate for the dispersion of soil 

colloids and the detachment of root segments. Roots were separated by sieving. Total root 

37

surface was derived by scanning (Epson Perfection 1600 Photo) using a special software of DT 

Scan, Delta Devices. 

Yields and yield components 

The plots were harvested on 152, 168, and 205 days after sowing in the first, second, and third 

seasons respectively and grain yields were determined after natural drying. 

Meteorological observations 

Daily values of mean air temperature, relative humidity, photosynthetically active radiation and 

precipitation during all three seasons were taken from the National Observatory of Athens 

located in a distance of about one kilometer to the east of the experimental site. 

RESULTS AND DISCUSSION 

The meteorological conditions prevailed during observations (i.e., after day 90) differed among 

the three seasons (Table 2). 

38 

Table 2. The time integrals of average daily temperature (∫T), relative humidity (∫RH) 

and PAR in specific periods (0-90 days, >91 days, all season) in the three 

seasons. Total rainfall (P) before and after the installation of the rainout 

shelters is also shown 

Seasons 

∫T 

( o C) 

0-90 >91 Total 

∫RH 

(%) 

0-90 >91 Total 

∫PAR 

(W m -2 ) 

0-90 >91 Total 

P 

(mm) 

bef. after 

2002-3 7.90 20.60 13.11 72,56 55,32 65,51 121,33 251,93 172,33 130,0 60,0 

2003-4 9,01 16,97 12,71 68,22 54,34 62,16 100,64 216,21 155,70 293,0 55,0 

2004-5 8,95 15,86 12,71 72,01 58,20 64,53 86,86 213,72 156,13 277,0 52,4 

The first season was considerably drier than the other two in terms of the rainfall before the 

installation of the rainout shelters, this resulting in smaller amounts of water stored in the soil. 

Higher values of air temperature, PAR and low relative humidity induced a higher evaporative 

demand in the first season, which offset to a certain extent the beneficial effects of the rainfall 

in February and March and brought plants to a water status similar to that in the second season. 

On the other end, lower temperatures and PAR were the main characteristics of the third 

season, which resulted in lower evaporative demands and less intense water stress. 

Thus, seasonal effects were reflected in the values of the WPI, which were more negative in the 

first and second season and less negative in the third one (Table 3). Both, the imposed 

irrigation treatments and the variability of seasons provided the ground for an adequate 

differentiation in plant water status necessary for evaluating the performance of the landraces 

under drought.

Table 3. The average values of the WPI (MPa) of all landraces of bread and durum 

wheat in the three seasons and irrigation treatments (W1, W2, W3, W4). The 

different letters indicate statistically significant differences (p

The values of the regression coefficients (b) between grain yields and the WPI for each 

landrace indicate their sensitivity to drought. Lower values of b denote a smaller decrease in 

yield for a given increase in water stress (i.e., a fall in WPI to more negative values), namely a 

higher degree of adaptation to water stress and vice versa. Table 5 shows that the values of b 

varied significantly among the landraces in many cases. It also shows that the regressions 

differed among seasons for the majority of the examined landraces. This means that, apart from 

water stress, other environmental factors decisively affected the relationships between yield 

and WPI and, therefore, the comparisons among the landraces concerning their drought 

susceptibility had to be performed separately for each season. 

40 

Table 5. The regression (b) and correlation coefficients (r) between the grain yields and 

the WPI for the bread (A) and durum wheat (B) landraces in the three seasons. 

Values of b followed by the same letter denote non-significant differences at 

the 5% level. *:p

In order to classify the landraces into groups of different sensitivity to water stress we have to 

take into account primarily the sign and the significance of the correlation coefficients as well 

as the significant differences in b among landraces. In this context, the consistency of the 

behaviour among seasons is also important. Table 6 presents the classification of the landraces 

in the three seasons and their overall ranking for their sensitivity to drought. 

Table 6. Classification of the examined wheat landraces in groups and ranking in 

decreasing order of sensitivity of their grain yield to water stress, based on the 

sign and significance of the correlation coefficients as well as on the values 

and levels of significance of the respective regression coefficients between 

their grain yield and WPI. The groups of the landraces are separated by blank 

lines 

Bread wheat Durum wheat Sensitivity 

Grinias Zante Moudros 5 High 

Hasiko 

Atheras 184 

Zoulitsa 

Grinias 148 

Skylopetra 

Atheras 186 

Atheras 137 

Giulio 138 

Asprostaro 

Kontopouli 

Mavrotheri 

Moudros 11 

Heraclion 


Atsiki 6 

Romanou 10 

Lemnos 


The Table shows some clear differences in the responses of the landraces to water stress in 

both species. In bread wheat, there were some landraces with definitely high (‘Grinias Zante’ 

and ‘Hasiko’), low (‘Giulio 138’ and ‘Asprostaro’) and intermediate sensitivity. In durum 

wheat, ‘Moudros 5’ exhibited the highest, whereas ‘Kontopouli 17’ consistently the lowest 

sensitivity. In fact, ‘Kontopouli 17’ showed quite a striking response by increasing its yield 

(significantly in the first season) with falling WPI in all seasons. 

Table 7 summarizes the intensity of the expression of the drought resistance traits examined in 

this work for each landrace, expressed comparatively as a ranking number. The degree of 

osmotic adjustment was derived from the average difference in ψso between the two extreme 

irrigation treatments (W1 and W4) in all seasons for each landrace; stomatal sensitivity from 

the degree of seasonal and daily fluctuations in stomatal resistance, as well as from the 

differences observed between irrigation treatments; leaf rolling from the correlations between 

visual rolling intensity and Ψl ; leaf temperature depression from the average values over all 

irrigation treatments and seasons; leaf senescence from the regressions between the rate of leaf 

Low 

41

senescence and WPI; root activity from the average values of root surface density in the top 

25cm over all treatments and seasons. The average grain yield over the three seasons for the 

well-watered treatment W1 is also included. 

42 

Table 7. A summary of the intensity of the various traits related to drought resistance, 

expressed as ranking numbers (in decreasing order, 1 to 10; for stomatal 

sensitivity 1 to 4) for each landrace. The criteria used for describing the 

intensity of each trait are described in the text. The average grain yield over 

the three seasons in the W1 treatment with the relevant ranking number is 

shown in the last column. The landraces are presented in decreasing order of 

their grain yield sensitivity to water stress (Table 6). (A) Bread wheat. (B) 

Durum wheat. 

Landraces 

Osmotic 

adjust. 

Stomatal 

sensitivity 

(A) 

Leaf 

Cooling 

Leaf 

rolling 

Leaf 

senescence 

Root 

density 

Max. Grain 

yield (t ha -1 ) 

Ranking 

Grinias Zak. 5 1 4 5 10 7 2.74 2 

Hasiko 1 4 2 4 1 9 2.52 5 

Atheras 184 3 3 7 1 4 10 2.58 4 

Zoulitsa 10 1 3 5 8 2 2.77 1 

Grinias 148 6 3 9 8 7 8 2.38 8 

Skylopetra 9 3 1 2 6 6 2.72 3 

Atheras 186 8 4 5 2 4 3 2.46 6 

Atheras 137 7 2 6 10 2 5 2.39 7 

Giulio 138 1 1 10 9 3 1 2.20 9 

Asprostaro 4 4 8 7 9 4 2.04 10 

Moudros 5 2 2 

(B) 

5 6 1 10 2.39 4 

Kontopouli 6 2 7 2 3 4 2.32 7 

Mavrotheri 1 2 1 3 6 7 2.33 5 

Moudros 11 7 4 5 9 9 9 2.33 5 

Heraclion 3 1 8 5 8 6 2.66 1 

Kontopouli 16 8 2 4 4 6 3 2.52 2 

Atsiki 6 9 3 10 10 5 8 2.32 7 

Romanou 10 5 2 9 6 4 5 2.31 9 

Lemnos 4 1 3 1 10 1 2.18 10 

Kontopouli 17 10 2 2 6 2 2 2.45 3 

As a rule, no association between high yields and yield stability determined through WPI is 

evident in Table 7. The highest yielding landraces exhibited intermediate or high sensitivity to 

water stress in both bread and durum wheat, with the exception of ‘Kontopouli 17’ which 

remarkably combined high yields with the ability to produce more under water stress 

conditions. Therefore, in contrast with the findings of Fischer & Maurer (1978) stating that

yield stability under drought is related with high potential yields of wheat cultivars, our results 

are closer to those of Hurd (1974) and Clarke et al. (1981) showing poor relationships between 

high yields and yield stability under drought. Instead, low-yielding landraces, such as ‘Giulio 

138’, ‘Asprostaro’, ‘Romanou 10’, and ‘Lemnos’ are exhibiting low sensitivity (i.e., high 

adaptability) to water stress. 

The association of yield stability to water stress with the parameters of drought resistance 

examined in this study may help understand possible mechanisms involved in the overall 

performance of any landrace to drought. Karamanos (1984) and Turner (1986) attempted to 

evaluate the mechanisms of adaptation to water shortage in terms of their influence on 

productive processes. Among these mechanisms, those referring to osmotic adjustment are 

considered as ‘low cost’ mechanisms because do not affect photosynthesis, crop growth and 

yield. McCree (1986) found no metabolic cost of osmotic adjustment in sorghum plants 

subjected to drought. Nevertheless, the maintainance of water uptake by the enhancement in 

root growth will maintain the assimilation rate in leaves, but may reduce the above ground 

plant productivity by diverting dry matter to the roots (Passioura, 1983). Conversely, the 

reduction of water loss by early leaf senescence and shedding and/or stomatal closure are 

considered as ‘costly’ mechanisms by consuming dry matter and reducing carbon assimilation 

respectively. 

Apart from its well understood physiological role, osmotic adjustment is also related to high 

yields in wheat and sorghum under limited water supply (Morgan, 1983, 1984; Morgan et al., 

1986; Ludlow & Muchow, 1990). In our work, however, only a few landraces exhibited an 

association between high degrees of osmotic adjustment and high yields (‘Atheras 184’, 

‘Heraclion’); in most cases, the landraces showing intense osmotic adjustment were low in the 

rank of yields (‘Giulio 138’, ‘Asprostaro’, ‘Lemnos’), or, conversely, landraces with a low 

degree of adjustment were high in the rank of yields (‘Zoulitsa’, ‘Skylopetra’, ‘Kontopouli 17’, 

‘Kontopouli 16’). Furthermore, with the exception of ‘Giulio 138’, the degree of osmotic 

adjustment was not associated with yield stability to water stress for the majority of the 

landraces examined. 

Root surface density in the layer of 0-25 cm appeared to be related with yield stability more 

than the other traits for many landraces; as a rule, landraces with a higher sensitivity to water 

stress had a less dense root system in the top soil layer and vice versa. Higher root densities 

were associated with higher water extraction and higher yields in wheat (Wright & Smith, 

1983; Morgan & Condon, 1986). However, the lack of information for deeper soil layers in this 

work makes any conclusion concerning the whole root system risky. 

The traits associated with a reduction in transpiration (efficiency of stomatal control, leaf 

rolling) did not reveal a clear relationship with the degree of yield sensitivity to water stress. 

There were landraces exhibiting high yield sensitivity with either efficient (‘Grinias Zante’, 

‘Zoulitsa’) or inefficient stomatal control (‘Hasiko’, ‘Atheras 184’, ‘Moudros 11’). Conversely, 

landraces with low yield sensitivity were found to exhibit either efficient (‘Lemnos’, ‘Giulio 

138’) or inefficient stomatal control (‘Asprostaro’). Leaf rolling acted either as an alternative 

43

(mostly in bread wheat) or an additive mechanism (in some durum wheat landraces) to the 

stomatal control of transpiration. Only in a few cases (‘Atsiki 6’, ‘Moudros 11’ and 

‘Asprostaro’) was observed a low sensitivity of the stomatal mechanism and a low rolling 

tendency. Drought-induced leaf senescence, a drastic way to save water, was not consistently 

related to yield sensitivity. 

Despite the view that leaf temperature depression is a reliable indicator of the degree of water 

stress experienced by crops (Ehrler et al., 1978; Idso et al., 1981; Blum et al., 1982), no 

consistent association of leaf-air temperature difference with grain yield sensitivity to water 

stress was detected. Some highly yield-sensitive landraces tended to show more intense leaf 

cooling (‘Hasiko’, ‘Grinias Zante’, ‘Zoulitsa’, ‘Mavrotheri’), but intense cooling was also 

observed in less yield-sensitive landraces (‘Kontopouli 17’, ‘Lemnos’). Moreover, less intense 

cooling was observed in less sensitive bread wheat landraces (‘Giulio 138’, ‘Asprostaro’). 

The sensitivity of the landraces to drought, evaluated by means of the regression analysis of 

yield against WPI, provides a very useful information for the plant breeder. The data presented 

above showed considerable differentiation among landraces concerning the sensitivity of their 

yield to water shortage. However, a larger differentiation exists when considering the 

parameters of drought resistance examined in this study. Yield is a complex character, 

determined collectively by a wide range of factors throughout the life cycle of a plant. 

Accordingly, it is unrealistic to expect that the use of a single or even a small number of 

morphological and ecophysiological traits associated with drought resistance could provide 

reliable information on the performance of crops under drought. The involvement of a 

considerable number of interacting factors affecting water supply, water loss, drought escape 

and drought tolerance, expressed in different combinations among landraces, makes a 

generalized interpretation of the observed yield performances of all landraces to drought very 

complicated. Instead, an ecophysiological interpretation of the yield performance on the basis 

of individual landraces may be more reliable and useful. 

REFERENCES 

Blum A ; Mayer J ; Gozlan G (1982). Infrared thermal sensing of plant canopies as a screening 

technique for dehydration avoidance in wheat. Field Crops Research 5, 137-146. 

Clarke J M ; Karamanos A J ; Simpson G M (1981). Case examples of research progress in 

drought stress physiology. In: Water Stress on Plants, ed. G M Simpson, pp. 140-272, 

Praeger: N. York. 

Cosgrove W J; Rijsberman F R (2000). World Water Vision, Earthscan Publications: London. 

Eberhart S A; Russell W A (1966). Stability parameters for comparing varieties. Crop Science 

6, 36-40. 

Ehrler W L; Idso S B; Jackson R D; Reginato R J (1978). Wheat canopy temperature: Relation 

to plant water potential. Agronomy Journal 70, 251-256. 

Finlay K W; Wilkinson G N (1963). The analysis of adaptation in a plant-breeding programme. 

Australian Journal of Agricultural Research 14, 742-754. 

44

Fischer R A; Maurer R (1978). Drought resistance in spring wheat cultivars. Australian 

Journal of Agricultural Research 29, 897-912. 

Hanks R J; Keller J; Rasmussen V P; Wilson G D (1976). Line source sprinkler for continuous 

variable irrigation-crop production studies. Soil Science Society of America 

Proceedings 40, 426-429. 

Hurd E A (1974). Phenotype and drought tolerance in wheat. Agricultural Meteorology 14, 39- 

55. 

Idso S B; Reginato R; Reicosky D; Hatfield J (1981). Determining soil-induced plant water 

potential depressions in alfalfa by means of infrared thermometry. Agronomy Journal 

73, 826-830. 

Karamanos A J (1984). Ways of detecting adaptive responses of cultivated plants to drought. 

An agronomic approach. In: Being Alive on Land. Tasks on Vegetation Science, vol.13, 

eds N S Margaris, M Arianoutsou-Farragitaki & W C Oechel, pp. 91-101, Dr. W. Junk 

Publ.: The Hague. 

Karamanos A J (2003). Leaf water potential. In: Encyclopedia of Water Science, pp. 579-587, 

Marcel Dekker: N. York. 

Karamanos A J ; Papatheohari A Y (1999). Assessment of drought resistance of crop 

genotypes by means of the water potential index. Crop Science 39, 1792-1797. 

Linn C S; Binns M R (1988). A superiority measure of cultivar performance for cultivar x 

location data. Canadian Journal of Plant Science 68, 193-198. 

Ludlow M M; Muchow R C (1990). A critical evaluation of traits from improving crop yields 

in water-limited environments. Advances in Agronomy 43, 107-153. 

McCree K J (1986). Whole plant carbon balance during osmotic adjustment to drought and 

salinity stress. Australian Journal of Plant Physiology 13, 33-43. 

Morgan J M (1983). Osmoregulation as a selection criterion for drought tolerance in wheat. 

Australian Journal of Agricultural Research 34, 607-614. 

Morgan J M (1984). Osmoregulation and water stress in higher plants. Annual Review of Plant 

Physiology 35, 299-319. 

Morgan J M; Condon A G (1986). Water use, grain yield and osmoregulation in wheat. 

Australian Journal of Plant Physiology 13, 523-532. 

Morgan J M; Hare R A; Fletcher R J (1986). Genetic variation in osmoregulation in bread and 

durum wheats and its relationship to grain yield in field environments. Australian 

Journal of Agricultural Research 37, 449-457. 

Palutikof J (1993). Mediterranean land use and desertification –The MEDALUS project. In: 

Climate Change and Impacts, ed. I. Troen, pp. 165-172, European Commission: 

Brussels. 

Passioura J B (1983). Roots and drought resistance. Agricultural Water Management 7, 265- 

280. 

Ritchie J T; Nesmith D S (1991). Temperature and crop development. In: Modelling Plant and 

Soil Systems, eds J Hanks & J T Ritchie, pp. 5-29, American Society of Agronomy: 

Madison, Wisc. 

Schollander P F; Hammel H T; Hemmingsen E A; Bradstreet E D (1964). Hydrostatic pressure 

and osmotic potential in leaves of mangroves and some other plants. Proceedings of the 

National Academy of Sciences of USA, 52, 119-125. 

45

Sullivan C Y; Ross W M (1979). Selecting for drought and heat resistance in grain sorghum. 

In: Stress Physiology in Crop Plants, eds H Mussell & R C Staples, pp.262-281, J. 

Wiley & Sons: N. York. 

Turner N C (1986). Crop water deficits: a decade of progress. Advances in Agronomy 39, 1-51. 

Tyree M T; Hammel H T (1972). The measurement of the turgor pressure and the water 

relations of plants by the pressure-bomb technique. Journal of Experimental Botany 23, 

267-282. 

Wright G C; Smith R C G (1983). Differences between two grain sorghum genotypes in 

adaptation to drought stress. 2. Root water uptake and water use. Australian Journal of 

Agricultural Research 34, 627-636. 

46

Mezeyová I, Šiška B, Mezey J, Paulen O: Spatial Presentation (Gis) of Winter Apple Tree Phenology in 

Conditions of the Slovak Republic influenced by expected Climate Change. In: Feldmann F, Alford D V, Furk C: 

Crop Plant Resistance to Biotic and Abiotic Factors (2009), 47-52; ISBN 978-3-941261-05-1; © Deutsche 

Phytomedizinische Gesellschaft, Braunschweig, Germany 

2-3 Spatial Presentation (GIS) of Winter Apple Tree Phenology in 

Conditions of the Slovak Republic Influenced by Expected Climate 

Change 

Mezeyová I, Šiška B, Mezey J, Paulen O 

Department of Fruit Production, Viticulture, and Enology, Department of Biometeorology and 

Hydrology, Slovak University of Agriculture in Nitra, Trieda A. Hlinku 2, 949 76, Nitra, Slovak 

Republic, tel.: 004237 641 4718 

Email: ivana_mezeyova@centrum.sk 

ABSTRACT 

Phenological observations have become an important biological indicator of 

changes in environmental conditions. The aim of this study was the spatial 

processing of winter apple varieties under regional agroclimatic condition in the 

Slovak Republic with the help of trend analyses, modeling and the Arc View 3.2. 

Program (GIS). Phenological data related to unchanged climate conditions, 

represented by climate norms from 1961 to 1990, were obtained from the Slovak 

Hydrometeorological Institute. Scenarios used here, and usually used for the Slovak 

Republic, have been developed by Lapin et al. 2000. There was chosen two 

phenological stages: BBCH 61 = beginning of flowering, and BBCH 87 = fruit 

ready for picking. Two facts were confirmed. Firstly, there was a strong correlation 

between the onset of various phenological stages and altitude; secondly, in the 

model tested, the onset of these growth stages tended to become earlier. The 

strongest shift related to BBCH 87, with the onset of fruit ripening becoming earlier 

in response to changes in climate. While at present, in lowland sites the onset of 

fruit ripening (i.e. BBCH 87) ranged from 26 September to 6 October (on 1,063,875 

ha), under conditions of climate change harvest might be in advance of 17 August 

(on 635,125 ha) and range from 17 to 28 August (on 715, 775 ha), depending on the 

altitude. This represents a predicted shift of about 5 weeks in Slovakian lowlands 

less than 200 m above sea level 

47

INTRODUCTION 

Phenological observations have become an important biological indicator of environmental 

conditions changes. Impact of changing climate on various agricultural crops (field crops, 

vegetables, fruits, and grape) has been studied for years at the Slovak Agricultural University 

in Nitra, and its scientific co-workers lead by one of the authors of this report contributed to the 

program awarded by the Norwegian Nobel Committee. Regarding the fact that climate change 

influence has been experienced in different branches of agriculture there has been paid strong 

attention to the study of the processes related to it e.g. crop potential, regionalization, impact 

on pest and disease infectious pressure etc.. The aim of the report was spatial processing of 

winter apple tree varieties agroclimatic regionalization in condition of Slovak Republic by the 

help of trend analyzes, modeling and Arc View 3.2. Program (GIS). 

MATERIAL AND METHODS 

Phenological data for task solving related to unchanged climate conditions, represented by 

climate normal 1961 – 90, were obtained from the database of the Slovak Hydrometeorological 

Institute. There were chosen 13 phenological stations for detailed covering of the Slovak 

Republic territory in horizontal and vertical directions. Phenological data for broad set of 

winter apple tree varieties were used. There were elaborated trend analyzes of winter apple tree 

varieties of chosen phenological stages onset. There were chosen two phenological stages: 

BBCH 61 – Beginning of flowering and BBCH 87 – Fruit ripe for picking. For the 

determination of onset trends there was used 2 nd order polynomial equation. For phenological 

analyses there was counted hypothetic onset datum of observed phenological stages of winter 

apple tree varieties in conditions of changed climate according to climate change scenarios. 

The scenarios used in the report and usually used for the Slovak Republic territory, have been 

developed by Lapin et al. 2000. Consequently, spatial analyzes of winter apple tree varieties 

onsets were elaborated in Arc View 3.2. Program, which was a part of the Geographical 

information systems (GIS). Maps were created with the help of map algebra, which is used as a 

programming language for processing and analyzing of grids (screens) (Šimonides 2000). 

Priming digitized map – DMR – digital model of Slovak Republic Relief (Geomodel 2005) 

was used as an input object. 

RESULTS 

Slovak republic is extended in 4,903,347 ha and it includes 2,436,879 ha (49.7%) of 

agricultural soils, 2,004,100 ha (41%) of forests soils, 92,895 ha (2%) of water surfaces, 

224,670 ha (5%) of built-up areas and 144,844 ha (3%) of other surfaces (Ministry of 

Agriculture of SR 2007). Spatial results were concerned to entire Slovak republic territory; 

there was not selected agricultural soil or specific intensive orchards territory. All surfaces of 

Slovak republic until observed 730 m above sea level take 4,103,125 ha. This territory was 

consequently divided in to monitored categories. 

48

Winter varieties of present apple tree intensive growing get in BBCH 61 - Beginning of 

flowering - on majority of Slovak territory from April 14 th till April 29 th (1,639,325 ha), in 

higher situated localities from April 29 th till May 14 th (1,101,300 ha) and in altitudes from 350 

m.s.l. till 730 m.s.l. after May 14 th (1,362,500 ha). In climate change conditions there is 

prediction of earlier BBCH 61 onset, because winter varieties get to observed phenological 

stage before April 14 th on territory of 1,245,400 ha, what is the biggest surface in comparison 

with other predicted intervals (Figure 1, Table 1). Even stronger move in stage onset have been 

found in case of BBCH 87 - Fruit ripe for picking in climate change conditions. 

Table 1. Spatial expression of BBCH 61 onset for winter apple tree varieties in Slovak 

republic territory in unchanged and changed climate conditions in ha 

BBCH 61 – WINTER VARIETIES AREAS in ha 

Phenological stage onset 1 x CO2 2 x CO2 

< 14.4. 0 1 245 400 

14.4. – 29.4. 1 639 325 931 850 

29.4. – 14.5. 1 101 300 850 000 

> 14.5. 1 362 500 1 075 875 

While in present regionalization it is for lowlands localities characteristic BBCH 87 onset in 

interval from September 26 th till October 6 th (1,063,875 ha), in climate change conditions there 

is prediction of harvest duration before August 17 th (635,125 ha) and from August 17 th till 

August 28 th (715,775 ha) for mentioned altitudes. That means there is a predicted shift about 5 

weeks in lowlands lower than 200 meters about sea level. BBCH 87 onset typical for highest 

altitudes of Slovak Republic is after October 16 th on territory of 1 804 400 ha in climate 

unchanged conditions. Predicted term of harvest in climate change conditions belongs to 

interval from September 6 th till September 16 th (Figure 2, Table 2). 

Table 2. Spatial expression of BBCH 87 onset for winter apple tree varieties in Slovak 

republic territory in unchanged and changed climate conditions in ha 

BBCH 87 – WINTER VARIETIES AREAS in ha 

Phenological stage onset 1 x CO2 2 x CO2 

< 17.8. 0 635125 

17.8. - 27.8. 0 715775 

27.8. – 6.9. 0 947825 

6.9. – 16.9. 0 1 804 400 

26.9. – 6.10. 1 063 875 0 

6.10. – 16.10. 1 336 750 0 

> 16.10. 1 702 500 0 

49

50 

Figure 1. Mean datum of onset of phenological stage BBCH 61 – Beginning of 

flowering (winter varieties) in unchanged climate conditions in Slovak 

Republic territory

Figure 2. Mean datum of onset of phenological stage BBCH 87 – Beginning of 

flowering (winter varieties) in unchanged climate conditions in Slovak 

Republic territory 

51

CONCLUSIONS 

Two facts have been confirmed according to statistical methodology and GIS analyzes: strong 

dependence between phenological stages onsets and altitudes and phenological stages onset 

shifting towards to earlier terms in model situation. There is a presumption of possible apple 

tree growing in higher altitudes in consequence of these facts. It could help to extend intensive 

orchards in case of apple fruit request. 

ACKNOWLEDGEMENTS 

Regionalization of apple tree phenological stages in conditions of unchanged and changed 

climate was supported by grant agency of Slovak republic – VEGA 1/4427/07: Design of new 

agroclimatic regionalization of plant production in condition of changing climate in Slovakia 

and by grant within APVV (Slovak Research and Development Agency) No. -151/06. 

REFERENCES 

Digital Terrain Models (2005). GeoModel s.r.o, 

http://www.geomodel.sk/sk/download/download.htm, (19.03.2005) 

Lapin M; Melo M; Damborská I; Gera M; Faško P (2000). New climate change scenarios for 

Slovak Republic on the base of outputs from Global Circulation Models (GCM). In: 

National Climate Program SR 8/2000, Ministry of environment SR, pp. 5 – 35. Slovak 

Hydrometeorological Institute: Bratislava. 

Ministry of Agriculture of the Slovak Republic (2007). National Rural Development Plan of 

the Slovak Republic. http://archiv.mpsr.sk/slovak/dok/nsprv_prilA/0.1.pdf 

Šimonides I (2000). Geographical Information Systems. Slovak Agricultural University: Nitra, 

114 p. ISBN 80-7137-740-6. 

52

Grundmann B M, Roloff A: Use of Forest Tree Species Under Climate Change. In: Feldmann F, Alford D V, Furk 

C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 53-65; ISBN 978-3-941261-05-1; © Deutsche 


2-4 Use of Forest Tree Species Under Climate Change 

Grundmann B M, Roloff A 

Technische Universität Dresden; Institute of Forest Botany and Forest Zoology; Chair of 

Forest Botany, Pienner Str. 7; 01737 Tharandt 

roloff@forst.tu-dresden.de, www.forst.tu-dresden.de/Botanik 

ABSTRACT 

The aim of this study was to evaluate various forest tree species according to their 

applicability for forest ecosystems, bearing in mind future climate change. This was 

achieved by the integrative interpretation and evaluation of publications concerning 

the natural distribution areas and the physiological and ecological potential of tree 

species. In this context drought tolerance plays a major role, as does resistance 

against frost. To evaluate an average frost resistance for each species, the general 

tolerances against winter frosts and also late frosts were considered. Forty-seven 

tree species were evaluated at four sites with different soil water conditions, ranging 

from wet to very dry. The KLAM-Wald (German: KLimaArtenMatrix für 

Waldbaumarten; climate species matrix for forest trees) clearly summarises the 

evaluated species. Most of the indigenous tree species tend to build into stable 

forest ecosystems on adequate sites, and this is likely to continue in future. When 

choosing suitable tree species, additional factors such as nutrients, altitude and, for 

some species, the pathogenic risks should also be considered as a routine. An 

evaluation of forest tree species in climate change comparable to this study has not 

yet been published; it therefore represents a completely new approach. 

INTRODUCTION 

During a period from 1906-2005 the global average surface temperature has increased by 

0,74 °C (IPCC 2007). Until the year 2100 it may rise by again 2-4 °C, relative to the period of 

1980-1999. Naturally accompanied with warming is a change of the precipitation regime. 

Whilst precipitation in Europe will increase in winter, it will decrease during the vegetation 

period by about 10-25% (SRES A1B scenario). Though, there are numerous uncertainties 

about regional precipitation distributions. Droughts, hurricanes, intense rainfalls and floodings 

53

already occure more frequently. As well as the number of heavy storms is increasing in Europe 

(Leckebusch & Ulbrich 2004; Fuhrer et al. 2006; Leckebusch et al. 2006). 

Modification of forest ecosystems due to climate change can only be predicted restrictedly, 

because regional and local stand characteristics as watercapacity mainly influence the climate 

factors (Ammer & Kölling 2007). Forests have to readapt in an unprecedented way (Bolte & 

Ibisch 2007). There have been alternations between cold and warm stages in earth history, but 

these changes never took place in such a rate (Rahmstorf & Schellnhuber 2006). 

Extreme sites as the dry inner-alpine valley in the canton Wallis in Switzerland, already show 

changes in tree species composition (Rigling et al. 2006). Changes in the growth stages 

(flowering, regeneration, longitudinal and radial growth) will take influence on the competition 

between tree species (Ammer & Kölling 2007; Menzel 2006). The natural distribution ranges 

of species will move horizontically and also vertically (Felbermeier 1994; Walther et al. 2005; 

Zimmermann et al. 2006). 

To find out which tree species will be suitable for forest ecosystems in future is a main task for 

foresters and forest scientists. The tolerance against extreme atmospheric conditions, as 

droughts or late frosts, will be of highest interest, as well as the adaptation to low winter 

temperatures. This individual tolerance depends on the species but also on the provenance due 

to different local adaptation (Tognetti et al. 1995; Schraml & Rennenberg 2002; Czajkowski & 

Bolte 2006). Silviculture will have to select species with best possible adaptability. Warming in 

the vegetation period and shifting of main precipitation to winter (Ammer & Kölling 2007; 

Jacob et al. 2007) will increase stress for the trees. This might lead to a higher resistance of the 

plants, but it may also cause growth depressions, visible damages and in the end culminating in 

death of single plants or extinction of a species. On some kinds of sites tree species will 

become unsuitable and will be replaced by others (Walther 2003, 2006; Wohlgemuth et al. 

2006). Furthermore it is assumed that other aspects of forest utilisation might gain in 

importance, e.g. ecologigal, recreational or aesthetical functions. 


To achieve an evaluation of main tree species according to their applicability for forest 

ecosystems in future an integrative interpretation and evaluation of publications concerning the 

natural distribution areas and the physiological and ecological potential of tree species was 

conducted. The main attention was turned to drought tolerance (Krüssmann 1977, 1983; Sakai 

& Larcher 1987; Ellenberg 1996; Walther & Breckle 1999; Warda 2001; Breckle 2005; Roloff 

2006; Roloff & Bärtels 2006; Roloff & Rust 2007; Schütt et al. 1994-2008). In addition the 

applicability as tree for urban areas, was interpreted as metaphor for drought tolerance (GALK 

2006; Roloff 2006; Roloff & Pietzarka 2007; Roloff et al. 2008a-d). In cities the climatic 

conditions affect trees directly and intensified. 

The ecologically oriented silviculture always had to select tree species with regard to the 

specific site conditions. Therefore the potential occurrence of species in natural vegetation 

stands, which displays the potential of species to grow on sites of different soil water 

54

conditions, was included in the evaluation (Schmidt 1995; Ellenberg 1996; Schütt et al. 1994- 

2008). Following four different types of sites will be considered: 

− wet to very fresh 

− fairly fresh to fresh 

− moderate fesh to moderate dry 

− dry to very dry. 

Evaluating the ability of species to colonize sites of different water conditions includes their 

degree of drought resistance. This general evaluation was conducted by the integrative 

interpretation and evaluation of publications concerning the natural distribution areas and the 

physiological and ecological potential of tree species. Degrees of 1 to 4 were defined as 

follows: 

1 = very suitable 

2 = suitable 

3 = limited suitable 

4 = not suitable 

To evaluate the frost tolerance of each species, the above mentioned literature was used to 

interpret the species frost tolerance and the resistance against late frost. These two 

characteristics were averaged to a general frost tolerance. If this tolerance was evaluated as 

very high or high, it had no influence on the conclusive result; in case of a decreased tolerance 

against winter frost or late frost (degree 3 or 4) the result was degraded by one degree. The 

degrees were defined the following: 

1 = extremely frost resistent 

2 = frost resistent 

3 = limited frost resistent 

4 = frost sensitive. 


Consisting forest stands can be adapted to future conditions by means of adjusted silvicultural 

treatments as shortened rotation periods or wide spatial stem distribution. Risk minimization by 

ecologically oriented forest reconstruction will be one of the main strategies (Leitgeb & 

Englisch 2006). Therefore a minimum of mixture and the cultivation of species, which are 

mostly suitable for the coming conditions, will be required and of course the choice of siteadapted 

species. Natural regeneration holds another option for silviculture. The natural 

selection favours individuals with highest tolerance for changing conditions (Ammer & 

Kölling 2007). In general, climate change will have no influence on tree growth on sites with 

optimal conditions. But on sites, were the trees reach their physiological limits, forestry has to 

intervene (Döbbeler & Spellmann 2002). 

55

Secondary tree species 

The role of secondary and admixed tree species like Norway maple (Acer plantanoides), smallleaved 

lime (Tilia cordata), European walnut (Juglans regia), wild cherry (Prunus avium) or 

wild service tree (Sorbus torminalis) will gain in importance, due to their high climatic 

applicability (Künanz 1949; Buttenschon & Buttenschon 1999; Müller-Kroehling & Franz 

1999; Steffens & Zander 2001; Studhalter et al. 2001; Schulte 2003, 2005; Breckle 2005; 

Häner et al. 2005; Schuster 2007; Küster 2008; Nickel et al. 2008). In general, these species 

need mesotrophic soils, so that species selection has to be site adequate. In mixed decidious 

forests the competitiveness of some of these species might decrease against dominating beech 

trees. But service tree or chess-apple might get established very well in the understorey 

(Schulte 2003). Natural regeneration is strongly recommended, due to the natural selection of 

individuals, which bare high tolerance against changing conditions. Active planting of suitable 

secondary species at forest edges will also lead to an increasing proportion in the medium- to 

long-term (“biological automation”). Additionally the selection of suitable provenances will 

take influence on the merchantable qualities. 

Tree species of the Mediterranean region 

An increasing competitiveness of tree species of the Mediterranean region like downy oak 

(Quercus pubescens), sweet chestnut (Castanea sativa), holly (Ilex aquifolium) or Turkey oak 

(Quercus cerris) has been observed for the last decades (Walther et al. 2005; Walther 2006; 

Lang 2007). But changing of the climatic conditions might proceed more rapidly than these 

species will migrate northwards forming stands (Lischke et al. 2006). In general, forest 

ecosystems show a time lag in their development. Singular tree species could fail more quickly 

than other species can substitute them (Wagner 2004). Furthermore the drought tolerance is 

extremely depending on the provenance even with species of southern origin. This is reported 

on sweet chestnut (Barthold et al. 2004), which showed brownish leave colouring in July in 

2003 at the southern Alps, but only on extreme sites. Shallow soils with low water retaining 

capacity can induce drought damages at chestnuts, which might not only lead to a growth 

reduction, but also to a susceptibility for cancer diseases, which have already been observed at 

the northern side of the Alps (Heiniger et al. 2007). Therefore the importance of the choice of 

the provenance should be stressed. 

The wide spread assumption the climate north of the Alps might conform to the Mediterranean 

conditions in the long-term, should be considered carefully. High proportions of sites will 

probably obtain comparable atmospheric conditions, but low winter temperatures will still 

interfere the spread of Mediterranean species. Especially late frosts will affect flowering, 

fructification and generally natural regeneration. The future climate in Central Europe and 

especially during the transition period, will not be comparable to any conditions spatially 

observable in Europe. It might become similar to regions in the Southern-east of Europe like 

Hungary where summers are warm and dry and winters are wet and cold. Further the climatic 

conditions will become highly regionally differentiated. 

56

Foreign tree species 

Several foreign tree species are well established in Central Europe for decades, like Douglas fir 

(Pseudotsuga menziesii), red oak (Quercus rubra) or grand fir (Abies grandis) (Gulder 1999; 

Gossner 2004; Gossner & Ammer 2006; Asche 2007; Möhring 2007; Wezel 2008). These 

species are characterised by an outstanding adapting potential to changing climate conditions 

and their economical relevance might increase by importing even more suitable provenances. 

Foreign species, like already established or new species with high drought tolerance could 

prove to be a reasonable addition to the existing pool of species. Cultivation of unknown 

foreign but drought tolerant species, should be carried out tentatively, because many facts have 

to be analysed; e.g. the reaction to late frosts, the productivity and the effects on soil and 

environment (Ammer & Kölling 2007). 

The cultivation of lodgepole pine (Pinus contorta) in the 1920s in Sweden explains the 

problematic of a possibly invasive potential of foreign species (Engelmark et al. 2001). 

Introduced into an existing ecosystem, Lodgepole pine spread uncontrollably, suppressed local 

species and brought foreign pests along. The natural balance between local fauna and flora can 

be disturbed by new species. Due to a lack of experience with tree species from America or 

Asia, which had not been exposed to the competition structure of European forest ecosystems, 

it should be handled carefully to introduce species from these regions. Furthermore, foreign 

species should only be cultivated on extreme sites, where local species merely survive with 

difficulties. 

Challenge for silviculture under climate change 

Controlling the game density will still be essential to reach the target of growing stock in 

future. Often game interferes biodiversity but also may catalize it, depending on the selection 

pressure. Therefore foresters have to regulate the selection. 

In contrast, pest outbreaks will become an incalculable risk for forest ecosystems, because the 

future development can not be assessed. Due to increasing temperatures several species might 

shift in their development periods (Steyrer & Tomiczek 2007). But the determining growth 

factor is in many cases the photoperiod, for that the situation might stay constant or even 

develop to the disadvantage of some species. Likewise, foreign species could become 

established (Peñuelas & Boada 2003; Krehan & Steyrer 2006; Blaschke & Cech 2007; Hoyer- 

Tomiczek 2007; Perny 2007). Therefore the risks for some tree species cannot be assessed. In 

natural vegetation stands often a balance exists between pests and hosts; new pathogenes could 

cause epidemics (Heiniger 2003). Climate change might benefit the development of 

thermophile pathogenes and increase the defense of tree species due to stress (drought, stagnant 

moisture, soil acidification). One solution to minimize epidemic risks is the formation of 

horizontically and vertically structured, species-rich mixed forests and the choice of suitable 

provenances. 

57

Physiological adaptivity 

To give the best possible indication for the applicability of forest tree species for stable forest 

ecosystems, was the aim of this study. But this cannot be stated resting only on the evaluation 

of the species potentials. The exclusive estimation of the susceptibility to plant-physiological 

stress factors as temperature extremes, drought or storm, does not lead to a conclusion if entire 

ecosystems will cope with future conditions. Forest ecosystems have to be evaluated as a 

whole. Factors as tree species composition, competition, site adaptablity and game 

management determine the basic parameters and thus the sensitivity of forests (Kätzel 2008). 

The adaptivity of tree populations describes the potential to be responsive to environmental 

parameters, common and unknown. This physiological effort is genetically determined and the 

extent of tolerance for individual survival is described by the species-specific and genetical 

reaction norm. A wide physiological (genetical) reaction norm therefore is the basis for a high 

adaptivity. Due to changing environmental conditions this norm has to vary or expand. This 

might take place at the individual or the population level through genetical recombination or 

mutation. But facing climate change Savolainen et al. (2007) suspect these genetical processes 

of adaptation too slow. Positive effects of mutations in forest tree species are not yet described. 

Generally, in situ-studies of the physiological adaptivity of tree species indicate a high 

variability in their reaction to selection, that can be attributed to a high genetical diversity on 

the population level. Tree species with pioneer character might take their early and frequent 

fructification as advantage. This strategy of reproduction constantly produces new kinds of 

genotypes, which are exposed to climatically induced selective pressure, thus, best suited 

individuals are favoured (Kätzel 2008). 

The evaluation of tree species potentials in this study, as well as in most related publications, 

are based on a static approach. Therefore the status quo of the adaptivity potential is evaluated. 

The possible modification of the genetical norm of reaction in space and time remains 

unconsidered. Essential factors as mutation rates, recombination and especially regeneration 

are included into dynamical (evolutive) approaches, which, however, remain too indefinite. 

Evaluation of the tree species 

As result the frost resistance of each tree species was evaluated on the basis of extensive 

literature studies. By means of their natural distribution and physiological potential, their 

ability to colonize sites of different water conditions was included. This evaluation was 

performed by markings from 1 = very suitable to 4 = not suitable. Tree species with a limited 

winter frost or late frost resistance were downgraded by one marking. To exemplify, the 

evaluation of Black alder and European silver fir will be illustrated in the following. 

Firstly the frost resistance was determined (tab. 1, col. 3). Further, the ability to grow on 4 sites 

of different soil water condition was investigated analysing the natural distribution of the 

species and its ecophysiological potential (col. 2). The frost resistance of Black alder was 

58

evaluated with mark 2, so the ability growing on these sites is not limited. Thus, Black alder is 

very suitable to suitable for wet to fresh sites, but for moderate fresh to very dry not suitable. 

Table 1. Evaluation of the potential of Alnus glutinosa for four sites of different water 

conditions, the total result depends on the applicability to the sites and the frost 

resistance 

Site Potential for site Average frost resistence Result 

wet to very fresh 1 2 1 

fairly fresh to fresh 2 2 2 

moderate fresh to m. dry 4 2 4 

dry to very dry 4 2 4 

European silver fir is relatively resistant against winter frosts, but it is especially sensitive to 

late frosts. Therefore its general frost resistance is limited and the ability for each site has to be 

downgraded. By means of this evaluation European silver fir is a tree species, which is not 

suitable for wet as for very dry sites, but it is suitable for moderate fresh to moderate dry sites, 

prior on sites of higher elevation (tab. 2). 

Table 2. Evaluation of the potential of Abies alba for four sites of different water 

conditions, the total result depends on the applicability to the sites and the frost 

resistance 

Site Potential for site Average frost resistence Result 

wet to very fresh 4 3 4 

fairly fresh to fresh 1 3 2 

moderate fresh to m. dry 1 3 2 

dry to very dry 4 3 4 

CONCLUSIONS AND KLAM-WALD 

Forest ecosystems in Central Europe will endure and fulfil its multiple functions, even though 

the tree species composition and structure might change or rather should be diversified. In 

particular the importance of these days rare secondary tree species as Norway maple, Smallleaved 

lime, Wild apple, European walnut, Wild cherry, Service tree or Wild service tree will 

increase. These species are especially qualified due to its properties. To increase the 

proportion, any natural regeneration should be preserved but also active planting at forest edges 

will support the autonomous spreading. Both solutions are cost-saving in contrast to seeding or 

planting and also benefit from climatically induced selectivity. 

Equal to the importance of the choice of suitable species is the choice of suitable provenances. 

Former experiences with assumed drought tolerant species of the Mediterranean as Downy oak 

or Sweet chestnut proved this property to extremely dependend on the provenance. The 

suitable choice of foreign tree species, which are already established in Central Europe for 

59

decades, as Douglas fir and Red oak, might improve its already high valence. Introducing new 

foreign tree species should be carried out especially deliberately, because American or Asian 

tree species were not exposed to competition with European species. There is a lack of 

experience with effects of new species on present ecosystems concerning the invasive 

potential, soil interferences or coincidental introduction of novel pests, which might affect the 

balance within the indigenous flora and fauna. Foreign tree species and species of the 

Mediterranean region should only be cultivated on sites with extreme conditions, where 

indigenous species show difficulties. 

To give a practicable overview of suitable tree species for four sites of different water 

conditions, the following matrix for forest tree species (KLAM-Wald) (tab.3) shows a ranking, 

which allows a deliberate choice of suitable species in accordance to local site conditions. The 

species are ranked from very suitable over suitable and limited suitable to not suitable for each 

type of site. Further, the species are arranged alphabetically to prevent a ranking within the 

categories. 

This evaluation on the basis of frost and drought tolerance represents a new approach, which is 

to be discussed. The results have to be confirmed with further systematic analyses and 

experiments. 

60 

Table 3. KLAM-Wald; Ranking list of suitable tree species for wet to very dry sites 

wet to very fresh fairly fresh to fresh m. fresh to m. dry dry to very dry 

very suitable very suitable very suitable very suitable 

Alnus glutinosa Acer pseudoplatanus Acer campestre Acer campestre 

Alnus incana Betula pendula Acer platanoides Acer platanoides 

Betula pubescens Populus nigra Acer pseudoplatanus Betula pendula 

Populus nigra Populus tremula Betula pendula Carpinus betulus 

Prunus padus Prunus padus Carpinus betulus Pinus nigra 

Salix alba Quercus petraea Larix decidua Pinus strobus 

Sorbus aucuparia Quercus robur Pinus cembra Pinus sylvestris 

suitable Quercus rubra Pinus nigra Populus tremula 

Fraxinus excelsior Salix alba Pinus sylvestris Prunus avium 

Quercus robur Sorbus aucuparia Populus tremula Quercus petraea 

Ulmus laevis Tilia cordata Prunus padus Robinia pseudoacacia 

Ulmus minor Tilia platyphyllos Quercus petraea Sorbus aria 

limited suitable Ulmus glabra Quercus rubra Sorbus domestica 

Acer pseudoplatanus suitable Robinia pseudoacacia Sorbus torminalis 

Betula pendula Abies alba Sorbus aria Tilia cordata 

Carpinus betulus Abies grandis Sorbus aucuparia suitable 

Pinus sylvestris Acer campestre Sorbus domestica Abies grandis 

Populus tremula Acer platanoides Sorbus torminalis Acer pseudoplatanus 

Quercus petraea Alnus glutinosa Taxus baccata Buxus sempervirens

Tilia cordata Alnus incana Tilia cordata Castanea sativa 

Tilia platyphyllos Betula pubescens Tilia platyphyllos Fraxinus ornus 

not suitable Carpinus betulus suitable Juglans regia 

Abies alba Fagus sylvatica Abies alba Larix decidua 

Abies grandis Fraxinus excelsior Abies grandis Malus sylvestris 

Acer campestre Juglans regia Alnus incana Pyrus pyraster 

Acer platanoides Larix decidua Betula pubescens Quercus cerris 

Buxus sempervirens Picea abies Buxus sempervirens Quercus pubescens 

Castanea sativa Pinus cembra Castanea sativa Quercus robur 

Fagus sylvatica Pinus strobus Fagus sylvatica Quercus rubra 

Fraxinus ornus Pinus sylvestris Fraxinus excelsior Sorbus aucuparia 

Ilex aquifolium Pseudotsuga menziesii Fraxinus ornus Taxus baccata 

Juglans regia Robinia pseudoacacia Ilex aquifolium Tilia platyphyllos 

Larix decidua Sorbus domestica Juglans regia Ulmus glabra 

Malus sylvestris Sorbus torminalis Malus sylvestris limited suitable 

Picea abies Ulmus laevis Pinus strobus Alnus incana 

Pinus cembra Ulmus minor Populus nigra Betula pubescens 

Pinus nigra limited suitable Prunus avium Fagus sylvatica 

Pinus strobus Buxus sempervirens Pseudotsuga menziesii Fraxinus excelsior 

Prunus avium Fraxinus ornus Pyrus pyraster Ilex aquifolium 

Pseudotsuga menziesii Ilex aquifolium Quercus cerris Pinus cembra 

Pyrus pyraster Malus sylvestris Quercus pubescens Prunus padus 

Quercus cerris Pinus nigra Quercus robur Pseudotsuga menziesii 

Quercus pubescens Prunus avium Ulmus glabra Ulmus laevis 

Quercus rubra Pyrus pyraster Ulmus laevis Ulmus minor 

Robinia pseudoacacia Quercus pubescens Ulmus minor not suitable 

Sorbus aria Sorbus aria limited suitable Abies alba 

Sorbus domestica Taxus baccata Picea abies Alnus glutinosa 

Sorbus torminalis not suitable Salix alba Picea abies 

Taxus baccata Castanea sativa not suitable Populus nigra 

Ulmus glabra Quercus cerris Alnus glutinosa Salix alba 


We like to thank Stiftung Wald in Not for the funding of this study. 

REFERENCES 

Ammer C; Kölling C (2007). Waldbau im Klimawandel - Strategien für den Umgang mit dem 

Unvermeidlichen. Unser Wald 4, 12-14. 

Asche N (2007). Fremdländische (neophytische) Baumarten in der Waldwirtschaft. Forst und 

Holz 62 (10), 30-32. 

61

Barthold F; Conedera M; Torriani D; Spinedi F (2004). Welkesymptome an Edelkastanien im 

Sommer 2003 auf der Alpensüdseite der Schweiz. Schweiz. Z. Forstwesen 155, 

392-399. 

Blaschke M; Cech T L (2007). Absterbende Weißkiefern - eine langfristige Folge des 

Trockenjahres 2003? Forstschutz aktuell 40, 32-34. 

Bolte A; Ibisch P L (2007). Neun Thesen zu Klimawandel, Waldbau und Waldnaturschutz. 

AFZ/Der Wald 62, 572-576. 

Breckle S W (2005). Möglicher Ein-uss des Klimawandels auf die Waldvegetation 

Nordwestdeutschlands? LÖBF-Mitteilungen 2, 19-24. 

Buttenschon R M, Buttenschon J (1999). Population dynamics of Malus sylvestris stands in 

grazed and ungrazed, semi-natural grasslands and fragmented woodlands in Mols 

Bjerge, Denmark. Ann. Bot. Fennici 35, 233-246. 

Czajkowski T; Bolte A (2006). Unterschiedliche Reaktion deutscher und polnischer Herkünfte 

der Buche (Fagus sylvatica L.) auf Trockenheit. Allg. Forst- u. J.-Ztg. 177 (2), 30-40. 

Döbbeler H; Spellmann H (2002). Methodological approach to simulate and evaluate 

silvicultural treatments under climate change. Forstw. Cbl. (121, Suppl. 1), 52-69. 

Ellenberg H (1996). Vegetation Mitteleuropas mit den Alpen - in ökologischer, dynamischer 

und historischer Sicht. Ulmer: Stuttgart. 

Engelmark O; Sjöberg K; Andersson B; Rosvall O; Ågren G I; Baker W L; Barklund P; 

Björkman C; Despain D G; Elfving B; Ennos R A; Karlman M; Knecht M F; Knight 

D H; Ledgard N J; Lindelöw Å; Nilsson C; Peterken G F; Sörlin S; Sykes M T (2001). 

Ecological effects and management aspects of an exotic tree species: the case of 

lodgepole pine in Sweden. Forest Ecology and Management 141, 3-13. 

Felbermeier B (1994). Arealveränderungen der Buche infolge von Klimaänderungen. Allg. 

Forstzeitschrift 5, 222-224. 

Fuhrer J; Beniston M; Fischli, A; Frei C; Goyette S; Jasper K; Pfister C (2006). Climate risks 

and their impact on agriculture and forests in Switzerland. Climatic Change 79, 79–102. 

GALK (2006). Straßenbaumliste 2006. Gartenamtsleiterkonferenz des Deutschen Städtetages, 

http://www.galk.de 

Goßner M (2004). Diversität und Struktur arborikoler Arthropodenzönosen fremdländischer 

und einheimischer Baumarten. Ein Beitrag zur Bewertung des Anbaus von Douglasie 

(Pseudotsuga menziesii (Mirb.) Franco) und Roteiche (Quercus rubra L.). Neobiota 5, 

1-324. 

Goßner M; Ammer U (2006). The effects of Douglasfir on tree-specific arthropod communities 

in mixed species stands with European beech and Norway spruce. European Journal of 

Forest Research 125, 221-235. 

Gulder H-J (1999). Standörtliche Ansprüche von Douglas', Grandis, Strobe und Co. LWF 

aktuell 20, 17-23. 

Heiniger U (2003). Das Risiko eingeschleppter Krankheiten für die Waldbäume. Schweiz. Z. 

Forstwesen 154, 410-414. 

Heiniger U; Graf R; Rigling D (2007). Der Kastanienrindenkrebs auf der Alpennordseite. Wald 

und Holz 5, 50-53. 

Hoyer-Tomiczek U (2007). Braunau am Inn: Asiatischer Laubholzbockkäfer weitet sein 

Befallsgebiet aus. Forstschutz aktuell 40, 21-23. 

62

Häner R; Hoebee E; Holderegger R (2005). Wildbirnenbestände - klein aber fein? Wald und 

Holz 5, 29-32. 

IPCC (2007). Climate Change 2007: Mitigation of climate change. IPCC (Intergovernmental 

Panel on Climate Change), Genf. 

Jacob D; Göttel H; Lorenz P (2007). Hochaufgelöste regionale Klimaszenarien für 

Deutschland, Österreich und die Schweiz. Mitteilungen der Deutschen 

Meteorologischen Gesellschaft 1, 10-12. 

Krehan H; Steyrer G (2006). Klimaänderung - Schadorganismen bedrohen unsere Wälder. 

BFW-Praxisinformation 10, 15-17. 

Krüssmann G (1977). Handbuch der Laubgehölze. Parey: Berlin, Hamburg. 

Krüssmann G (1983). Handbuch der Nadelgehölze. Parey: Berlin, Hamburg 

Kätzel R (2008). Klimawandel - Zur genetischen und physiologischen Anpassungsfähigkeit der 

Waldbaumarten. Archiv f. Forstwesen u. Landsch.ökol. 42, 9-15. 

Küster H (2008). Die Verbreitungsgeschichte der Walnuss. LWF Wissen 60, 11-15. 

Künanz H (1949). Ein Landeskulturgesetz für die Forstwirtschaft - Beitrag zu Neuaufbau und 

Sicherung des oberhessischen Hochwaldes. Forstwiss. Cbl. 68 (2), 114-123. 

Küster H (2008). Die Verbreitungsgeschichte der Walnuss. LWF Wissen 60, 11-15. 

Lang W (2007). Die Edelkastanie - wiederentdeckt im Zeitalter des Klimawandels. AFZ/Der 

Wald 62, 923-925. 

Leckebusch G C; Ulbrich U (2004). On the relationship between cyclones and extreme 

windstorm events over Europe under climate change. Global and Planetary Change 44, 

181–193. 

Leckebusch G C; Koffi B; Ulbrich U; Pinto J G; Spangehl T; Zacharias S (2006). Analysis of 

frequency and intensity of European winter storm events from a multi-model 

perspective, at synoptic and regional scales. Climate Research 31 (1), 59-74. 

Leitgeb E; Englisch M (2006). Klimawandel - Standörtliche Rahmenbedingungen für die 

Forstwirtschaft. BFW-Praxisinformation 10, 9-11. 

Lischke H; Zimmermann N E; Bolliger J; Rickebusch S; Löffler T J (2006). TreeMig: A forestlandscape 

model for simulating spatio-temporal patterns from stand to landscape scale. 

Ecological Modelling 199, 409-420. 

Menzel A (2006). Zeitliche Verschiebung von Austrieb, Blüte, Fruchtreife und Blattverfärbung 

im Zuge der rezenten Klimaerwärmung. Forum für Wissen, Wald und Klimawandel, 

47-53. 

Möhring C (2007). Ein Neubürger auf dem Prüfstand. Küstentannen aus Nordamerika als 

leistungsstarke Bäume, die dem Klimawandel trotzen? Laborgespräch 4, 1-8. 

Müller-Kroehling; Franz C (1999). Elsbeere und Speierling in Bayern - Bemühungen um ihren 

Erhalt, Anbau, Waldbau und Holzverwertung. Corminaria 12, 3-8. 

Nickel M; Steinacker L; Klemmt H.-J; Pretzsch H (2008). Wachstum verschiedener 

Nussbaumarten in Bayern. LWF Wissen 60, 37-43. 

Perny B (2007). Verstärktes Auftreten saugender Schädlinge im Wald und Stadtgebiet. 

Forstschutz aktuell 40, 14-16. 

Peñuelas J; Boada M (2003). A global change-induced biome shift in the Montseny mountains 

(NE Spain). Global Change Biology 9(2), 131-140. 

Rahmstorf S; Schellnhuber H J (2006). Der Klimawandel. C.H. Beck Wissen: München. 

63

Rigling A; Dobbertin M; Bürgi M; Feldmeier-Christe E; Gimmi U; Ginzler C; Graf U; Mayer 

P; Zweifel R; Wohlgemuth T (2006). Baumartenwechsel in den Walliser 

Waldföhrenwäldern. Forum für Wissen, Wald und Klimawandel, 23-33 

Roloff A (2006). Bäume in der Stadt - was können sie fernab des Naturstandortes ertragen? 

Forst und Holz 61, 350-355. 

Roloff A; Bonn S; Gillner S (2008a). Baumartenwahl und Gehölzverwendung im urbanen 

Raum unter Aspekten des Kimawandels. Forstwiss. Beitr. Tharandt / Contr. For. Sc. 

Beiheft 7, 92-107. 

Roloff A; Bonn S; Gillner S (2008b). Klimawandel und Baumartenwahl. Deutsche Baumschule 

3, 36-38. 

Roloff A; Bonn S; Gillner S (2008c). Klimawandel und Baumartenwahl in der Stadt - Als 

Straßenbäume geeignete Arten bei verändertem Klima. AFZ/Der Wald 63, 398-399. 

Roloff A; Bonn S; Gillner S (2008d). Konsequenzen des Klimawandels – Vorstellung der 

Klima-Arten-Matrix (KLAM) zur Auswahl geeigneter Baumarten. Stadt+Grün 57, 

53-60. 

Roloff A; Bärtels A (2006). Flora der Gehölze - Bestimmung, Eigenschaften, Verwendung. 

Ulmer: Stuttgart. 

Roloff A; Pietzarka U (2007). Zur Baumartenwahl am urbanen Standort. Welche Bedeutung 

hat die Unterscheidung von Pionier- und Klimaxbaumarten. Jahrbuch der Baumpflege, 

Thalacker, 157-168. 

Roloff A; Rust S (2007). Reaktionen von Bäumen auf die Klimaänderung und Konsequenzen 

für die Verwendung. In: Urbane Gehölzverwendung im Klimawandel und aktuelle 

Fragen der Baumpflege, eds A Roloff, D Thiel, H Weiß, pp. 16-28. Forstw. Beitr. 

Tharandt / Contrib. For. Sc., Beiheft 6. 

Sakai A; Larcher W (1987). Frost Survival of Plants - Responses and adaptation to freezing 

stress. Ecological Studies 62. Springer-Verlag: Berlin, Heidelberg. 

Savolainen O; Bokma F; Knürr T; Kärkkainen ??; Pyhäjärvi T; Wachowiak W (2007). 

Adaptation of forest trees to climate change. Climate change and forest genetic 

diversity. Koskela, J. et al; Rom ??? 

Schmidt P A; Freistaat Sachsen, Staatsministerien für Ernährung und Landwirstchaft (Hrsg.) 

(1995). Übersicht der natürlichen Waldgesellschaften Deutschland. Schriftenreihe der 

Sächsischen Landesanstalt für Forsten 4. 

Schraml C; Rennenberg H (2002). The different reactions of beech tree (Fagus sylvatica L.) 

ecotypes to drought stress. Forstw. Cbl. 121, 59-72. 

Schulte U (2003). Waldökologische Strukturveränderungen - 30 Jahre Dauerbeobachtung in 

sechs Naturwaldzellen der Nordeifel. LÖBF-Mitteilungen 2, 35-39. 

Schulte U (2005). Biologische Vielfalt in nordrhein-westfälischen Naturwaldzellen. LÖBF- 

Mitteilungen 3, 43-48. 

Schuster D (2007). Die Suche nach der idealen Baumart. Augsburger Allgmeine, 08. August. 

Schütt P; Weisgerber H; Lang U M; Roloff A; Stimm B (1994-2008). Enzyklopädie der 

Holzgewächse: Handbuch und Atlas der Dendrologie. Landberg am Lech: Ecomed. 

Steffens R; Zander M (2001). Untersuchungen zur Verbreitung, Ökologie und genetischen 

Variation des Speierlings (Sorbus domestica L.) in Sachsen-Anhalt. Mitt. florist. Kart. 

Sachsen-Anhalt 6, 7-24. 

64

Steyrer G; Tomiczek C (2007). Orkanschäden und Witterung begünstigen Borkenkäfer. 

Forstschutz aktuell 40, 3-5. 

Studhalter S; Ulber M; Bonfils P (2001). Förderungsstrategie für eine seltene und wertvolle 

Baumart. Wald und Holz 11, 31-32. 

Tognetti R; Johnson J D; Michelozzi M (1995). The response of European beech (Fagus 

sylvatica L.) seedlings from two Italian populations to drought and recovery. Trees 9, 

348-354. 

Wagner S (2004). Klimawandel - einige Überlegungen zu waldbaulichen Strategien. Forst und 

Holz 59(8), 394-398. 

Walther G-R (2003). Plants in a warmer world. Perspectives in Plant Ecology, Evolution and 

Systematics 6(3), 169-185. 

Walther G-R (2006). Palmen im Wald? Exotische Arten nehmen in Schweizer Wäldern bei 

wärmeren Temperaturen zu. Forum für Wissen, Wald und Klimawandel, 55-61. 

Walther G-R; Berger S; Sykes M T (2005). An ecological 'footprint' of climate change. Proc. 

R. Soc. B 272, 1427-1432. 

Walther H; Breckle S-W (1999). Vegetation und Klimazonen: Grundriss der globalen 

Ökologie. Ulmer: Stuttgart. 

Warda H D (2001). Das große Buch der Garten- und Landschaftsgehölze. Bruns Pflanzen: Bad 

Zwischenahn. 

Wezel G (2008). Die Douglasie auf dem Markt - Anzucht, Anbau und Versorgung. LWF 

Wissen 59, 27-31. 

Wohlgemuth T; Bugmann H; Lischke H; Tinner W (2006). Wie rasch ändert sich die 

Waldvegetation als Folge von raschen Klimaveränderungen? Forum für Wissen, Wald 

und Klimawandel, 7-16. 

Zimmermann N E; Bolliger J; Gehrig-Fasel J; Guisan A; Kienast F; Lischke H; Rickebusch S; 

Wohlgemuth T (2006). Wo wachsen die Bäume in 100 Jahren? Forum für Wissen, 

Wald und Klimawandel, 63-71. 

65

Profft I, Frischbier N: Forestry in a Changing Climate − The Necessity of Thinking Decades Ahead. In: Feldmann 

F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 66-74; ISBN 978-3-941261- 

05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

2-5 Forestry in a Changing Climate − the Necessity of Thinking Decades 

Ahead 

Profft I, Frischbier N 

Thuringian State Institute for Forestry, Game and Fishery, D – 99867 Gotha, Germany 

E-mail: ingolf.profft@forst.thueringen.de 

Forest ecosystems play a decisive role in the global and regional climate change debate. Forest 

ecosystems are the biggest terrestrial carbon sink, taking up significant quantities of carbon 

dioxide (CO2) and storing the carbon for long periods of time in woody biomass and soil, 

thereby reducing levels of atmospheric CO2. These effects become even more pronounced as 

use of sustainable timber, rather than other energy intensive materials or fossil fuels, can lead 

to a significant reduction in greenhouse gas emissions. 

However, forests and their species distribution are directly and indirectly affected by climate 

change, via abiotic and biotic disturbances: Storm and drought events can have as great an 

impact on stand stability and vigour, as occurrences of disease and the actions of pests. 

The following article presents an overview of the current efforts being made to develop an 

adaptation strategy for the forest sector in Thuringia. 

As with other land use in the public and commercial sectors, forestry must consider the threats 

resulting from climate change. Adaptation strategies and measurements to mitigate potential 

risks have to be developed with a long term vision, while still fulfilling all roles expected from 

forests by society. However, compared with other sectors, forestry in general faces particular 

difficulties, such as: 

− Strong dependency on existing site conditions that can not by modified or changed (i.e. 

not practical/possible to use fertiliser, irrigation or use of greenhouses). 

− Significant generation length of tree species and therefore long term consequences of 

decisions, often over large areas. 

− Existing stand conditions exposed to nitrogen depositions, acidification and nutrient 

fluxes as well as present age structure, dominated by age classes below 80 years, that can 

not be changed rapidly without economic losses. 

− Multiple demands and expectations by society that have a direct influence on 

management strategies and decision making by all forest owners. 

66

− Insufficient knowledge about the specific effects climate change can have on tree growth 

and vigour as well as interactions between climate change and forest ecosystem 

functioning. 

Since the Pleistocene, climate conditions have been regarded as stable, despite slight variations 

occurring in Europe and elsewhere. However, climate change is expected to increase the 

variability of climatic conditions in the next decades, particularly in spatial and temporal 

temperature patterns and precipitation levels. Despite a long tradition, forestry, and its 

associated research activities, has hitherto been unable to provide sufficient and satisfactory 

information on this complex issue. Because of the immediate need to develop adaptation 

strategies for forestry, it is not viable to postpone decisions until more and better information is 

available. For these reasons, an adaptation strategy in forestry has to be based on current bestavailable 

knowledge, accompanying latest research activities, and has to be in two parts: 

1. Spatial analyses of potential risks for present forest ecosystems and their services and 

functions based on actual tree species distributions and present climate conditions. 

2. The development of prospective tree species recommendations, considering their long 

term suitability and management strategies which take regional-scale climate changes 

into account. 

Since the growing conditions for forests can rarely be modified for economic, practical or legal 

reasons, soil and climate parameters are the key factors for all forest management decisions 

regarding tree species recommendation, growing potential and silvicultural treatment. The 

climate of Thuringia is characterized by the transition from maritime Western Europe to 

continental Eastern Europe. Despite its relatively small size, Thuringia covers a broad range of 

ecological conditions from dry and warm plains in the centre to the cold and wet mountainous 

regions of the Thuringian forests and includes the Rhön, typified by even more extreme 

weather conditions. 

Under human influence, Norway spruce became the most abundant tree species in Thuringia, 

due to its outstanding growth rate and yield per hectare. Currently, it is of central importance to 

the Thuringian forestry and timber economy and covers about 54% of the forested area. Beech, 

which grows in 26% of the forested area, is the most common deciduous tree species. Oak 

forests account for around 4%, and pine forests for approximately 16% of forested area. 

Work on part I of the two-part adaptation strategy, involves risk analyses which concentrate on 

the main tree species. Therefore, emphasis is placed on the evaluation of Spruce and its 

potential risk. As the name of this species suggests, the Norway spruce prefers the moist and 

cool climates typical of its natural origin, which are mainly to be found in the middle and high 

altitudes of the mountain ranges in Thuringia. Nonetheless, spruce has been planted outside its 

ecologically preferred areas and even sometimes in unsuitable soil types – with significant 

consequences. Due to its high susceptibility to several biotic and abiotic risk factors, e.g. bark 

beetle, drought, wind-throw, and snow-break, stability issues have to be given more 

consideration than has been the case in the past. To assess the risk potential, a multi level 

scheme has been under development taking not only soil and climate conditions into account 

67

ut also other factors such as insect monitoring data. These monitoring data can provide 

essential information about reduced vigour and health of present forest stands caused by 

unsuitable soil conditions and insufficient water regime (availability). These stands are highly 

susceptible to permanent and annual bark beetle infestations and can be distinguished from 

stands with occasional infestation in extreme dry and hot years. Such permanent monitoring 

during the growing season over many years can help detect areas at higher risk. This 

information can be used as a proxy for spatial and temporal analyses of impacts of climate 

change for spruce forest ecosystems. Like other insects, bark beetles are poikilothermic 

organisms. Their body temperature is directly tied to the temperature of their environment. 

Global warming with a prolonged and warmer growing season can have a significant influence 

on bark beetle’s life cycle and thus on infestation patterns and outbreaks. Particularly in the 

case of spruce, such monitoring provides useful information about the regional distribution of 

damage caused by eight-toothed spruce bark beetle (Ips typographus) and six-toothed spruce 

bark beetle (Pityogenes chalcographus). The evaluation combined with additional climate 

information represents vigour deficits and can be a useful parameter within the risk analysis. 

Based on soil and macro-climate classification combined with the age class of present spruce 

stands, regions placed at higher risk of severe damage can be selected and stand-specific 

management options, such as shorter rotation periods or moderate thinning regimes, can be 

implemented. The consequent improvement of stand stability and structure due to thinning 

from above (“high thinning”), driven by a high crown percentage of 50% minimum, h-:-d ratio 

as an indicator of tree stability, is becoming a primary factor in the management of present and 

future spruce forests in Thuringia (Fig. 1). 

This scheme is however still lacking a storm component. Therefore, the next stages will be a 

storm analysis based on a digital elevation model and finally, for economic reasons, a financial 

module to evaluate the financial consequences of changes in management options. 

Subsequently, the scheme is expected to be adapted to produce a comparable risk scheme for 

beech. 

The second part of the adaptation strategy is complex and comprises more uncertainties and 

difficulties. Due to the slow adaptation potential of forest ecosystems compared to the expected 

rate of climate change, one of the key problems forestry must currently resolve is how to 

accurately evaluate the long term suitability of tree species in the context of climate change. 

Several questions must be answered before further steps can be taken: 

− Is the use of climate scenarios (e.g. IPCC SRES), rather than historical measurements 

and their extrapolation, suitable for prospective tree species recommendations? 

− What planning horizon has to be chosen to illustrate a realistic approach for such 

recommendations? 

− What forest-specific climate parameters are suitable indicators for tree growth? 

− What spatial resolution represents an acceptable balance between regional and site-level 

analysis? 

68

Figure 1. Present spruce stands in Thuringia being at potential risk due to climate 

change based on a multi level scheme. Data are given in percentage of the total 

spruce area per macroclimatic unit. 

Scenario data are derived from global circulation models fitted with many parameters in order 

to give a realistic picture of atmospheric processes. Depending on the definition of chosen 

parameters, a set of climate projections for different scenarios is always provided. However, 

while all of these scenarios share an equal probability of actually becoming reality, they are 

able to determine regional trends of climatic change. Additionally, they present information 

about climate conditions that have not been experienced in the past. Conversely, historical 

measurements can only be modified in a very subjective way without sound background and 

consideration of regional differentiations. Even with present uncertainties, climate scenarios 

can therefore provide helpful information for the decision making process regarding long term 

strategies in forestry. Thus, all prospective analyses for the forest sector in Thuringia focus on 

the use of scenario data. In current research in this area, the SRES scenario A1B is mainly 

used. However, when using scenario data one must always be aware of the pre-definitions and 

specific defaults the scenario is based on and take into account what level or range of 

uncertainty is given with the data set. 

69

The question of an appropriate planning horizon for tree species recommendations and 

therefore the chosen reference period used for climate projections is even more complex. Since 

trees can potentially last more than 200 years, those planted at present will be exposed to 

climate conditions for a long period of time, even longer than reliable climate data are available 

for at the moment. Additionally, increasing variability leads to faster changes in growing 

conditions than trees are adapted to. In any case, climate data presently only cover this century 

up to the year 2100. There are three options for defining a planning horizon from a forestry 

perspective (see Fig. 2): 

70 

Figure 2. Options for long term adaptation strategies in forestry 

Option A – present as planning horizon (period 1971-2000): 

(+) optimal climate conditions for the planting stage 

(-) suboptimal climate conditions during the best-growing stage with high increments 

(-) climate conditions becoming more and more unsuitable for planted trees at the end of the 

century 

Option B – middle of the century as planning horizon (2041-2070): 

(+) optimal climate conditions during the best-growing stage with high increments 

(-) suboptimal climate conditions for the planting stage 

(-) climate conditions becoming more and more suboptimal at the end of the century

Option C - end of the century as planning horizon (2071-2100): 

(+) climate conditions becoming more and more optimal at the end of the century, leading to an 

increasing certainty to reach the final stand stage 

(–) unsuitable growing conditions for the planting stage 

(-) suboptimal climate conditions during the best-growing stage 

Growing conditions on site can be modified by silvicultural measurements, e.g. preliminary 

planting instead of clear cutting followed by replanting on open fields. With this strategy, the 

canopy of the upper storey provides shelter against frost and drought for the new stand 

generation. This leads to a milder stand climate with less exposure to weather extremes. 

Additionally, shortening rotation periods and replanting the next generation earlier than 

planned can reduce risks at the end of the century. Consequently, in prospective analyses for 

Thuringian, option B (period 2041-2070) is chosen as planning horizon for the long-term 

adaptation strategy of the forest sector. 

Changes in climate conditions have not have been an important factor of forestry and forest 

research in the past: Despite the many cultivation and provenance tests, only a little 

information is available regarding growing requirements for tree species, both native and 

introduced. In the 1980s, first prediction models for plant distributions based on natural 

vegetation surveys and coupled phytogeographic and climate information were established. 

The resulting climate envelopes for plant and tree species have recently been revived. Kölling 

(2007) developed new climate envelopes, for relevant tree species in Germany, by using data 

for natural vegetation distribution in Central Europe and also the Worldclim data set 

(http://www.waldclim.org). These envelopes give a rough idea of areas of potential for certain 

tree species and can be used for first prospective evaluations through integration with scenario 

climate data. However, this approach neglects soil-specific parameters and therefore can not be 

used for detailed analyses at stand level. Alternatively, more complex vegetation models and 

associated vegetation surveys can be used to derive potential tree species suitability for a 

certain region. In Thuringia, the BERN model (Bioindication for Ecosystem Regeneration 

towards natural conditions) has recently been applied to derive potential tree species suitability 

for Thuringia. Within this model, the existence of plant species and their dependence on 

various site factors is assessed and potential distribution functions of plant communities and 

their related species are developed (Fig. 3). 

71

72 

Figure 3. Ecogram for nutritional class M and specific site classes MS2 (mesotrophic 

moderately moist sandstone), MS3 (mesotrophic moderately dry sandstone), 

MG2 (mesotrophic moderately moist less skeletal silicate) and MG3 

(mesotrophic moderately dry less skeletal silicate) according to the German 

soil classification 

These functions have been applied to present and future conditions to define forest 

communities and related tree species for specific soil and climate conditions. The results will 

be used as starting point for an in-depth evaluation process to develop tree species 

recommendations for Thuringia that not only fulfil ecosystem services and functions in an 

optimal manner, but also meet the demands and expectations of society while continuing to 

make an invaluable contribution to the economy. 

Additional to the work, presented here, knowledge transfer and environmental education must 

be a major task for all institution to improve the knowledge about climate change and people´s

attitude. For this reason, the internet portal “Forest & Climate” was developed in 2004 and 

launched in 2005 under the internet domain www.forestandclimate.net. The portal covers the 

whole issue of climate change and forestry including carbon sequestration aspects. It should 

serve as an open platform for other institutions, associations and groups working in the field of 

forestry, ecosystem research, timber use and climate change, where they can present their work 

and results in a popular scientific manner. Currently more than 200 articles of about 35 

different institutions are online and permanent extensions as well as updates with latest news 

will ensure a sustainable transfer of recent research findings. Every institution, public initiative 

and project is invited to join this activity and to provide articles and information so the internet 

portal “Forest & Climate” (Fig. 4). 

Figure 4. Logo of the internet portal “Forest & Climate” via www.forestandclimate.net 

REFERENCES 

Kölling C (2007). Klimahüllen für 27 Waldbaumarten. AFZ-Der Wald, 23-2007, 1242-1245. 

73

74 

SESSION 3: POSTER PRESENTATIONS

Oldenburg E, Manderscheid R, Erbs M, Weigel H J: Interaction of free air carbon dioxide enrichment (FACE) and 

controlled summer drought on fungal infections of maize. In: Feldmann F, Alford D V, Furk C: Crop Plant 

Resistance to Biotic and Abiotic Factors (2009), 75-83; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 

Gesellschaft, Braunschweig, Germany 

3-1 Interaction of free air carbon dioxide enrichment (FACE) and 

controlled summer drought on fungal infections of maize 

Oldenburg E 1 , Manderscheid R 2 , Erbs M 2 , Weigel HJ 2 

1 

Julius Kühn-Institute, Messeweg 11/12, D- 38104 Braunschweig, Germany 

2 Institute of Biodiversity, Johann Heinrich von Thünen-Institute, Bundesallee 50, D-38116 


Email: elisabeth.oldenburg@jki.bund.de 

ABSTRACT 

The recent report of the Intergovernmental Panel on Climate Change predicts an 

increase of atmospheric CO2 concentration, air temperature and summer drought 

conditions during the next decades. This will influence maize growth and also affect 

its susceptibility to fungal infection. Therefore, a two year experimental study was 

conducted, in which maize was grown in the field under ambient (ca. 380 ppm) and 

elevated atmospheric (550 ppm) CO2 concentrations and two watering regimes 

(well-watered or restricted water supply during summer). Moreover, in the second 

year all variants were combined with two mulching treatments (bare soil, straw 

mulch). In the first year, the rainy summer prevented the initiation of drought stress, 

which was assured in the second year by installation of rain shelters. Smut disease 

symptoms from Ustilago maydis were quite small and not influenced by the 

treatments. Infection by Exserohilum turcicum could be detected in the wellwatered 

treatments in the second year only. Leaf blight was slightly enhanced under 

elevated CO2 concentrations. Fusarium disease symptoms (ear rot, stem rot) could 

not be visually detected or were at a very low level and unrelated to the 

experimental treatments. However, the Fusarium mycotoxin deoxynivalenol (DON) 

indicating latent Fusarium infection was detected in whole plant samples and maize 

kernels. Under well-watered conditions no obvious influence on DON 

concentrations in whole maize samples was observed at elevated atmospheric CO2 

(550 ppm), but DON in kernels was lower at 550 ppm than at 380 ppm atmospheric 

CO2 concentration. Under summer drought conditions, reduced DON levels were 

observed at 550 ppm CO2 and both mulching treatments compared to 380 ppm CO2 

indicating a positive effect on the health status of maize at elevated atmospheric 

CO2 concentration when suffering from water deficit. 

75

INTRODUCTION 

According to the recent report of the Intergovernmental Panel on Climate Change atmospheric 

CO2 concentration ([CO2]) will continue to increase up to 600 ppm until the middle of the 

century and the severity of summer drought will be intensified (Meehl et al. 2007). There is 

evidence that a rise in [CO2] decreases the transpiration demand of C4 plants like maize and 

thus mitigates the negative effects of water shortage (Leakey et al. 2004). Furthermore, 

climatic changes may induce stress effects in cultivated plants thus affecting their susceptibility 

to microbial plant pathogens (Garrett et al. 2006, Burdon et al. 2006). 

There has been no field study in Europe, in which the potential interactive effects of [CO2] and 

summer drought on plant growth and health of maize has been investigated. Therefore a free 

air carbon enrichment (FACE) experiment (Manderscheid et al. 2008) has been conducted 

which investigated the interaction of future [CO2] and summer drought on fungal infections of 

maize under real field conditions. The study was focused on the most relevant pathogenic fungi 

of Fusarium spp., Ustilago maydis and Exserohilum turcicum infecting maize cultivated in 

Europe (Munkvold 2003, Martinez-Ezpinoza et al. 2002, Smith & White 1988) and inducing 

diseases like Fusarium stem and ear rot, smut and leaf blight. Furthermore, Fusarium-infected 

maize is frequently contaminated with Fusarium mycotoxins (Logrieco et al. 2002, Oldenburg 

et al. 2005). Therefore the occurrence of the Fusarium mycotoxin deoxynivalenol in maize was 

also investigated in this study. 


FACE Experiment 

The FACE trial was carried out at an experimental field in Braunschweig (Johann Heinrich von 

Thünen-Institute) during two seasons in the years of 2007 and 2008. Maize was grown in three 

experimental ring areas (20 m diameter) at ambient (380 ppm) and elevated (550 ppm), [CO2] 

respectively, using a free air CO2 enrichment (FACE) system. Details of the field conditions 

and the FACE system have already been published (Weigel et al. 2006). Each ring was 

divided in two half circles with different water regimes (well-watered, i.e. plant available soil 

water content (PAW) > 50%; and drought stress during summer, i.e. PAW

Crop cultivation measures 

In both experimental years the maize variety “Romario” was cultivated on each plot of the 

FACE trial. The maize seed was drilled on 30th April in 2007 and 9 th May in 2008 to establish 

a plant density of about 10 individual plants per m 2 . Herbicides (Callisto, Certrol B, Gardo 

Gold, Peak and Milagro) were applied ca. 10-20 days after crop emergence. The plants were 

fertilized according to local farm practice at sowing (K, N, P, S) and approximately one month 

later (N, Mg) in order to prevent nutrient deficiency. Nitrogen fertilization amounted to 170 

and 200 kg N ha -1 in 2007 and 2008, respectively. 

The CO2 fumigation was started on 9th and 11th June in 2007 and 2008 respectively, when the 

crop had a leaf area index of ca. 0.6, and lasted until final harvest at the end of September. 

Evaluation of fungal disease symptoms 

Ustilago maydis infection were evaluated by monitoring the number of single plants showing 

smut disease symptoms within 30 plants grown in a single row just before harvest and 

integrating the data into the BSA- rating system (see: official guidelines of the 

Bundessortenamt). 

Exserohilum turcicum (syn.=Helminthosporium turcicum ) leaf blight was evaluated by 

visually examining the spread of typical greyish spindle-shaped spots on the upper 3 rd leaf of 

the plants sampled at 7th October one week after final harvest. Plants from the drought stress 

plots were omitted since most leaves were already dead (leaf area index < 0.4). From each of 

the two (with and without straw mulch) well watered quarters of the six experimental ring 

areas a sample containing six leaves was taken. Subsequently, the lamina of the leaves was 

separated with a trimmer into different fractions (greyish spindle-shaped spots, brown area, 

yellow area, and green area). The area of each fraction per sample was determined with a leaf 

area meter (Model LI-3100 from LICOR). 

Fusarium disease symptoms were evaluated by either monitoring the number of single plants 

showing stem rot within 30 plants grown in a single row just before harvest and integrating the 

data into the BSA-rating system (see: official guidelines of the Bundessortenamt) or by 

visually inspecting harvested ears for showing typical Fusarium mycelium and/or conidia. 

Furthermore, the samples were analysed for the Fusarium mycotoxin deoxynivalenol. 

Harvest and sample preparation 

At the growth stage of silo maturity (BBCH 85; see BBCH scale of Meier 2001), 15 whole 

plants per variant were harvested by hand, subsequently chopped and oven dried at 105 °C for 

at least 48 hours. The ears of further 5 single plants per variant were harvested by hand and, 

after removing the husks, oven dried at 105°C for at least 48 hours. After drying, the kernels of 

the ears were separated from spindles. All samples of whole plants and kernels were ground to 

pass through a 1 mm sieve before being analysed for deoxynivalenol. 

77

Determination of deoxynivalenol 

Deoxynivalenol (DON) was determined by use of the competitive ELISA test kit “Ridascreen 

Fast DON”, product No. R 5901 from R-Biopharm, Darmstadt, Germany. The sample 

extraction procedure was carried out as follows: 2.5 g of sample was suspended in 50 ml 

distilled water and shaken for 30 min using a horizontal shaker at 160 motions per minute. The 

extracts were 

first filtered through a fluted filter and then centrifuged at 15.000 rev min -1 for 5 min at 10°C to 

remove solid particles. The resulting supernatants were directly applied in the ELISA test (two 

replicates), which was performed according to the manufacturer’s procedure. The limit of 

quantification was 0.22 mg DON kg -1 . 

RESULTS 

Treatment effects on smut disease 

Ustilago maydis (smut) disease symptoms were observed in a very low extent at BSA-ratings 

of 1-2 (0-2%) in 2007, but in 2008 no smut was detected in any variant of the FACE trial. 

Treatment effects on Exserohilum turcicum leaf blight 

Exserohilum turcicum leaf blight was detected in the second year of the FACE experiment 

only. The area of greyish spindle-shaped spots on the upper 3 rd leaf due to leaf blight was 

lowest under ambient [CO2] and increased slightly under elevated (CO2] (Table 1). 

78 

Table 1: Effect of different atmospheric CO2 concentrations (380 ppm, 550 ppm) and 

mulching treatments (bare soil, straw mulch) on green, greyish (due to leaf 

blight), brown and yellow portion of the area of the upper 3 rd leaf of maize in 

October. Data represent means and standard error (n=3) is given in 

parenthesis. 

CO2 380 ppm 550 ppm 

Mulching bare soil straw bare soil straw 

Green area (cm) 146 (7) 153 (17) 114 (16) 136 (32) 

Grayish area (cm) 17 (5) 12 (6) 23 (13) 30 (18) 

Brown area (cm) 94 (8) 69 (13) 108 (2) 101 (17) 

Yellow area (cm) 5 (2) 12 (1) 9 (3) 13 (6) 

Total leaf area (cm) 262 (4) 245 (8) 255 (4) 280 (7) 

However, the extent of leaf blight infection was small and ranged from 5-10% of the total leaf 

area. The brown coloured area of the leaf, which represented some 40% of the total leaf area,

was higher at 550 ppm than at 380 ppm [CO2] and mainly responsible for a slight decrease in 

the portion of the green area of the plants grown under elevated [CO2]. No specific effects 

resulting from the mulching treatment was visible. 

Treatment effects on Fusarium stem and ear rot 

Symptoms of Fusarium stem rot were not found in 2007, whereas in 2008 were occasionally 

detected at very low BSA ratings of 1-2 (0-2%). In both experimental years, stem rot was 

unimportant and could not be related to any treatment applied in the FACE trial. 

Visible symptoms of Fusarium infections of other organs of the maize plant, especially ear 

infection, were not observed in both experimental years. However, the Fusarium mycotoxin 

DON was detected in both whole plant samples and maize kernels in 2007, indicating latent 

Fusarium infection of the plants. The mean concentrations of DON found in whole plant 

samples were about 5-fold higher in comparison with those found in the kernels (Fig.1), but 

ranged at the same level at both ambient and elevated [CO2]. 

DON 

(mg kg -1 ) 

4 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

380 

Whole plant 

Kernels 

550 380 550 

Atmospheric CO2 concentration ( ppm) 

Figure 1. DON concentrations in whole plant and kernel samples of maize grown under 

well watered conditions at different atmospheric CO2 concentration (380 ppm, 

550 ppm) in the 1 rst year (2007). Data represent means and standard error 

(n=12). 

In contrast, the DON concentrations in the kernels were considerably lower when the plants 

were cultivated at 550 ppm compared to 380 ppm [CO2]. In 2008, DON was not detected in the 

maize kernels, so that a specific effect of CO2 treatment could not be verified in the second 

experimental year. 

Contents of DON detected in whole plant samples in 2008 in dependence of [CO2] and water 

availability are summarized in Fig. 2. At well-watered conditions, higher DON concentrations 

79

were detected at elevated [CO2] compared to ambient [CO2], but this difference remains be 

uncertain due the variability of the results at 550 ppm [CO2]. At conditions of summer drought, 

DON content in whole plants was about 3-fold lower at elevated [CO2] than at ambient [CO2]. 

80 

DON 

(mg kg -1 ) 

Figure 2. DON concentrations in whole plant samples of maize cultivated on bare soil 

under well watered and summer drought conditions at different atmospheric 

CO2 concentrations (380 ppm, 550 ppm) in the 2 nd year (2008). Data represent 

means and standard error (n=3). 

DON 

(mg kg -1 ) 

3,5 

3 

2.5 

2 

1.5 

1 

0,5 

0 

3 

2,5 

2 

1,5 

1 

0,5 

0 

380 550 

380 550 

Atmospheric CO2 concentration (ppm) 

Well watered 

Drought stressed 

Well watered 

Drought stressed 

380 550 

380 550 

Atmospheric CO2 concentration (ppm) 

Figure 3. DON concentrations in whole plant samples of maize cultivated on straw 

mulch under well watered and summer drought conditions at different 

atmospheric CO2 concentrations (380 ppm, 550 ppm) in the 2 nd year (2008). 

Data represent means and standard error (n=3).

When the soil surface was covered with straw under well watered conditions, mean DON 

concentrations in whole plants ranged at a low level at both ambient and elevated [CO2] 

(Fig. 3). 

In contrast drought stress conditions caused about 4-fold higher levels of DON (Fig. 3), but 

mean DON value was somewhat lower at 550 ppm [CO2] than at 380 ppm [CO2]. 

DISCUSSION 

In this project the effect of elevated [CO2] combined with intensified summer drought as 

predicted for the middle of the century by the IPCC (Meehl et al. 2007) was simulated over 

two years in a maize field and the effects on fungal infection of the crop was monitored. This is 

to our knowledge the first study, in which [CO2] and water supply have been manipulated in a 

maize field while the fungal infection of the crop was being investigated. 

Smut disease symptoms from Ustilago maydis were quite small and not influenced by the 

treatments. 

The cultivar “Romario” used in this study is known to be susceptible to infection by 

Exserohilum turcicum. However, leaf blight symptoms were clearly visible only in the second 

year. In order to facilitate a quantification of leaf blight infection, the area of typical greyish 

spindle-shaped spots were measured in the 3 rd upper leaf of maize, which explicitly showed 

these typical symptoms. In the drought stress plots these leaves were already dead and did not 

show leaf blight infection. Under well-watered conditions, leaf blight seems to be slightly 

enhanced under elevated [CO2]. However, the leaf portion with greyish spots was almost below 

10% of the total leaf area and therefore may not have had an effect on plant growth. In 

addition, we observed a bay-coloured area, which represented ca. 40% of total leaf area and 

was also slightly increased under elevated [CO2]. It is not known whether this leaf discolouring 

resulted from any fungal infection. 

Although visible symptoms of Fusarium ear and stem rot were not evident or ranged at a low 

level in both years of investigation, the Fusarium mycotoxin deoxynivalenol indicating latent 

Fusarium infection has been detected in whole plant samples as well as in kernels of maize. 

There was evidence that Fusarium ear infection might be reduced at elevated [CO2] of 550 

ppm, as DON concentrations detected in maize kernels (2007) were significantly reduced 

compared to DON in kernels at [CO2] of 380 ppm. However, this effect could not be verified in 

2008, as no positive DON concentrations have been detected in kernel samples of 2008. 

Under well-watered conditions, elevated [CO2] seems to have no obvious influence on the 

Fusarium infection of whole maize plants; either no differences in DON concentrations 

compared to ambient [CO2] were observed (2007) or DON concentrations exhibited high 

variability at 550 ppm [CO2] (2008). However, when water deficit affects the plants, an 

elevated [CO2] might reduce the susceptibility of maize against Fusarium infection, as 

considerably lower DON concentrations were detected in drought-stressed plants at 550 ppm 

than at 380 ppm [CO2]. A similar effect was observed under drought stress conditions, when 

81

the soil surface was mulched with straw to reduce evaporation, but the decrease in DON 

contamination at 550 ppm [CO2] was not as high as compared to bare soil. This is thought to 

result from a higher infection pressure of Fusarium spores originating from the straw layer, as 

plant residues like wheat straw are known to bepotential sources of plant pathogens, e.g. 

Fusarium (Pereyra et al. 2004). 

The results of this study showed evidence of a positive effect on the health status of maize 

plants suffering from drought stress at elevated atmospheric CO2 concentration, as fungal 

infection with Fusarium spp. causing the contamination with the Fusarium mycotoxin DON 

was observed to be reduced under these conditions. 


Technical assistance by Andrea Kremling, Jürgen Liersch and Evelin Schummer is gratefully 

acknowledged. This experiment was linked to a subproject of LANDCARE2020 and was 

funded by BMBF and the Federal Ministry of Food, Agriculture and Consumer Protection. 

REFERENCES 

Burdon JJ; Thrall PH; Ericson L (2006). The current and future dynamics of disease in plant 

communities. Annual Review of Phytopathology 44, 19-39. 

Garrett KA; Dendy SP; Frank EE; Rouse MN; Travers SE (2006). Climate change effects on 

plant disease: Genomes to Ecosystems. Annual Review of Phytopathology 44, 489-509. 

Leakey A D B; Bernacchi C J; Dohleman F G; Ort D R; Long S P (2004). Will photosynthesis 

of maize (Zea mays) in the US Corn Belt increase in future [CO2] rich atmospheres? An 

analysis of diurnal courses of CO2 uptake under free-air concentration enrichment 

(FACE). Global Change Biology 10, 951-962. 

Logrieco A; Mule G; Moretti A; Bottalico A (2002). Toxigenic Fusarium species and 

mycotoxins associated with maize ear rot in Europe. European Journal of Plant 

Pathology 108, 597-609. 

Manderscheid R; Erbs M; Nozinski E; Weigel H J (2008). Interaction of free air carbon dioxide 

enrichment and controlled summer drought on soil and plant water relations and on 

canopy growth in a maize field. Italian Journal of Agronomy/ Rivista di Agronomia, 3 

Suppl., 615-616. 

Martinez-Espinoza AD; Garcia-Pedrajas MD; Gold SE (2002). The Ustilaginales as plant pests 

and model systems. Fungal Genetics and Biology 35, 1-20. 

Meehl G A; Stocker T F; Cllins W D; Friedlingstein P; Gaye A T; Gregory J M; Kitoh A; 

Knutti R; Murphy J M; Noda A; Raper S C B; Watterson I G; Weaver A J; Zhao Z C 

(2007). Global Climate Projections. In: Climate Change 2007: The Physical Science 

Basis. Contribution of Working Group I to the Fourth Annual Assesment Report of the 

Intergovernmental Panel on Climate Change, eds S Solomon, D Qin, M Manning, Z 

Chen, M Marquis, K B Averyt, M Tignor & H L Miller. Cambridge University Press: 

New York. 

82

Meier U (2001). Growth stages of mono- and dicotyledonous plants. BBCH Monograph. 2 nd 

Edition, Federal Biological Research Centre for Agriculture and Forestry, 

Braunschweig. 

Munkvold GP (2003). Epidemiology of Fusarium diseases and their mycotoxins in maize ears. 

European Journal of Plant Pathology 109, 705-713. 

Oldenburg E; Höppner F; Weinert J (2005). Distribution of deoxynivalenol in Fusariuminfected 

forage maize. Mycotoxin Research 21, 196-199. 

Pereyra SA; Dill-Macky R; Sims AL (2004). Survival and inoculum production of Gibberella 

zeae in wheat residue. Plant Disease 88, 724-730. 

Smith DR; White DG (1988). Diseases of Corn. In: Corn and Corn Improvement, eds GF 

Sprague & JW Dudley, pp.687-766. Madison:Wisconsin. 

Weigel H J; Manderscheid R; Burkart S; Pacholski A; Waloszczyk K; Frühauf C; Heinemeyer 

O (2006). Responses of an arable crop rotation system to elevated [CO2]. In: Managed 

Ecosystems and CO2 Case Studies, Processes, and Perspectives, eds J Nösberger; S P 

Long; R J Norby; M Stitt; G R Hendrey & H. Blum, Ecological Studies, Vol. 187, pp. 

121-137. 

83

Chikh-Rouhou H, González-Torres R, Álvarez JM: Plant tissue colonization by the fungus race 1.2 of Fusarium 

oxysporum f. sp. melonis in resistant melon genotypes. In: Feldmann F, Alford D V, Furk C: Crop Plant 

Resistance to Biotic and Abiotic Factors (2009), 84-86; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 


3-2 Plant tissue colonization by the fungus race 1.2 of Fusarium oxysporum 

f. sp. melonis in resistant melon genotypes 

Chikh-Rouhou H, González-Torres R, Álvarez JM 

Centro de Investigación y Tecnología Agroalimentaria de Aragón. Unidad de Tecnología en 

Producción vegetal. Unidad de Sanidad Vegetal. P.o. Box 727, 50080 Zaragoza. Spain. 

Email: Hela.chikh.rouhou@gmail.com 

INTRODUCTION 

Fusarium wilt of muskmelon caused by Fusarium oxysporum f.sp. melonis (Fom) is one of the 

most threatening diseases of melon crops in Spain and elsewhere (Mas et al. 1981, Appel & 

Gordon 1994). Since 1976 four physiological races of Fom have been described, namely races 

0, 1, 2 and 1.2. Race 1.2 was further divided into two patotypes: 1.2Y, which causes leaf 

yellowing before wilting, and 1.2W nonyellowing strains where wilting occurs without prior 

yellowing symptoms (Risser et al. 1976). 

Once introduced into a field, Fom can persist even after rotation to non host crops because the 

fungus survives in the soil as chlamydospores, and is able to colonize crop residues and roots 

of most crops (Banihashmi & DeZeeuw 1975, Gordon et al. 1989). Because of the persistence 

of the pathogen in the soil, Fusarium wilt of melon can only be properly controlled by the use 

of resistant cultivars or hybrids. No genes have been identified in melons that confer high 

levels of resistance to either 1.2Y or 1.2W. However, it have been found resistance to race 1.2 

in Piboule genotypes, this potential resistance is under polygenic recessive control. This type of 

resistance is difficult to introduce into commercial cultivars, and only a few ones have been 

developed incorporating resistance to Fom, most of them are only used as rootstocks 

(Ficcadenti et al. 2002, Perchepied et al. 2005). 

Nowadays many sources of resistance to Fom races 0, 1 and 2 are known (Cohen & Eyal 1987, 

Zink & Thomas 1990, Pitrat et al.1996, Álvarez et al. 2005), but it does not occur the same for 

race 1.2. For this reason, during 2003-2006,110 melon accessions in CITA (Zaragoza) were 

screened and a relatively high resistance to race 1.2 was found in four accessions, 3 of them 

from Japan and the fourth a Portuguese accession (Chikh-Rouhou et al. 2007). 

The objective of this research was to determine whether the resistant accession plants were able 

to stop fungal invasion of their root or stem. 

84


The plant material used was the susceptible accession ‘Piel de Sapo’ and the resistant ones 

‘Shiroubi Okayoma’, ‘C-211’, ‘K.N.M’ and ‘BG-5384’. The Fom isolates used to prepare the 

inoculum were 37mls and Fom0125 belonging to 1.2W y 1.2Y respectively. 

To test which plant regions were invaded by Fom race 1.2, and to examine the relationship 

between resistance and presence of the pathogen in the plant tissue, seedlings of the susceptible 

genotype and the resistant ones were inoculated with the two pathotypes of Fom race 1.2, and 

after 20 days, three plants of each accession were collected, surface-sterilized in sodium 

hypochlorite for 2min, followed by rinsing during 2 min in sterile water and then dried on 

sterile filter paper. 

Three slices were cut from the lower, middle and upper parts of the hypocotyl respectively 

from each plant of the above genotypes, and then were plated on Petri dishes containing sterile 

V8 medium for 7 days at 25ºC, to determine which parts of the plants were colonized. The 

diameter of the fungus mycelium developed from each section of the hypocotyls was 

measured. These data were ANOVA analyzed and the means were separated using the LSD 

test. 

DISCUSSION 

The results showed, that seven days after plating on sterile V8 medium, a massive growth of 

Fom developed from all segments of the susceptible genotype (‘Piel de Sapo’). All the slices of 

the ‘Piel de Sapo’ hypocotyls were colonized by the fungus, and the mycelium that emerged 

was dense and intensely colored. Most of the hypocotyl slices of the resistant genotypes were 

also colonized, but the diameter and the density of the mycelial mass was significantly smaller 

than those of the mycelium that emerged from the susceptible one. It appears that the extent of 

the colonization by the fungus in the upper segment was somewhat smaller than that in the 

lower and middle hypocotyl. Similar results were obtained by Ficcadenti et al. (2002) who 

found that the fungus was present in the hypocotyls of the resistant plants in a small proportion. 

In our study, the susceptible genotype and the resistant ones differed in colonization of the 

hypocotyl by the pathogen Fom race 1.2, being the diameter of the mycelium produced from 

the hypocotyl slices of ‘Piel de Sapo’ significantly greater than that produced from the resistant 

ones. Indeed, we appreciated a decrease of the mycelium diameter in the upper part of the 

hypocotyl, for the resistant genotypes, which can indicate a restriction of the fungus to the 

lower parts of the plant. So it appears that resistant plants were able to restrict, to some extent, 

on their hypocotyls, the fungal invasion. 

REFERENCES 

Álvarez J M; González-Torres R; Mayor C; Gómez-Guillamón M L (2005). Potential sources 

of resistance to Fusarium wilt and Powdery mildew in melons. HortScience 40 (60), 

1657-1660. 

85

Appel D J; Gordon T R (1994). Local and regional variation in population of Fusarium 

oxysporum from agriculture field soils. Phytopathology 84, 786-791. 

Banihashmi Z ; DeZeeuw D J (1975). The behavior of Fusarium oxysporum f. sp. melonis in 

the presence and absence of host plants. Phytopathology 65, 1212- 1217. 

Chikh-Rouhou H; Álvarez J M; González-Torres R (2007). Differential interaction between 

melon cultivars and Race 1.2 of Fusarium oxysporum f.sp melonis. Communications in 

Agricultural and Applied Biological Sciences 72 (4), 825-829. 

Cohen Y; Eyal H (1987). Downy mildew, powdery mildew and Fusarium wilt resistant 

muskmelon breeding line ‘PI-124111F’. Phytoparasitica 15 (3), 187-195. 

Ficcadenti N; Sestili S; Annibali S; Campanelli G; Belisario A; Maccaroni M; Corazza L 

(2002). Resistance to Fusarium oxysporum f. sp. melonis race 1,2 in muskmelon lines 

‘Nad-1’ and ‘Nad-2’. Plant Disease 86 (8), 897-900. 

Gordon T R; Okamoto D; Jacobson D J (1989). Colonization of muskmelon and non-host 

crops by Fusarium oxysporum f. sp. melonis and other species of Fusarium. 

Phytopathology 79, 1095–1100. 

Mas P; Molot P M; Risser G (1981). Fusarium wilt of muskmelon. In: Fusarium: Disease, 

Biology and Taxonomy, eds P E Nelson; T A Toussen & R J Cook, pp 169-177. 

Pennsylvania State University Press. University Park, PA, USA. 

Perchepied L ; Dogimont C ; Pitrat M (2005). Strain specific and QTLs involved in the control 

of partial resistance to Fusarium oxysporum f.sp melonis race 1.2 in a recombinant 

inbred line population of melon. Theor. Appl. Genet 111, 65-74. 

Pitrat M; Risser G; Bertrand F; Blancard D; Lecoq H (1996). Evaluation of a melon collection 

for disease resistances. Cucurbits Towards 2000. Proceedings of the VIth Eucarpia 

Meeting on Cucurbit Genetics and Breeding, pp 49-58. 

Risser G; Banihashimi Z; Davis D W (1976). A proposed nomenclature of Fusarium 

oxysporum f.sp. melonis races and résistance genes in Cucumis melo. Phytopathology 

66, 1105-1106. 

Zink F W; Thomas C E (1990). Genetics of resistance to Fusarium oxysporum f.sp. melonis 

races 0, 1, and 2 in muskmelon line MR-1. Phytopathology 80, 1230-1232. 

86

Beckuzhina S, Kochieva E: Wheat double haploid lines with improved salt tolerance: in vitro selection and RAPD 

analysis. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 87-90; 

ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-3 Wheat double haploid lines with improved salt tolerance: in vitro 

selection and RAPD analysis 

Beckuzhina S 1 , Kochieva E 2 

1 

Astana Agricultural University, Prospect Pobedy 62, 010011, Astana, Kazakhstan, sarabek@yandex.ru; 

2 

Moscow Agricultural Academy, Timiryazevskaya 49, 127550, Moscow, Russia, 

ekochieva@yandex.ru 

In the major grain crops anther culture is the commonly used method to develop haploids and 

double haploids (DH). Double haploid plants have been increasingly used by breeders to 

develop and release new cultivars with improved agronomic traits. Combination of microspore 

embryogenesis with in vitro selection can provide an efficient screen for desired gametoclonal 

variants. In this method the selection agent is introduced into culture medium. Surviving 

embryos/plants are doubled and grown in the greenhouse. Verification takes place in the in the 

next generation. Several double haploid lines resistant to herbicides have been developed in 

rapeseed by this process (Swanson et al., 1988). Similar systems of in vitro mutagenesis and 

selection were developed for generating DH lines with improved tolerance to Sclerotinia 

sclerotiorum in B. napus (Liu et al., 2005) and Erwinia carotovora in B. campestris (Zhang & 

Takahata, 1999). 

Table 1. Screening of wheat gametoclonal variants in selective conditions. 

Genotype NaCI 

concentration (%) 

No anthers No 

embryoids 

Embryogenesis 

efficiency (%) 

First cycle 

Tselinnaya-Jubileinaya 0.01 2000 16 1.1 

0.05 1180 13 0.52 

0.1 1200 9 1.0 

Second cycle 

U-580 0.01 500 17 3.4 

0.05 500 12 2.4 

0.1 580 13 2.2 

87

In addition to herbicide and disease resistance, mutants for seed quality traits in rapeseed (Kott, 

1998) and for salt tolerance in rice (Rahman et al.. 1995) have been selected. An essential 

component of this system is the molecular characteristic of selected genotypes. Several 

techniques of molecular biology are available for detection of genetic polymorphism at the 

DNA level. The randomly amplified polymorphism (RAPD) method has been widely used to 

estimate genetic diversity (Araujo et al., 2001; Bocianowski et al., 2003). The objectives of 

this study were (I) to screen salt tolerant digaploid wheat lines via anther culture and (II) to 

investigate the genetic diversity of anther-derived plants by RAPD analysis. 

88 

Table 2. Crop yield (centner/ha) in field tests under saline conditions 

Year 

Genotype 2004 2005 2006 Mean 

control 20.2 21.7 26.0 22.6 

U-580 24.1 22.3 26.2 24.2 

LGV-1 24.6 21.9 26.1 24.2 

LGV-3 28.1 20.8 29.6 26.2 

LGV-20 24.7 19.6 25.6 23.3 

The spring wheat cv. Tselinnaya-Jubileinaya was used in the experiments. To screen salt 

tolerant embryoids wheat anthers were cultivated on the selective media containing 0.01, 0.05 

and 0.1% NaCI (Table 1). The selection was performed in the population of 4,380 anthers. The 

anther response varied from 0.52% to 1.1%. The spontaneous digaploid line U-580 was 

selected and grown in the greenhouse to maturity. The F1 generation of this line was subjected 

to the second cycle of in vitro anther culture. We were able to screen three gametoclonal lines 

LGV-1, LGV-3 and LGV-20 under selective conditions (NaCI). The response of selected lines 

to salt salinity was investigated at the field site in the Agricultural Research Centre, 

Kazakhstan (Table 2). There was a significant difference between the control wheat cultivar 

and double haploids. The gametoclonal line LGV-3 demonstrated the highest yield in saline 

conditions. The field test has revealed that stress tolerance was manifested at the level of whole 

plant and inherited. 

After observing the inheritance of salt tolerance in field trails RAPD analysis was performed to 

investigate the genetic basis of this variation. The 9 decamber primers amplified 24 

polymorphic fragments. The RAPD profiles of three gametoclonal lines LGV-1, LGV-3, LGV- 

20 differentiated this group from parental U-580 line: 13 polymorphic lines were scored. The 

dendrogram generated by cluster analysis of RAPD polymorphism using coefficient of 

similarity of Jaccard for investigated genotypes can be divided into two groups (Fig. 1). The 

first one includes LGV-1 and LGV-20 gametoclones. The original cv. Tselinnaya-Jubileinaya, 

DH line U-580 and gametoclone LGV-3 belong to the second subgroup.

Figure 1. Dendrogram generated by cluster analysis of RAPD polymorphism showing 

genetic divergence between wheat cultivars Akmola-2 (1), Tselinnaya- 

Jubileinaya (3) and gametoclonal lines U-580 (2), LGV-1 (4), LGV-3 (6) and 

LGV-20 (5). 

The data presented here provide further evidence that the anther culture technique has the 

potential to increase wheat stress tolerance. The phenotypic variation for salt tolerance was 

related to genetic variability between the parental cultivar (U-580) and gametoclonal variants 

(LGV-1, LGV-3 and LGV-20), as was shown by RAPD analysis. Double haploid lines 

designed in this study can be used in breeding programmes to design salt-tolerant genotypes 

and in basic research to study the mechanisms of salt tolerance. 

REFERENCES 

Araujo L G; Prabhu A S; Filippi M C & Chaves L J (2001). RAPD analysis of blast resistant 

somaclones from upland rice cultivar IAC 47 for genetic divergence. Plant Cell Tissue 

and organ Culture. 67, 16-172. 

Bocianowski J; Chelkowski J; Kuczynska A; Wisniewska H; Surma M & Adamski T. (2003). 

Assessment of RAPD markers for barley doubled haploid lines resistant and susceptible 

to Fusarium culmorum at seedling and adult plant growth stages. Applied Genetic 44, 

355-360. 

Kott L (1988). Application of doubled haploid technology in breeding of oilseed Brassica 

napus. AgBiotech News Inform 10, 69N-74N. 

Liu S; Wang H; Zhang J; Fitt B D L; Xu Z; Evans N; Liu Y; Yang W & Gao X (2005). In 

vitro mutation and selection of doubled haploids Brassica napus lines with improved 

resistance to Sclerotinia sclerotorium. Plant Cell Reports 24, 133-144. 

Rahman M H; Krishinaraj S & Thorpe T A (1995). Selection for salt tolerance in vitro using 

microspore-derived embryos of Brassica napus CV Topas, and the characterization of 

putative tolerant plants. In vitro cellular and developmental biology. Plant 31, 116-121. 

89

Swanson E B; Coumans M P; Brown G L; Patel J D & Beversdorf W D (1988). The 

characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection 

of microspores and protoplasts. Plant Cell Reports 7, 83-87. 

Zhang & Takahata Y (1999). Microspore mutagenesis and in vitro selection for resistance to 

soft rot disease in Chinese cabbage (Brassica campestris L. ssp. Pekinesis). Breeding 

Science 49, 161-166. 

90

Wegener C B, Jansen G: Antioxidants in wild and cultivated potato species. In: Feldmann F, Alford D V, Furk C: 



3-4 Antioxidants in wild and cultivated potato species 

Wegener C B, Jansen G 

Julius Kuehn Institute, Institute for Resistance Research and Stress Tolerance, Experimental 

Station for Potato Research, D-18190 Sanitz, Germany 

Email: christina.wegener@jki.bund.de 

ABSTRACT 

Wild potatoes are a valuable gene pool that is of increasing interest in potato 

breeding. In this study, 17 accessions of cultivated Solanum tuberosum subsp. 

andigena and three wild potato species (S. bulbocastanum, S. chacoense, S. 

pinnatisectum) were examined for their contents of soluble phenols including 

chlorogenic acid in tuber tissue and their antioxidant capacity. Among them, S. 

pinnatisectum accessions exhibited on average the highest quantities of phenols in 

their tuber tissue, coincident with an enhanced antioxidant activity. S. tuberosum 

subsp. antigena, S. chaconese and S. bulbocastanum accessions all expressed lower 

levels concerning these quality traits. 

INTRODUCTION 

During metabolism plants continuously generate reactive oxygen species (ROS), whose 

formation is accelerated under varying types of environmental stress (Noctor & Foyer 1998), 

such as pathogen attack, wounding, high light intensity and heavy metal concentrations, low 

and high temperature, drought etc. ROS include superoxide -, hydroxyl - and peroxyl radicals, 

hydrogen peroxide as well as singlet oxygen ( 1 O2). At low concentrations these intermediates 

have useful functions as signalling molecules, linked to a cascade of plant responses to biotic 

and abiotic stresses (Desikan et al. 2005). However, increased levels of active oxygen species 

as caused by environmental stresses are associated with oxidation of DNA, proteins and 

membrane lipids, altogether toxic processes that lead to disruption of metabolism and 

destruction of cells (Desikan et al. 2005). In order to provide protection against such oxidative 

stress, plants have evolved inducible antioxidant mechanisms that keep the active oxygen 

under control (Noctor & Foyer 1998). The antioxidative system of plants comprises numerous 

enzymes such as superoxide dismutase, ascobate peroxidase, catalase etc. and compounds of 

low molecular weight, e.g. ascorbate, glutathione, α-tocopherol, and carotenoids. In addition, 

plant phenols function as radical scavengers (Grace 2005). The phenolics, including 

flavonoids, tannins, hydroxycinnamates and lignin, are mainly derived from cinnamic acid via 

91

the phenylpropanoid metabolism (Hahlbrock & Scheel 1989). Similar to reactive oxygen 

species, phenylpropanoids are inducible by various environmental stresses (Dixon & Paiva 

1995). The study of stress-inducible responses in plants is a prerequisite for the development of 

stress tolerant crop species that are able to produce high yield under stress conditions (Jansen et 

al. 2008). Also for potato breeding a high level of tolerance to biotic and abiotic stresses is a 

major challenge for the future. Another goal is enhancing positive health-related quality traits 

like vitamins, antioxidants and anti-cancer compounds (Bamberg & del Rio 2007). In this 

context the rich genetic resource comprised by wild potatoes is a valuable source which should 

increasingly be explored and exploited in order to improve cultivated potatoes. 

In the present study, a cultivated (S. tuberosum subsp. andigena, adg) and three wild potato 

species (Solanum bulbocastanum, blb; S. chacoense, chc; S. pinnatisectum, pnt), each 

comprising several accessions (Table 1 and 2), were examined for contents of soluble phenols 

as well as chlorogenic acid in tuber tissue, and their antioxidant capacity. The role of phenolic 

compounds as an important component of the antioxidant system in plants is highlighted. 


Seed tubers of wild (blb, chc, pnt) and cultivated potato species (adg) were supplied by the 

Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Potato Genebank, 

Groß Lüsewitz. Ten plants per accession were grown in pots under a shelter from April to 

October 2007. Fertilizer, insecticides and all other treatments were conducted according to 

local agronomic practice. After harvest, tubers were stored in a controlled environment at 5 °C. 

The analyses described below were performed in duplicate (SD ≤ 5%); 20 tubers were taken as 

an average sample for each accession. 

Soluble phenol analyses 

Preparation of extracts from tuber tissue for assaying total soluble phenols and chlorogenic 

acid was carried out as detailed in Wegener et al. (2009). The total amount of phenols present 

in the extracts was determined according to Cahill & Mc Comb (1992). Standards were 

prepared from p-coumaric acid. Amounts of soluble phenols were expressed in grams per 

kilogram of fresh weight (fw). 

Measurement of chlorogenic acid was performed as described by Griffiths et al. (1992). 

Amounts of chlorogenic acid were calculated as grams per kilogram of freeze dried matter 

(fdm). 

Assay of antioxidant capacity 

Measurement of the antioxidant activity by means of a photochemiluminescent method (PCL) 

was performed on a Photochem instrument, utilizing an ACW-kit for water soluble and an 

ACL-kit for lipid soluble antioxidants (Instrument and kit reagents: AnalytikJena AG), as 

92

detailed by Wegener et al. (2009). The antioxidant activity was calculated by means of an 

ascorbic acid calibration curve for hydrophilic antioxidants and a trolox calibration curve for 

lipid soluble antioxidants, using the Photochem software package. Results were expressed in 

microgram equivalents in antioxidant activity of the reference compound, i.e. as ascorbic acid 

(ACE) or trolox equivalents (TXE) per microgram of fresh weight (fw). 

Statistic analyses: The differences in view of phenol contents and antioxidant activity between 

S. pinnatisectum and the other three potato species were valued by means of t-test for unpaired 

samples, whereby P< 0.05 was considered significant. 


Within the group of wild and cultivated potatoes tested here, S. pinnatisectum accessions 

revealed on average the highest quantity of phenols including chlorogenic acid in their tuber 

tissue (Table 1), while S. bulbocastanum accessions ranked on a lowest level concerning both. 

Table 1. Concentrations of soluble phenols and chlorogenic acid in tuber tissue of wild 

and cultivated potato species (Significance of the difference between pnt and 

the other potato species * P < 0.05) 

Species 

Number of Chlorogenic acid (g kg -1 fdm) Soluble phenols (g kg -1 fw) 

accessions Average Range Average Range 

adg 3 0.63 0.44 - 0.91 0.57 0.50 - 0.66 

blb 3 0.19 0.18 - 0.19 0.32 0.26 - 0.36 

chc 5 1.01 0.56 - 1.65 0.54 0.27 - 0.90 

pnt 6 1.64* 0.60 - 2.68 1.55* 0.58 - 2.76 

Particularly, accession pnt 98-2 exceeded all the other genotypes in its phenol (2.76 g kg -1 fw) 

and chlorogenic acid content (2.68 g kg -1 fdm), a tendency that had been observed one year 

before. Generally, amounts of phenols found in 2007 were in a good agreement with the results 

of the year 2006 (Wegener & Jansen, unpublished data). It should be mentioned, that all 

accessions of S. pinnatisectum were comparable in their phenol values with purple fleshed 

potato breeding clones that revealed on average notably higher phenol quantities (2.6-times) in 

tuber tissue than white/yellow fleshed cultivars (Wegener et al. 2009). The high level of 

soluble phenols in pnt accessions coincided with an enhanced antioxidant activity, including 

water (ACE) and lipid soluble (TXE) antioxidants (Table 2). Again the accession pnt 98-2 was 

outstanding in both, i.e. its ACE (3.64 µg mg -1 fw) and TXE value (4.52 µg mg -1 fw) were 

considerably higher than that of all the other accessions tested here. 

93

94 

Table 2. Water (ACE) and lipid soluble (TXE) antioxidant capacity of wild and 

cultivated potato species (Significance of the difference between pnt and the 

other potato species * P < 0.05) 

Species 

Number of ACE equivalent (µg mg -1 fw) TXE equivalent (µg mg -1 fw) 

accessions Average Range Average Range 

adg 3 0.19 0.09 - 0.40 0.37 0.19 - 0.73 

blb 3 0.03 0.01 - 0.04 0.07 0.03 - 0.10 

chc 5 0.74 0.40 - 1.86 1.01 0.56 - 1.80 

pnt 6 2.09* 0.77 - 3.64 2.62* 0.92 - 4.52 

Moreover, a significant (P< 0.01, n=17) correlation could be observed between ACE and TXE 

values (r=0.98), ACE and phenols (r=0.95) as well as TXE and phenols (r=0.96). All this may 

underline the special role of plant phenols as scavengers of free radicals. In addition, these 

results demonstrate that wild potatoes could be a prominent source for valuable quality traits in 

future potato breeding. For example, an involvement of S. pinnatisectum accessions could be 

beneficial to boost the level of the antioxidant system in new cultivars. However, in 

comparison to the breadth of material available in genebanks worldwide, relatively little has 

been used up to now (Bamberg & del Rio 2007). This may change in future, when the 

international potato genome sequencing project will discover interesting genes, and the 

announced new tools such as transformation of potatoes with cisgenes (Jacobsen & Schouten 

2008) will be successfully introduced into potato breeding. 

REFERENCES 

Bamberg J B; del Rio A H (2007). Genetic diversity and genebanks. Potato Research 50, 207- 

210. 

Cahill D M; McComb J A (1992). A comparison of changes in phenylalanine ammonia-lyase 

activity, lignin and phenolic synthesis in the roots of Eucalyptus calophylla (field 

resistant) and E. marginata (susceptible) when infected with Phytophthora cinnamomi. 

Physiological and Molecular Plant Pathology 40, 315-332. 

Desikan R; Hancock J; Neill S (2005). Reactive oxygen species as signalling molecules. In: 

Antioxidants and reactive oxygen species in plants, ed N Smirnoff, pp. 169-196. 

Blackwell Publishing Ltd: Oxford. 

Dixon R A; Paiva N L (1995). Stress-induced phenylpropanoid metabolism. The Plant Cell 7, 

1085-1097. 

Grace S C (2005). Phenolics as antioxidants. In: Antioxidants and reactive oxygen species in 

plants, ed N Smirnoff, pp. 142-168. Blackwell Publishing Ltd: Oxford. 

Griffiths D W; Bain H; Dale M F B (1992). Development of a rapid colorimetric method for 

determination of chlorogenic acid in freeze-dried potato tubers. Journal of the Science 

of Food and Agriculture 58, 41-48.

Hahlbrock K; Scheel D (1989). Physiology and molecular biology of the phenylpropanoid 

metabolism. Annual Review Plant Physiology and Plant Molecular Biology 40, 347- 

369. 

Jacobsen E; Schouten H J (2008). Cisgenesis, a new tool for traditional plant breeding. Potato 

Research 51, 75-88. 

Jansen M A K; Hectors K; O’Brien N M; Guisez Y; Potters G (2008). Plant stress and human 

health: Do human consumers benefit from UV-B acclimated crops? Plant Science 175, 

449-458. 

Noctor G; Foyer C H (1998). Ascorbate and Glutathione: Keeping active oxygen under control. 

Annual Review Plant Physiology and Plant Molecular Biology 49, 249-279. 

Wegener C B; Jansen G; Jürgens H U; Schütze W (2009). Special quality traits of coloured 

potato breeding clones: Anthocyanins, soluble phenols and antioxidant capacity. 

Journal of the Science of Food and Agriculture 89, 206-215. 

95

Kopertekh L, Schiemann J: Removal of a selectable marker in transgenic potato by PVX-Cre virus vector. In: 

Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 96-99; ISBN 978-3- 

941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-5 Removal of a selectable marker in transgenic potato by PVX-Cre virus 

vector 

Kopertekh L, Schiemann J 

Julius Kühn Institute, Federal Research Centre for Cultivated Plants (JKI), Institute for 

Biosafety of Genetically Modified Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 

Potato (Solanum tuberosum) is the most important non-cereal food crop and ranks fourth in 

world production after wheat, maize and rice. A number of new potato genotypes with 

improved nutritional value (Chakraborty et al., 2000) and enhanced tolerance against 

pathogens (Missiou et al., 2004; Meiylaghan et al., 2006) and environmental stresses (Tang et 

al., 2008) have been designed by genetic transformation. Generation of transgenic plants is 

usually based on the selection of transgenic events using marker genes. Selectable marker 

genes are not required for the expression of the trait gene and could be removed from the 

characterized transgenic plants. The reasons and strategies for generating marker-free 

transgenic plants have been discussed in several reviews (Hare & Chua, 2002; Miki & 

McHugh, 2004). One of the approaches is based on site-specific recombination. The Cre/lox 

site-specific recombination system turned out to be very efficient in different plant species and 

has become the most commonly used site-specific recombination system for the elimination of 

marker genes. We report here on the application of the transient Cre/lox system based on PVX 

virus vector to potato plants. 

The system that we designed includes two components: lox-transgenic plants and PVX-Cre 

virus vector. In the lox-containing pLH-35S-lx-nptII-lx-gfp construct for plant transformation 

the nptII marker gene is flanked by two lox sites in direct orientation. Delivery of Cre 

recomobinase by plant virus vector should result in the removal of the nptII sequence and 

subsequent expression of the gfp reporter gene (Fig. 1). We utilized a PVX plus-strand RNA 

virus that replicates extra chromosomally, moves quickly from cell to cell from a site of local 

infection and can redirect protein synthesis of host cells to express high levels of protein of 

interest throughout the plant to transiently express Cre protein in potato. In this vector the cre 

sequence was inserted into a PVX virus cDNA clone between PVX movement and coat protein 

genes. 

The pLH-35S-lx-nptII-lx-gfp construct was transferred into potato (cv. Tomensa) by 

agrobacterium-mediated transformation. A total of 89 independent transgenic potato lines were 

selected on the basis of their ability to grow and root on kanamycin selective medium. PCR 

analysis confirmed the presence of the nptII marker and gfp reporter genes in these lines. 

96

Southern blot analysis for seven transgenic lines revealed that they contained 1 to 3 copies of 

the T-DNA. Three transgenic lines with single T-DNA copy (2, 12 and 31) were chosen for 

subsequent experiments and propagated under sterile conditions. 

Figure 1. Schematic representation of the pLH-35S-lx-nptII-lx-gfp plasmid and PVX-Cre 

expression vector. PLH-35S-lx-nptII-lx-gfp is a gene expression construct to 

drive the nptII and gfp gene expression in potato plants. It contains the loxflanked 

nptII selectable marker gene and the gfp reporter gene in inactive state. 

Once PVX-Cre-mediated gene excision between two lox sites occurs, the nptII 

sequence is removed and the reporter gene will be activated. The expected 

PCR products before and after marker gene excision are indicated, and the 

expected size (bp) of the products is shown. PVX-Cre expression vector 

includes RNA polymerase (165K), triple gene block sequences (8K, 12K, 

25K), cre recombinase gene (cre) and coat protein (cp) gene. 

At the next step potato leaf explants were infected with PVX-Cre vector via particle 

bombardment. To enhance the Cre-expression level the RNA silencing suppressor p19 was codelivered 

together with PVX-Cre vector. Infected explants were allowed to regenerate without 

selection pressure. One important aspect of this strategy is the elimination of the virus from 

infected tissue. To this end the regeneration medium was supplemented with 5 mg/l of the 

nucleoside analog ribavirin (1, beta-D-ribofuranosyl-1,2,4-tirazole-3-carboxamide). Numerous 

plants regenerated after 6-8 weeks of cultivation did not demonstrate any morphologic 

abnormalities. Selection of marker-free potato plants was done by PCR analysis. This approach 

allowed us to separate regenerants with complete nptII gene excision from chimeric plants. 

Designed primers amplify the large PCR fragment of 1754 bp (Fig. 1) from the unrecombined 

pLH-35S-lx-nptII-lx-gfp construct and the small fragment of 660 bp after site-specific 

recombination. About 73 plants yielded a PCR product according to the elimination of the nptII 

gene (Fig. 2, Table 1). 

97

98 

Figure 2. PCR analysis of PVX-Cre-mediated gene excision in potato regenerants. 

Genomic DNA was extracted from potato plants (line 2 (lanes 1 and 2; line 12 

(lanes 3 and 4) and line 31 (lanes 5 and 6)) regenerated from PVX-Cre 

infected leaf explants and subjected to PCR analysis. Forward and reverse 

primers amplify either a 660 bp PCR product indicating Cre-mediated nptII 

gene excision or a 1754 bp PCR product indicating unexcised lox-flanked 

sequence. DNA of non-infected line 2 (Lane 7), wild type potato (lane 8), and 

plasmid pLH-35S-lx-nptII-lx-gfp was used as control. Molecular weight 

marker (M). 

The recombination efficiency expressed as a ratio of plants with complete gene excision to the 

total number of investigated plants varied from 20% for line 2 and 31 to 27% for line 12. These 

data indicate that PVX-Cre-mediated marker gene excision in potato was more efficient than 

the self-excision heat inducible Cre/lox system (Cuellar et al., 2006). The presence of virus 

was examined in leaf tissue of the regenerants by Western blot analysis using antibody to PVX 

coat protein. The analysis showed that only one plant from 73 investigated plants was infected. 

This result is similar to that in an earlier report, where it was shown that ribavirin eliminated 

virus from PVX infected tobacco very efficiently (Kopertekh et al., 2005). 

Table 1. Efficiency of PVX-Cre-mediated marker gene elimination in potato. 

Line 

Regenerants 

Tested No excision Excision Recombiantion 

efficiency, % 

2 100 80 20 20 

12 113 83 30 27 

31 112 89 23 20

In conclusion, the results reported here demonstrate the successful excision of the antibiotic 

resistance nptII gene in potato. Cre-virus vectors should provide a useful tool, especially for 

vegetatively propagated species to select transformants that have lost the resistance markers or 

any other undesirable transgene sequence from transgenic plants. 

REFERENCES 

Chakraborty S; Chakraborty N & Datta A (2000). Increased nutritive value of transgenic potato 

by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. 

Proceedings of the National Academy of Sciences 97, 3724-3729. 

Cuellar W; Gaudin A; Solorzano D; Casas A; Nopo L; Chudalayandi P; Medrano G; Kreuze J 

& Ghislain M (2006). Self-excision of the antibiotic resistance gene nptII using a heat 

inducible Cre-loxP system from transgenic potato. Plant Molecular Biology 62, 71-82. 

Hare P D & Chua N-H (2002). Excision of selectable marker genes from transgenic plants. 

Nature Biotechnology. 20, 575-580. 

Kopertekh L; Jüttner G & Schiemann J (2004). PVX-Cre-mediated marker gene elimination 

from transgenic plants. Plant Molecular Biology 55, 491-500. 

Meiyalaghan S; Jacobs J M E; Butler R C; Wratten S D & Conner A J (2006). Transgenic 

potato lines expressing cry1Ba1 or cry1Ca5 genes are resistant to potato tuber moth. 

Transgenic Research 49, 203-216. 

Miki B. & McHugh S. (2004). Selectable marker genes in transgenic plants: applications, 

alternatives and biosafety. Journal of Biotechnology 107, 193-232. 

Missiou A; Kalantidis K; Boutla A; Tzortzakaki S; Tabler M & Tsagris M (2004). Generation 

of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA 

silencing. Molecular Breeding 14, 185-197. 

Tang L; Kim M D; Yang K-S; Kwon S-Y; Kim S-H, Kim J-S; Yun D-J; Kwak S-S & Lee H-S 

(2008). Enhanced tolerance of transgenic potato plants overexpressing nucleoside 

diphosphate kinase 2 against multiple environmental stresses. Transgenic Research 17, 

705-715. 

99

Jansen G: Effects of temperature on yield parameters of Lupinus angustifolius and Pisum sativum cultivars. In: 

Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 100; ISBN 978-3- 


3-6 Effects of temperature on yield parameters of Lupinus angustifolius 

and Pisum sativum cultivars 

Jansen G 

JKI, Institute for Resistance Research and Stress Tolerance, OT Groß Lüsewitz, Rudolf-Schick- 

Platz 3, 18190 Sanitz 

gisela.jansen@jki.bund.de 

100 

Abstract 

The future of agricultural productivity depends on the ability of different plant 

species to grow in changing environments. One of the important changes that will 

occure with global warming is rising temperature during the growing period. This 

study is focused on the analysis of the effects of rising temperature on yield 

parameters such as pod setting, seeds per pod and whole seed yield of Lupinus 

angustifolius and Pisum Sativum cultivars.

Majic D: Antioxidative enzymes in buckwheat (Fagopyrum esculentum Moench) leaves subjected to flooding 

stress. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 101; 


3-7 Antioxidative enzymes in buckwheat (Fagopyrum esculentum Moench) 

leaves subjected to flooding stress 

Majic D 

Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O.Box 23, 

11010 Belgrade, Serbia 

Email: draganamajic@yahoo.com 

Abstract 

Due to the global climate changes, flooding became a widespread natural disaster, 

greatly reducing survival and yield of many important crops. Although the 

prominent consequence of flooding is oxygen deprivation, the most severe damage 

plant encounters during re-aeration. 

The behavior of the enzymatic antioxidant defense system was studied in 

buckwheat leaves subjected to flooding stress. The effects of flooding stress were 

analyzed during hypoxia as well as upon return to air. Oxidative damage was 

detected during flooding and in its aftermath, monitoring ROS and lipid 

peroxidation levels. In order to define the antioxidative status in the stressed leaves, 

activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase, 

and soluble peroxidases were measured. The results show that antioxidant 

enzymatic activities were enhanced during hypoxia, but the most prominent 

increases were noticed upon return to air, when the strongest oxidative stress occurs 

and the need for antioxidative defense is the highest. 

101

Witzel K, Hensel G, Kumlehn J, Hajirezaei M, Rutten T, Melzer M, Börner A, Mock H-P, Kunze G: Analysis of 

Barley Genotypes with Contrasting Response Towards Salinity Using Complementary Molecular and 

Biochemical Approaches. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 

Factors (2009), 102; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, 

Germany 

3-8 Analysis of Barley Genotypes with Contrasting Response Towards 

Salinity Using Complementary Molecular and Biochemical Approaches 

Witzel K, Hensel G, Kumlehn J, Hajirezaei M, Rutten T, Melzer M, Börner A, Mock H-P, 

Kunze G 

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 

Gatersleben, Germany 

Email: hensel@ipk-gatersleben.de 

102 

Abstract 

Salinity is one of the most severe abiotic stress factors, and there is a high interest in 

unraveling mechanisms leading to salt tolerance and improvement of crop plant 

performance on saline soils. Among the cereals, barley is considered to be notably 

salt-tolerant, and available accessions cover a wide range of responses towards 

salinity. Therefore, the analysis of mapping populations representing a huge natural 

genetic diversity is an appropriate tool to study line-specific stress responses. Here 

we report on the analysis of contrasting accessions from the Steptoe-Morex 

mapping population with molecular, biochemical and structural methods to obtain a 

full picture of salt stress response mechanisms. This experimental platform includes 

proteome analysis of root tissue, followed by MS-based identification of proteins 

that show differential expression between genotypes or upon salt treatment. A 

comparative profiling of primary metabolism compound (carbohydrates, amino 

acids and compatible solutes) is performed using HPLC and GC-MS 

instrumentation. Furthermore, the analysis of morphology and ultrastructure is 

investigated in order to assess the effect of salt treatment on cell structure. In order 

to isolate genes conferring salt tolerance, a heterologous gene expression system is 

utilized, where a cDNA library was constructed from root tissue of a salt-adapted 

tolerant barley line and transferred into salt sensitive yeast strains. Transformants 

with an enhanced tolerance towards salinity are isolated and the barley cDNA 

analyzed. For functional testing of candidates revealed by proteome and 

transcriptome approaches, stable over-expression experiments in barley are 

performed. By employing this integrative approach we want to identify mechanisms 

augmenting salt tolerance in barley.

Schum A , Balko C, Debnath M: Root Characteristics and N Uptake of Potato Genotypes Grown in vitro in 

Response to Nitrogen Deficiency Stress. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and 

Abiotic Factors (2009), 103; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, 


3-9 Root Characteristics and N Uptake of Potato Genotypes Grown in vitro 

in Response to Nitrogen Deficiency Stress 

Schum A 1 , Balko C 1 , Debnath M 2 

1 

Institute for Resistance Research and Stress Tolerance, JKI, Rudolf-Schick-Platz 3, Ortsteil 

Groß Lüsewitz, 18190 Sanitz 

2 

MGIAS, Shri Ram ki Nangal, Tonk Road, Jaipur, Rajasthan, Indien 

Email: christiane.balko@jki.bund.de 

Abstract 

The world's population is constantly growing while, at the same time, resources as 

energy and water become scarce. An increased efficiency of plant production per 

unit area under sustainable conditions is required to secure the provision of food 

and non food products. The ability for efficient nutrient uptake and utilisation 

differs between plant species due to specific morphological and physiological 

characteristics. Furthermore, genetically based differences have been demonstrated 

within one species. This indicates the feasibility of increasing the nutrient use 

efficiency by plant breeding. Roots play an important role in nutrient and water 

uptake and respond very directly to nitrogen stress. In our investigation an in vitro 

culture system was used to grow potato plantlets at different nitrogen levels. After 

two weeks of cultivation root parameters were determined by image analysis. All 

measured parameters, as for example root length, root diameter, and number of root 

tips, were negatively influenced by reduction of N supply to 1/8 of the original 

concentration. Some genotypes displayed a greater relative reduction in root length, 

others in root diameter. Kinetics of nitrogen uptake from the nutrient solutions has 

been determined as well. 

103

Jonsson A, Almquist C, Wallenhammar A-C: Correlation between soil characteristics and in-field variation of 

soil-borne pathogens. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 

(2009), 104; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-10 Correlation between soil characteristics and in-field variation of soilborne 

pathogens 

Jonsson A 1 , Almquist C 1 , Wallenhammar A-C 2 

1 

Division of Precision Agriculture, Department of Soil and Enviroment, SLU, PO Box 234, SE 

53223 Skara, Sweden 

2 

HS Konsult AB, Boställsvägen 4, SE 70227 Örebro, Sweden 

Email: anders.jonsson@mark.slu.se 

104 

Abstract 

Soil-borne diseases are responsible for annual yield losses in many agricultural 

crops. A prerequisite for successful development of a sustainable plant production is 

the availability of efficient analysis of soil- borne the diseases such as 

Aphanomyces eutheiches causing root rot in peas, Plasmodiophora brassicae 

causing club root in oil seed rape. Spatial variability within fields and variations 

between fields in the occurrence of Plasmodiophora brassicae and Aphanomyces 

euteiches were determined on farms in south and central Sweden using quantitative 

PCR-assays. The molecular methods developed are validated by traditional bioassay 

techniques. Soil was sampled using GPS from fields where the disease occurred and 

the results are presented as an interpolated disease map. Relations between the 

occurrence of pathogens and soil parameters such as pH-value, soil type, clay 

content, plant available macro- and micro nutrients were evaluated. The amount of 

pathogens were also correlated to electromagnetic conductivity (EM38).

Ryabushkina N, Askapuly A, Stanbekova G, Galiakparov N: First report of three grapevine viruses in Kazakhstan. 

In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 105; ISBN 978- 

3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-11 First report of three grapevine viruses in Kazakhstan 

Ryabushkina N, Askapuly A, Stanbekova G, Galiakparov N 

Timirjazev str, 45, Almaty, Kazakhstan 

Email: natrya7@yahoo.com 

Abstract: 

First survey for three economically important grapevine viruses, Grapevine Virus A 

(GVA), Grapevine leafroll - associated virus 3 (GLRaV-3) and Grapevine fanleaf 

virus (GFLV), has been done in Kazakhstan. For each virus pair of primers specific 

to the conservative regions of genome were designed and synthesized. These 

primers could be used simultaneously in reverse transcription reaction and PCR for 

detection of these viruses. Samples were randomly collected from each of 3 

different fields: two fields with Cabernet franc and one with Aligote. Total RNA 

extraction was done by modified CTAB method: samples were grinded in liquid 

nitrogen followed by addition of extraction buffer (0.1M Tris-HCl; 25mM EDTA; 

2M NaCl; 2% CTAB; 2% PVP) and chloroform, clarification, precipitation of RNA 

by ethanol. A reverse transcription reaction was done with a mix of reverse primers 

for all three viruses. A cDNA used for PCR with all three pairs of primers also in 

one tube. Analysis showed that approximately 37% of plants were infected. by at 

least one virus. Only GVA have been detected in samples collected from Aligote 

field, 7 out of 22. GFLV have been found in one Cabernet franc plant. GVA have 

been detected in 8 out of 24 and 6 out of 24 samples collected from two Cabernet 

franc fields, and GLRaV-3 in 9 and 10 samples, respectively. Most samples had a 

mixed infection with GVA and GLRaV-3. Positive samples were used for 

amplification and cloning part of genomes corresponding to the coat protein gene. 

Clones were sequenced, and nucleotide and deduced protein sequences were 

compared to known sequences in genebank. GFLV showed up to 86% identity at 

nucleotide sequence level and up to 93% at protein sequence level, GLRaV-3 - 97% 

and 98%, GVA - 91% and 96%, respectively. 

105

Fakhro A, Schwarz D, von Bargen S, Bandte M, Büttner C, Franken P: Influence of the fungal root endophyte 

Piriformospora indica on tomato growth and spread of Pepino mosaic virus. In: Feldmann F, Alford D V, Furk C: 

Crop Plant Resistance to Biotic and Abiotic Factors (2009), 106; ISBN 978-3-941261-05-1; © Deutsche 


3-12 Influence of the fungal root endophyte Piriformospora indica on 

tomato growth and spread of Pepino mosaic virus 

Fakhro A 1 , Schwarz D 2 , von Bargen S 1 , Bandte M 1 , Büttner C 1 , Franken P 2 

1 

Humboldt-University Berlin, Faculty of Agriculture and Horticulture, Institute for 

Horticultural Sciences, Section Phytomedicine, Lentzeallee 55/57, D-14195 Berlin, Germany. 

2 

Institute of Vegetable and Ornamental Crops, Großbeeren/ Erfurt e.V., Theodor- 

Echtermeyer-Weg 1, D-14979 Großbeeren, Germany 

Email: fakhroah@cms.hu-berlin.de 

106 

Abstract 

Pepino mosaic virus (PepMV) belonging to the genus Potexvirus was first identified 

in 1974 in pepino plants (Solanum muricatum Ait.) in Peru; (Jones et al., 1980). The 

virus caused in recent years worldwide a great damage in greenhouse and field 

production of tomato. The losses were up to 30% in the market yield and even up to 

50% concerning the quality of the fruits (Spence et al., 2006). The only method to 

control plant viruses in the greenhouse is the disinfection of all materials (Bosseur 

et al., 2004). The aim of the present work was to analyse whether a containment of 

this disease with root-endophytic fungi as biological agents is possible. As root 

colonising fungus the endophyte Piriformospora indica was selected. P. indica 

belongs to the Sebacinales (Basidiomycota). It has a broad host range and increased 

fresh weights of roots and shoots of many plants (Varma et al. 1999). P. indica 

induces resistance in barley against root and shoot pathogens (Waller et al. 2005), 

but has not been used up to now for inoculation of tomato. Tomato plants cultivar 

Hildares were grown in nutrient solution in hydroponic system and inoculated with 

spores and mycelium suspensions of the fungus. Three weeks later after controlling 

fungal colonisation of the roots, leaves were inoculated with PepMV. The spread of 

the virus was controlled using DAS-ELISA test system with the specific antibody 

AS-0554 (DSMZ, Braunschweig, Germany). At the end of the experiment plant 

growth parameters were monitored. P. indica promoted shoot growth and fruit fresh 

weights as it has been seen before with other plants on solid substrates. Concerning 

virus spread, the root endophyte showed a significant influence. In order to get a 

first insight into the molecular basis, RNA accumulation of a number of genes being 

related with virus infection in plants was analysed. The results will be presented.

Engelmann U, Kopahnke D, Ordon F: First results of mapping and exploitation of new sources of resistance to tan 

spot (Pyrenophora tritici-repentis) in wheat. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic 

and Abiotic Factors (2009), 107; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, 


3-13 First results of mapping and exploitation of new sources of resistance to 

tan spot (Pyrenophora tritici-repentis) in wheat 

Engelmann U, Kopahnke D, Ordon F 

Julius-Kühn Institut, Erwin-Baur-Str. 27, 06484 Quedlinburg 

Email: uta.engelmann@jki.bund.de 

Abstract 

Tan spot, caused by the fungus Pyrenophora tritici-repentis Drechs. (anamorph 

Drechslera tritici-repentis Shoem.), is one of the major foliar diseases in wheat. It 

appears worldwide with an increasing rate. This disease can cause a yield loss of up 

to 50% in susceptible wheat cultivars in combination with weather favourable to the 

fungus. The climate change in addition to the new famer management practise like 

reduction tillage or intensive wheat after wheat cultivation system lead to a fast 

spread of the pathogen. Therefore the development of resistant wheat cultivars is 

essential. The marker assisted selection is one tool in a fast and consumer-friendly 

plant breeding program. Phenotypic and genotypic data were collected to develop 

selective markers, which are allocated in an QTL-analysis. Seven DH-population 

(range from 80 to 231 genotypes) were investigated in a field test on two 

environments. The infection was provoked by man-made inoculum. In the season 

the development of the disease caused by Pyrenophora tritici-repentis was 

determined several times. The results were evaluated statistically. Additional the 

DH-populations were tested in the greenhouse by spray inoculating the whole plants 

using a spore suspension of monoconidial lines. The final concentration was 

adjusted to 3000 conidia/ml. The necrosis and chlorosis caused by the fungus were 

determinated in % leaf attack. The current results of the greenhouse and the field 

test are comparable. Furthermore we generated genotype data using SSR- and 

AFLP-analysis. Current we found in a parental screening 147 polymorphic 

combinations in 265 tested AFLP-primer combinations. The AFLP-analyse of one 

DH-Population is stared. Additional 267 SSR-makers were tested. 118 showed in 

the first researched DH-population between the partial lines polymohism. In future 

works we want to complete the genotyping and allocate this data with the 

phenotypic data. 

107

Babenko A, Mikhailova S, Chikin J, Nikolaeva I: The role of biotic factors in haricot (Phaseolus vulgaris L. Savi) 

cultivation on the south part of West Siberia. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic 



3-14 The role of biotic factors in haricot (Phaseolus vulgaris L. Savi) 

cultivation on the south part of West Siberia 

Babenko A 1 , Mikhailova S 1 , Chikin J 1 , Nikolaeva I 2 

1 

Tomsk State University, Lenina ave.,36, Tomsk 634050 Russia 

2 

Tomsk Garden Bed Ltd., Kirova str., 56, Tomsk 634020 Russia 

Email: dzedzin17@hotmail.com 

108 

Abstract 

Long-term experiments carried out in Tomsk State University have shown the 

ability of haricot being cultivated in the south of the forest zone of Western Siberia. 

The most promising grain crop sorts are characterized by stable fast-ripening and 

good crop capacity. The main factors decreasing ripening and quality of beans and 

haricot seeds are pests and some diseases caused by fungi. The most dangerous pest 

for haricot is Acanthoscelides obtectus Sav., and main source of haricot crops 

contamination is transportation of pests with seeds. This beetle gives only one 

generation in Siberia. Last years about 90 % of seeds are damaged by A. obtectus, 

and in some seeds display up to 20-25 signs of damage. The main ecological 

peculiarity of A. obtectus limited its normal development is its weak resistance to 

low temperature. Among fungal diseases some representatives of Fusarium have 

been selected from died haricot shoots and soil. On mature plants the affections of 

leaves and beans Botrytis cinerea Pers.Fr. and Sclerotinia sclerotiorum (Lib.) Mass. 

were more common. Haricot beans affections caused by Fusarium have been 

registered annually since mid-August up to end of September. Significant parts of 

haricot beans (up to 20%) were affected by fungal and bacterial diseases in latent 

form. Experiments on beans freezing against A. obtectus shown, that this methods 

not affected fungal diseases significantly.

Dinnesen S, Nedelev T, Hummel H E , Grozea I, Carabeţ A, Stef R, Ulrichs Ch: Diabrotica virgifera virgifera 

(Col.: Chrysomelidae) and its abundance in maize and neighbouring non-maize fields of West Romania. In: 

Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 109-116; ISBN 

978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-15 Diabrotica virgifera virgifera (Col.: Chrysomelidae) and its abundance 

in maize and neighbouring non-maize fields of West Romania 

Dinnesen S 1 , Nedelev T 1 , Hummel H E 2 , Grozea I 3 , Carabeţ A 3 , Stef R 3 , Ulrichs Ch 1 

1 

Humboldt-Universität zu Berlin, Urban Ecophysiology, Lentzeallee 55, D-14195 Berlin, 

Germany; 

2 

Justus-Liebig-University Giessen, Karl-Gloeckner- Strasse 21 C, D-35394 Giessen, Germany 

& Illinois Natural History Survey, Biodiversity and Ecological Entomology, Champaign, Ill. 

61820, USA 

3 

Banat’s University, Calea Aradului 119, RO-300645 Timişoara, Romania 

Email: sven@dinnesen.de 

INTRODUCTION 

Ever since the western corn rootworm (WCR) Diabrotica virgifera virgifera (Col.: 

Chrysomelidae) was first reported from Serbia in 1992, it constantly expanded to neighbouring 

countries. It is now common throughout South Eastern Europe and surpassed the economic 

threshold in a considerable number of states. In Romania, which has the largest acreage of 

grain maize in the EU-27 (2.5 million ha, Anonymus 2009), it was first reported in 1996 

(Vonica 1998). As a containment measure crop rotation plays an important role in managing 

this pest. Because of observation of a variant ecotype that lays eggs in soybean fields and thus 

can develop in next year maize in the Midwestern US (Spencer et al. 2005), more attention was 

paid to the limitations of this cultural method. It is known that the adults use a wide range of 

host plants as a food resource and even the larvae are basically able to develop on roots of 

plants other than maize, like grassy weeds or cereals (Breitenbach et al. 2006). The adults 

thereby fly up to 50 m and lay eggs up to 20 m into neighbouring fields (Igrc Barcic et al. 

2007). Therefore, we studied the incidence of WCR adults in fields of different alternative host 

plants bordering maize and compared it with the population dynamics of WCR in the maize 

fields. 

109

METHODS 

Metcalf sticky traps baited with MCA (4-methoxy-cinnamaldehyde) kairomone or female sex 

pheromone (8-methyl-decane-2-ol propanoate) were used to monitor WCR in maize fields and 

on fallow ground, which was mainly covered with grassy weeds like Setaria spp. and Sorghum 

halepense. In sunflowers (Helianthus annuus) and sorghum (Sorghum bicolor) only 

kairomone-baited traps were used. Mean values of beetle captures in the various host plants 

were compared using Kruskal-Wallis-test (H-test). Experiments were conducted in West 

Romania on field plots of Banat's University in Timişoara and on maize fields at the 

commercial production site near the village of Sag 10 km distant from town. For determining 

the changes of sex ratio of WCR during the examination period total captures of kairomone 

baited traps were collected separately for each week. Sexes were identified by the additional 

sclerite at the rather blunt apex of the male beetles. 

RESULTS 

Population dynamics in maize fields 

At both locations the population peak was observed in the end of July to beginning of August. 

In the maize fields near Sag a total of 8,000 and 996 beetles in 20 traps each baited with sex 

pheromone or MCA kairomone were caught, respectively (Fig. 1). 

mean daily captures of WCR per trap 

110 

140 

120 

100 

80 

60 

40 

20 

0 

1-Aug 

3-Aug 

5-Aug 

7-Aug 

9-Aug 

11-Aug 

13-Aug 

15-Aug 

17-Aug 

19-Aug 

21-Aug 

pheromone 

kairomone 

Figure 1. Mean daily captures of WCR per Metcalf sticky trap baited with female sex 

pheromone or MCA kairomone during the period August 1 to September 1 in 

the maize field near Sag, West Romania (error bars: + / - SD). 

23-Aug 

25-Aug 

27-Aug 

29-Aug 

31-Aug


160 

140 

120 

100 

80 

60 

40 

20 

0 

1-Aug 

3-Aug 

5-Aug 

7-Aug 

9-Aug 

11-Aug 

13-Aug 

15-Aug 

17-Aug 

19-Aug 

21-Aug 

23-Aug 

pheromone 

kairomone 

Figure 2. Mean daily captures of WCR per Metcalf sticky trap baited with female sex 

pheromone or MCA kairomone during the period August 1 to September 1 in 

the maize field in Timisoara, West Romania (error bars: + / - SD). 

At the maize field plots of Banat's University in Timişoara peak flight was observed one week 

earlier (Fig. 2), which may be caused by earlier hatching and accelerated larval development 

due to higher temperature sums accumulated in urban areas compared with the countryside. In 

comparison to Sag also the 3-fold higher ratio of captures in four kairomone baited traps with 

overall 1,232 beetles in contrast to 4,021 captures in four pheromone baited traps was 

noticeable. 

Sex ratio 

The ratio of females caught in kairomone baited traps increased during the first three weeks of 

the examination period and reached the maximum of 15 % in week 33 (Table 1). The last two 

weeks it went down again to 9 %. Potential reasons of the low proportion of females could be a 

generally lower fraction of females in the population, a lesser attractiveness of the MCA 

kairomone to females compared to males, or, more likely, a delayed flight peak of females later 

in the season. 

25-Aug 

27-Aug 

29-Aug 

31-Aug 

111

112 

Table 1. Sex ratio of WCR captures in kairomone baited Metcalf sticky traps between 

August 1 to September 1, 2008, in Timisoara and Sag, West Romania. 

week no. of ♂ no. of ♀ ratio males (%) ratio females (%) 

31 164 2 98,80 1,20 

32 205 11 94,91 5,09 

33 352 61 85,23 14,77 

34 143 18 88,82 11,18 

35 152 15 91,02 8,98 

Figure 3. Mean daily captures of WCR per Metcalf sticky trap baited with MCA 

kairomone within 12 days in the period of August 1 to August 12, 2008, in the 

maize and sunflower fields in Sag and Timisoara, West Romania (error bars: 

95 %-CI).


kairomone or sex pheromone within 12 days in the period of August 21 to 

September 1, 2008, in the maize field and on fallow ground in Timisoara, 

West Romania (error bars: 95 %-CI). 


kairomone within 12 days in the period of August 21 to September 1, 2008, in 

the maize and in the sorghum field in Timisoara, West Romania (error bars: 

95 %-CI). 

113

Abundance in neighbouring non-maize fields 

Sunflowers showed the lowest attractiveness for the adults of WCR with only 17 beetles in 

4 kairomone baited traps during August 2008 in the sunflower fields in Timişoara and Sag 

(Fig. 3). After 12 August 2008, no more beetles were caught due to the maturity reached by the 

sunflowers. The results of the monitoring on the fallow ground and in the sorghum field 

showed a significant number of adult WCR present in these fields. But on fallow ground they 

accounted only for about a third of the captures in the maize fields (Fig. 4). In contrast, the 

mean daily captures of more than 2 beetles per kairomone baited trap in S. bicolor were not 

significantly different compared to the captures of about 3 WCR caught per trap and day in the 

maize fields (Fig. 5). Also, at the end of the observation period, when the maize plants were 

matured and S. bicolor still provided green plant tissue, the captures exceeded those in the 

maize fields (Fig. 6). 


114 

8 

7 

6 

5 

4 

3 

2 

1 

0 

maize field 

sorghum field 

21-Aug 22-Aug 23-Aug 24-Aug 25-Aug 26-Aug 27-Aug 28-Aug 29-Aug 30-Aug 31-Aug 1-Sep 


kairomone within 12 days in the period of August 21 to September 1, 2008, in 

the maize and in the sorghum field in Timisoara, West Romania.

DISCUSSION 

These results indicate that WCR visiting neighbouring fields is no random event and depends 

on the phenological stage of the maize plants and the alternative hosts. Especially 

monocotyledoneous plants are attractive for the adults and provide the possibility of a hostplant 

shift which is most likely within the plant family Poaceae (Clark & Hibbard 2004). For S. 

bicolor larval development was demonstrated in laboratory trials by Moeser & Vidal (2005). 

Being well adapted to climatic conditions, Sorghum spp. (tolerant to drought and high 

temperatures) frequently occur in Romania. So, in this region, the high incidence of WCR on S. 

bicolor is particularly worrying. Furthermore some Setaria spp. showed the highest percentage 

of larval survivorship compared to other possible hosts (Breitenbach et al. 2006) and therefore 

could provide a refuge for populations of WCR. Although in our observations sunflowers were 

least attractive and dicotyledoneous plants generally are less suitable hosts for larvae, WCR 

could potentially damage economically important crops in Romania. In Hungary economic 

losses in sunflowers caused by WCR were reported (Horvath 2003). Also watermelons 

(Citrullus lanatus) could be a potential host for the adults. In North America WCR is known 

for attacking and damaging members of the plant family Cucurbitaceae (Rhodes et al. 1980) 

and also in Europe WCR visiting blossoms of oil pumpkins was observed (Hummel et al. 

2007). In summary, our observations and former findings are showing that egg laying into 

bordering fields and hatching in the next year maize or adaption to alternative host plants could 

reduce the effectiveness of the otherwise quite successful management of WCR by crop 

rotation. This unwanted side effect should therefore be monitored with special alertness. 


We would like to thank Schwarz Foundation in Neckarsulm, Germany, for financial support. 

REFERENCES 

Anonymus (2009). EUROPA – Eurostat – Home page. URL: http://epp.eurostat.ec.europa.eu 

(visited: 15 February 2009). 

Breitenbach S; Dehne H W; Gloyna K; Heimbach U; Thieme T (2006). Getreidearten und 

Ungräser als alternative Wirtspflanzen der Larven von Diabrotica virgifera virgifera 

LeConte, 1868 (Coleoptera, Chrysomelidae). Mitteilungsblatt der Deutschen 

Gesellschaft für Allgemeine und Angewandte Entomologie 15, 257- 258. 

Clark T L & Hibbard B E (2004). A comparison of non-maize hosts to support western corn 

rootworm (Coleoptera: Chrysomelidae) larval biology. Environmental Entomology 33, 

681-689. 

Horvath Z & Harvani A (2003). Diabrotica virgifera virgifera Leconte, a new sunflower pest 

from America. Helia 26 (39), 93-100. 

115

Hummel H E; Dinnesen S; Nedelev T; Ulrichs C; Modic S; Urek G (2007). Diabrotica virgifera 

virgifera in Confrontation Mood: Simultaneous Geographical and Host Spectrum 

Expansion in Southeastern Slovenia. In: Best Practice in Disease, Pest and Weed 

Management. The State of the Art, eds D V Alford, F Feldmann, J Hasler, A von 

Tiedemann, pp. 78-79. DPG – BCPC: Alton,UK. 

Igrc Barcic J; Bazok R; Edwards C R; Kos T (2007). Western corn rootworm adult movement 

and possible egg laying in fields bordering maize. Journal of Applied Entomology 

131(6), 400–405. 

Moeser J & Vidal S (2005). Nutritional resources used by the invasive maize pest Diabrotica 

virgifera virgifera in its new South-east-European distribution range. Entomologia 

Experimentalis et Applicata 114 (1), 55–63. 

Rhodes A M; Metcalf R L; Metcalf E R (1980). Diabroticite beetle responses to cucurbitacin 

kairomones in Cucurbita hybrids. Journal of American Horticultural Sciences 105 (6), 

838-842. 

Spencer J L; Levine E; Isard S A; Mabry T R (2005). Movement, dispersal and behaviour of 

Western Corn Rootworm adults in rotated maize and soybean fields. In: Western Corn 

Rootworm. Ecology and Management, eds S Vidal, U Kuhlmann, C R Edwards, pp. 

121-144. CABI Publishing: Wallingford, UK. 

Vonica I (1998). Auftreten und Verbreitung von Diabrotica virgifera virgifera LeConte in 

Rumänien. Pflanzenschutzberichte 57, 3-14. 

116

Dinnesen S, Hummel H E , Grozea I, Carabeţ A, Stef R, Ulrichs Ch: Monitoring Diabrotica virgifera virgifera 

(Col.: Chrysomelidae) with different lures and traps. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance 

to Biotic and Abiotic Factors (2009), 117-123; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 


3-16 Monitoring Diabrotica virgifera virgifera (Col.: Chrysomelidae) with 

different lures and traps 

Dinnesen, S 1 , Hummel H E 2 , Grozea I 3 , Carabeţ A 3 , Stef R 3 , Ulrichs C 1 

1 

Humboldt-Universität zu Berlin, Urban Ecophysiology, Lentzeallee 55, D-14195 Berlin, 

Germany; 

2 

Justus-Liebig-University Giessen, Karl-Gloeckner- Strasse 21 C, D-35394 Giessen, Germany 

& Illinois Natural History Survey, Biodiversity and Ecological Entomology, Champaign, Ill. 

61820, USA 

3 

Banat’s University, Calea Aradului 119, RO-300645 Timişoara, Romania 

Email: sven@dinnesen.de 

INTRODUCTION 

The western corn rootworm (WCR) Diabrotica v. virgifera, a major maize pest from North 

America, has been introduced into Europe around 1990. From its first reported invasion at 

Belgrade airport (Bača 1993), this alien invasive species expanded into all directions of South 

Eastern Europe. In some countries WCR already surpassed the economic threshold. The most 

important measure for making decisions about management strategies is monitoring the adults 

with pheromone traps. Thereby the economic threshold is defined by a certain number of 

beetles caught per trap per day or observed specimens per plant on a certain date. Because 

different trap and lure types are used and different intervals after which lure and trap bodies 

were changed, population densities from different areas and years are difficult to compare. 

Beside the availability of food resources the population dynamics of WCR is mainly influenced 

by climate through the impact of soil temperature on larval hatch and development. Further, 

indirect monitoring of adults with pheromone traps is affected by unfavourable weather 

conditions through reduced flight activity or effectiveness of the traps. Therefore, we examined 

trapping efficiency of different trap and lure types with regard to the impact of weather 

conditions in two heavily infested areas in South Eastern Europe, namely West Romania and 

East Slovenia. 

117

METHODS 

We tested three different trap and five lure types, three commercial long-term pheromone 

dispensers: (Temmen GmbH, type A, B and C) and chromatography paper squares which were 

treated with MCA (4-methoxy-cinnamaldehyde) kairomone or female sex pheromone (8methyl-decane-2-ol 

propanoate). Cup traps of the Metcalf type and the most commonly used 

Hungarian Csalomon ® PAL cloak traps served as traps of the sticky type. As a mass capacity 

trap of the Vario type the Bio-Pherotrap ® (Temmen GmbH) was used. For killing the beetles, 

this trap type was loaded with 0.5 g AL06, a silica dust based insecticide developed by 

Humboldt University Berlin (Ulrichs et al. 2006). Experiments were conducted on maize fields 

in Pince, East Slovenia, and in Sag, West Romania. On 21 July 2008, eight mass capacity 

traps, each equipped with the self-made paper square lures treated with kairomone or 

pheromone, were established in the maize field in Pince and these remained without changes 

for 43 days until 2 September. In Sag, 20 sticky cup traps each equipped with paper square 

lures and 16 mass capacity traps, each equipped with one of the pheromone dispensers or the 

sex pheromone applied on paper squares, were established in the maize field on 31 July. In 

addition, 10 sticky cloak traps each equipped with one of the dispensers or a paper square lure 

with kairomone or sex pheromone were arranged in the maize field on August 7. A minimum 

distance of 50 m was maintained between traps. Dependent on weather conditions the paper 

square lures in Sag were changed every 3 to 4 days. To compare the impact of weather 

conditions on captures in the different trap and lure combinations rank correlation coefficients 

(Spearman’s rho) were calculated. 

RESULTS & DISCUSSION 

Lures 

A comparison of the four pheromone types in the mass capacity traps in Romania showed no 

significant differences between the three commercial pheromone dispensers, with mean daily 

captures of 3.4 (Type A), 3.8 (Type B) and 4 (Type C) beetles per trap. In contrast, the sex 

pheromone applied on paper squares attracted significantly higher numbers with 19 beetles per 

trap and day (Fig. 1). This 5-fold higher number of beetle captures confirms former results that 

showed a higher sensitivity and trapping efficiency of this lure on Metcalf sticky cup traps 

compared with pheromone dispensers used with sticky cloak traps (PAL) (Wennemann & 

Hummel 2003, Bertossa & Hummel 2008). This lure type used in mass capacity traps in East 

Slovenia from 21 July to 2 September 2008, attracted an average of 761 and 248 beetles per 

trap baited with pheromone or kairomone lures, respectively (Fig. 2). The lures were not 

changed during this 43-day period and there were still living beetles found on the last day, 

showing an amazing long-term effect of this lure type. Maybe this unexpected long 

effectiveness is based on protection from rain and UV radiation under the roof of the Vario trap 

type. 

118

Traps 

For the three different trap types we found no significant differences in mean daily captures of 

22 (PAL), 19 (Metcalf) and 21 (Vario) beetles per trap and day (Fig. 3). In periods of 

decreasing captures in sticky traps an actual increase of captures in mass capacity traps was 

observed (Fig. 4). During the examination period the silica dust used as the insecticidal 

compound in the mass capacity traps showed no decrease in effectiveness. It killed the beetles 

by dehydration within 24 hours. 

Figure 1. Mean daily captures of WCR per mass capacity trap (vario type) baited with 

commercial rubber pheromone dispensers (type A, B & C) or sex pheromone 

attached on chromatography paper squares (type D) during the period 

August 1 to September 1 in the maize field in Sag, West Romania (error bars: 

95%-CI). 

Figure 2. Mean captures of WCR per mass capacity trap (Vario type) baited with MCA 

kairomone or female sex pheromone on chromatography paper squares within 

43 days in the period of July 21 to September 2, 2008, in the maize field in 

Pince, East Slovenia (error bars: + / - SD). 

119


120 

Figure 3. Mean daily captures of WCR per trap during the period August 8 to 

September 1 in the maize field in Sag, West Romania. All different trap types 

were baited with sex pheromone on chromatography paper squares (type D) 

(error bars: 95 %-CI). 

120 

100 

80 

60 

40 

20 

0 

8-Aug 

9-Aug 

10-Aug 

11-Aug 

12-Aug 

13-Aug 

Metcalf 

Vario 

PAL 

14-Aug 

15-Aug 

16-Aug 

17-Aug 

18-Aug 

19-Aug 

20-Aug 

21-Aug 

22-Aug 

23-Aug 

24-Aug 

25-Aug 

26-Aug 

27-Aug 

28-Aug 

29-Aug 

30-Aug 

31-Aug 

1-Sep 

Figure 4. Mean daily captures of WCR per trap during the period August 8 to 

September 1 in the maize field in Sag, West Romania. All different trap types 

were baited with sex pheromone on chromatography paper squares (type D).

Once the beetles came in contact with the silica dust, they refused or were unable to use their 

wings further on. Owing to the dryness and shelter from sunlight within the mass capacity 

traps, blackening and decay of the beetles was reduced. Therefore, in cases of long periods 

between collecting the beetles it is much easier to identify them in mass capacity traps 

compared with sticky traps. 

Impact of weather conditions 

The official monitoring by Banat’s University (Fig. 5) in Sag during the whole flight period 

shows significant correlations of WCR captures with temperatures (Table 1). This mainly 

reflects the temperature dependency of larval development and beetle flight activity. In 

contrast, during the comparison of the different trap types, the captures in Metcalf sticky traps 

and mass capacity traps also shows a significant negative correlation with precipitation and 

wind speed on both trap types and a variable influence of temperature on each trap type (Table 

2). The surprising negative correlation of captures in Metcalf sticky traps with temperature 

could be caused by reduced effectiveness of the pheromone through exposure of the lure to 

heat and UV radiation compared with the higher protection of the lure in the mass capacity 

traps. 

mean WCR captures per trap 

70 

60 

50 

40 

30 

20 

10 

0 

WCR 

soil temperature 

17-Jun 

21-Jun 

25-Jun 

29-Jun 

3-Jul 

7-Jul 

11-Jul 

15-Jul 

19-Jul 

23-Jul 

27-Jul 

31-Jul 

4-Aug 

8-Aug 

12-Aug 

16-Aug 

20-Aug 

24-Aug 

28-Aug 

1-Sep 

Figure 5. Mean daily captures of WCR in two PAL traps (bars), which were part of the 

official monitoring program of the Banat’s University, during the period 

17 June to 4 September in the maize fields in Sag, West Romania and mean 

soil temperature (C°) (line). 

40 

35 

30 

25 

20 

15 

10 

5 

0 

mean soil temperature (C°) 

121

122 

Table 1. Rank correlation coefficients (Spearman’s rho) of correlations between mean 

daily WCR captures in the two PAL traps during 17 June to 4 September in 

Sag and weather data. 

Correlation 

Coefficient 

mean 

air temp. 

min. 

air temp. 

max. 

air temp. 

mean 

soil temp. 

relative 

humidity precipitation wind speed 

,237 ** ,085 ,327 ** ,282 ** -,207 ** -,095 -,076 

Sig. (2-tailed) ,003 ,283 ,000 ,000 ,009 ,234 ,340 

N 160 160 160 160 160 160 160 

**. Correlation is significant at the 0.01 level (2-tailed). 

Table 2. Rank correlation coefficients (Spearman’s rho) of correlations between mean daily 

WCR captures in the Metcalf sticky traps and Vario mass capacity traps 

loaded with lure type D during the period 8 August to 1 September in Sag and 

weather data. 

Metcalf Correlation 

Coefficient 

Vario 

mean 

air temp. 

min. 

air temp. 

max. 

air temp. 

mean 

soil temp. 

relative 

humidity precipitation 

wind 

speed 

-,181 ** -,326 ** -,140 * -,076 ,158 * -,144 * -,197 ** 

Sig. (2-tailed) ,004 ,000 ,027 ,229 ,012 ,023 ,002 

N 250 250 250 250 250 250 250 

Correlation 

Coefficient 

,101 -,012 ,222 * ,160 -,166 -,203 * -,217 * 

Sig. (2-tailed) ,316 ,904 ,026 ,112 ,099 ,043 ,030 

N 100 100 100 100 100 100 100 

**. Correlation is significant at the 0.01 level (2-tailed). 

*. Correlation is significant at the 0.05 level (2-tailed). 

CONCLUSIONS 

The most important factor influencing trapping efficiency is the type of lure used. However, 

weather conditions also affect the flight activity of WCR, the effectiveness of the lure and thus 

the number of captures in pheromone traps. Mass capacity traps, therefore, could be an 

interesting addition to the commonly used sticky traps and could help to reduce the influence 

of weather on beetle captures and consequently on monitoring population dynamics of WCR. 

Thereby, the combination of long-term effectiveness and the preserving quality of the silica 

dust make the mass capacity traps especially suitable for long-time studies where an extended 

durability of traps is essential.


We would like to thank Temmen GmbH in Hattersheim, Germany, for providing pheromone 

dispensers and Schwarz Foundation in Neckarsulm, Germany, for financial support. 

REFERENCES 

Bača F (1993). New member of the harmful entomofauna of Yugoslavia Diabrotica virgifera 

virgifera LeConte (Coleoptera: Chrysomelidae). IWGO Newsletter 13 (1-2), 21-22. 

Bertossa M & Hummel HE (2008). Experiences with population dynamics of Diabrotica 

virgifera virgifera LeConte in the Swiss canton of Ticino up to 2007. Communications 

in Agricultural and Applied Biological Sciences 73 (3), 421-428. 

Wennemann L & Hummel HE (2004). Diabrotica virgifera virgifera LeConte: Langzeitstudien 

verschiedener Fallentypen in Ungarn. Mitteilungen aus der Biologischen Bundesanstalt 

für Land- und Forstwirtschaft 396, 377. 

Ulrichs C; Entenmann S; Goswami A; Mewis I (2006). Abrasive und hydrophil/lipophile 

Effekte unterschiedlicher inerter Stäube im Einsatz gegen Schadinsekten am Beispiel 

des Kornkäfers Sitophilus granarius L.. Gesunde Pflanzen 58 (3), 173-181. 

123

Zubek S, Stojakowska A, Kisiel W, Góralska K,Turnau K: Interactions between mycorrhizal fungi and medicinal 

plants. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 124-129; 


3-17 Interactions between mycorrhizal fungi and medicinal plants 

Zubek S 1 , Stojakowska A 2 , Kisiel W 2 , Góralska K 3 ,Turnau K 3 

1 

Department of Pharmaceutical Botany, Faculty of Pharmacy, Jagiellonian University 

Collegium Medicum, Medyczna 9, 30-688 Krakow, Poland 

2 

Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, 

Smetna 12, 31-343 Krakow, Poland 

3 

Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 

Kraków, Poland 

Email: katarzyna.turnau@uj.edu.pl 

124 

ABSTRACT 

Arnica montana and Inula ensifolia (Asteraceae), both containing several groups of 

biologically active secondary compounds, were cultivated under greenhouse 

conditions with arbuscular mycorrhizal fungi (AMF). The content of secondary 

metabolites was found to be correlated with mycorrhizal parameters and was also 

dependant on the particular fungus used for inoculation. The analysis of chlorophyll 

a fluorescence transients proved to be a valuable tool to evaluate the condition of 

investigated plant species influenced by different AMF. The results strongly 

encourage the use of AMF in optimization of cultivar conditions of medicinal 

plants. 

INTRODUCTION 

Arnica montana and Inula ensifolia (members of Asteraceae family) both contain several 

groups of secondary metabolites such as terpenoids and phenol compounds (Kohlmünzer 

2000). A. montana, which is a well-known medicinal plant, could be cultivated for industrial 

purposes, but cultivation is difficult and non-profitable. Both plants grow naturally in 

oligotrophic soils and according to previous research they are always mycorrhizal (Ryszka et 

al. 2009; Turnau et al. 2008). 

Plants, in general, differ in their dependence on mycorrhizal symbiosis, which is based on the 

plant species and particular soil conditions (Van der Heijden et al. 1998). They benefit by 

enhanced uptake of nutrients such as P and N and increased interface between roots and soil,

y means of the higher penetration of soil by mycelium (Smith & Read 1997). Arbuscular 

mycorrhizal fungi (AMF) positively increase the succession rate of plants (Turnau & 

Haselwandter 2002) and enhance their tolerance to heavy metals (Turnau et al. 2006), water 

stress, extreme salinity (Ruiz-Lozano & Azcon 2000), pathogenic fungi and nematodes 

(Azcon-Aguilar & Barea 1997; Pozo et al. 2002). Moreover, mycorrhizal plants may show 

larger biomass, faster growth rate and more effective photosynthesis than non-mycorrhizal 

ones (Strasser et al. 1995). Mycorrhiza induces many changes in plant physiology (Morandi 

1996; Strack et al. 2003) and was found to influence the level of secondary metabolites (Strack 

et al. 2003; Copetta et al. 2006; Khaosaad et al. 2006). The level of produced compounds may 

depend on root colonization by mycorrhizal fungi (Abu-Zeyad et al. 1999; Fester et al. 1999). 

The main aim of the present research was to compare non-mycorrhizal and mycorrhizal A. 

montana and I. ensifolia cultivated under greenhouse condition using selected AMF and to 

show whether inoculation could influence plant vitality and secondary metabolite production, 

namely sesquiterpene lactones, phenolic acids (A. montana) and thymol derivatives (I. 

ensifolia). 


Seeds of A. montana and I. ensifolia were germinated on wet filter paper in Petri dishes. Two 

week-old seedlings were transferred to containers with sterile substratum composed of a 

mixture of commercially available garden soil (A. montana) or soil collected from I. ensifolia 

natural habitats, sand and expanded clay at the rates 5:8:1 (v:v:v). The following treatments 

were prepared: 1. Control - without inoculum; 2. Glomus intraradices UNIJAG PL24-1; 3. G. 

intraradices UNIJAG PL-Kap; 3. Glomus clarum UNIJAG PL13-2; 4. AMF from natural 

stands isolated from both plants and multiplied in pot cultures. The plant seedlings were 

transferred to 200 ml pots and kept in sealed Sunbags (Sigma-Aldrich) under greenhouse 

conditions at 20 o C and the following light regime: 100–110 µmol×m –2 ×s –1 PAR, 12/12 h. 

Following the standard staining (Turnau et al. 2008), the mycorrhizal parameters were assessed 

in root samples according to Trouvelot et al. (1986). 

The plant material for the assessment of secondary metabolite content was divided into roots 

and shoots and analyzed separately. Extraction for the sesquiterpene lactone analysis was 

performed using the modified method described by Douglas et al. (2004). The analysis of 

phenolic acids was carried out according to the procedure described by Zidorn et al. (2005) and 

thymol derivatives according to Stojakowska et al. (2006). The extracts were analyzed by 

HPLC with direct injection of methanol-extracted samples. 

Chlorophyll a fluorescence transients of intact leaves of investigated species were measured 

with a Plant Efficiency Analyser (PEA) fluorimeter (Hansatech Instruments, GB). The 

transients, induced by a red light of 600 W×m -2 , were recorded for 1 s, starting 50 µs after the 

onset of illumination. Data were acquired every 10 µs for the first 2 ms and every 1 ms 

thereafter as described by Strasser et al. (1995). Each transient was analysed according to the 

OJIP-test (Strasser et al. 1995; Strasser et al. 2000). For the characterization of PSII behaviour 

125

the performance index (PI) was calculated according to Strasser et al. (2000). It combines three 

parameters: the density of reaction centers, the quantum yield of primary photosynthesis and 

the ability to transfer electrons into the electron chain between photosystem II and I. 

The data were assessed by ANOVA. Significance of differences between treatments was tested 

after Tukey (p

not contain any AMF. The knowledge of secondary metabolite production is important not 

only because of their therapeutic values. Due to their presence plants become less sensitive to 

various stresses. Phenolic compounds that were also studied presently are involved in many 

plant functions and several reports underline their protective role against oxidative stress that 

originate from various environmental factors (Rice-Evans et al. 1997; Santiago et al. 2000; 

Jung et al. 2003). 

Efficiency of the photosynthetic apparatus can be easily measured using Handy PEA 

equipment and the work reported here confirms the validity of this method. It can be also used 

while the optimization of plant cultivation condition is carried out. This is a fast and nondestructive 

way of obtaining data concerning plant performance (Strasser et al. 2000; Zubek et 

al. 2009). 

As shown above, the optimization of the medicinal plant cultivation should also take into 

account the effectiveness of the particular fungus to colonize roots. Strains that were more 

aggressive concerning mycorrhizal colonization might be less effective in secondary metabolite 

production while those that are less effective in root colonization might be accompanied by 

higher metabolite production. 

REFERENCES 

Abu-Zeyad R; Khan A G; Khoo C (1999). Occurrence of arbuscular mycorrhiza in 

Castanospermum australe A. Cunn. & C. Fraser and effects on growth and production 

of castanospermine. Mycorrhiza 9, 111–117. 

Azcon-Aguilar C; Barea J M (1997). Arbuscular mycorrhizas and biological control of soilborne 

plant pathogens – an overview of the mechanisms involved. Mycorrhiza 6, 457– 

464. 

Copetta A; Lingua G; Berta G (2006). Effects of three AM fungi on growth, distribution of 

glandular hairs and essential oil production in Ocimum basilicum L. var. Genovese. 

Mycorrhiza 16, 485–494. 

Douglas J A; Smallfield B M; Burgess E J; Perry N B; Anderson R E; Douglas M H; Glennie 

VL (2004). Sesquiterpene lactones in Arnica montana: A rapid analytical method and 

the effects of flower maturity and simulated mechanical harvesting on quality and yield. 

Planta Medica 70 (2), 166–170. 

Fester T; Maier W; Strack D (1999). Accumulation of secondary compounds in barley and 

wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and coinoculation 

with rhizosphere bacteria. Mycorrhiza 8, 241–246. 

Jung C; Maeder V; Funk F; Frey F; Sticher H; Frossard E (2003). Release of phenols from 

Lupinus albus L. roots exposed to Cu and their possible role in Cu detoxification. Plant 

and Soil 252, 301–312. 

Khaosaad T; Vierheilig H; Zitterl-Eglseer K; Novak J (2006). Arbuscular mycorrhiza increases 

the content of essential oils in oregano (Origanum sp., Lamiaceae). Mycorrhiza 16, 

443–446. 

Kohlmünzer S (2000). Farmakognozja. PZWL, Warszawa. 

Morandi D (1996). Occurrence of phytoalexins and phenolic compounds in endomycorrhizal 

interactions, and their potential role in biological control. Plant and Soil 185, 241–251. 

127

Pozo M J; Slezack-Deschaumes S; Dumas-Gaudot E (2002). Plant defence responses induced 

by arbuscular mycorrhizal fungi. In: Mycorrhizal technology in agriculture, eds S 

Gianinazzi, H Schüepp, J M Barea, K Haselwandter, pp. 103–111. Birkhauser 

Verlag/Switzerland. 

Rice-Evans C; Miller N J; Paganga G (1997). Antioxidant properties of phenolic compounds. 

Trends in Plant Sciences 2, 152–159. 

Ruiz-Lozano J M; Azcon R (2000). Symbiotic efficiency and infectivity of an autochtonous 

arbuscular mycorrhizal Glomus sp. from saline soil sand G. deserticola under salinity. 

Mycorrhiza 10, 137–143. 

Ryszka P; Błaszkowski J; Jurkiewicz A; Turnau K (2009). Arbuscular mycorrhiza of Arnica 

montana under field conditions - conventional and molecular studies. Mycorrhiza 

(submitted). 

Santiago L J M; Louro R P; de Oliveira D E (2000). Compartmentation of phenolic compounds 

and phenylalanine ammonia-lyase in leaves of Phyllanthus tenellus Roxb. and their 

induction by copper sulphate. Annals of Botany 86,1023–1032. 

Smith S E; Read D J (1997). Mycorrhizal Symbiosis. Academic Press: London. 

Stojakowska A; Michalska K; Malarz J (2006). Simultaneous quantification of eudesmanolides 

and thymol derivatives from tissues of Inula helenium and I. royleana by reversedphase 

high-performance liquid chromatography. Phytochemical Analysis 17, 157-161. 

Strack D; Fester T; Hause B; Schliemann W; Walter M H (2003). Arbuscular mycorrhiza: 

biological, chemical and molecular aspects. Journal of Chemistry and Ecology 29, 

1955–1979. 

Strasser R J; Srivastava A; Govindjee (1995). Polyphasic chlorophyll a fluorescence transient 

in plants and cyanobacteria. Photochememistry and Photobiology 61, 32–42. 

Strasser R J; Srivastava A; Tsimilli-Michael M (2000). The fluorescence transient as a tool to 

characterise and screen photosynthetic samples. In: Probing photosynthesis: 

mechanisms, regulation and adaptation, M Yunus, U Pathre, P Mohanty, pp. 445–483. 

Taylor & Francis: London. 

Trouvelot A; Kough J L; Gianinazzi-Pearson V (1986). Mesure du taux de mycorhization VA 

d’un systeme radiculaire. Recherche de methodes d’estimation ayant une signification 

fonctionnelle. In: Physiological and Genetical aspects of Mycorrhizae, eds V 

Gianinazzi-Pearson & S Gianinazzi, pp. 217–221. INRA: Paris. 

Turnau K; Anielska T; Ryszka P; Gawroński S; Ostachowicz B; Jurkiewicz A (2008). 

Establishment of arbuscular mycorrhizal plants originating from xerothermic grasslands 

on heavy metal rich industrial wastes - new solution for waste revegetation. Plant and 

Soil 305, 267-280. 

Turnau K; Haselwandter K (2002). Arbuscular mycorrhizal fungi, an essential component of 

soil microflora in ecosystem restoration. In: Mycorrhizal technology in agriculture, eds 

S Gianinazzi, H Schüepp, J M Barea, K Haselwandter, pp. 137–150. Birkhauser 

Verlag/Switzerland. 

Turnau K; Jurkiewicz A; Lingua G; Barea J M; Gianinazzi-Pearson V (2006). Role of 

arbuscular mycorrhiza and associated microorganisms in phytoremediation of heavy 

metal polluted sites. In: Trace elements in the environment: biogeochemistry, 

biotechnology, and bioremediation, eds M N V Prasad, K S Sajwan, R Naidu, pp. 235– 

252CRC Press: Boca Raton. 

128

Van Der Heijden M G A; Klironomos J N; Ursic M; Moutogolis P; Streitwolf-Engel R; Boller 

T; Wiemken A; Sanders IR (1998). Mycorrhizal fungal diversity determines plant 

biodiversity, ecosystem variability and productivity. Nature 396, 69–72. 

Zidorn C; Schubert B; Stuppner H (2005). Altitudinal differences in the contents of phenolics 

in flowering heads of three members of the tribe Lactuceae (Asteraceae) occurring as 

introduced species in New Zealand. Biochemistry, Systematic and Ecology 33, 855– 

872. 

Zubek S; Turnau K; Tsimilli-Michael M; Strasser R J (2009). Response of endangered plant 

species to inoculation with arbuscular mycorrhizal fungi and soil bacteria. Mycorrhiza 

19, 113–123. 

129

Abbasipour H, Askarianzadeh A: Identification of physical and biochemical agents related to resistance in 

different sugarcane cultivars to stalk borers, Sesamia spp. (Lep.: Noctuidae). In: Feldmann F, Alford D V, Furk C: 



3-18 Identification of physical and biochemical agents related to resistance 

in different sugarcane cultivars to stalk borers, Sesamia spp. (Lep.: 

Noctuidae) 

Abbasipour H, Askarianzadeh A 

Department of Plant Protection, College of Agricultural Sciences, Shahed University, Tehran, 

Iran Email: Abasipour@Shahed.ac.ir 

130 

ABSTRACT 

Sugarcane planting in Iran is threatened by stalk borers, Sesamia cretica and S. 

nonagrioides. Today, one of the strategies for control borers in sugarcane fields is 

usage of resistant cultivars. This study was conducted on twelve cultivars of 

sugarcane to identify physical and biochemical characters that cause resistance. 

Alkaloid and phenolic materials in two steps of tillering and harvesting times of 

stalks and millable cane were calculated. Percentage of POL (sugar solution 

particles) and Brix (sugar and nonsugar particles), juice and stalk fibre were 

measured, as were mineral elements including N, P, K, Si and Ca. Correlation of all 

measured factors with plant damage was calculated using SPSS software. 

Among the measured characters, phenolic compound in plant was inversely and 

significantly correlated with bored internodes (r = -0.53, P

species of stalk borer: Sesamia cretica and S. nonagrioides. Sesamia spp. damage a 

considerable number of sugarcane internodes annually in the province of Khuzestan (Danialy, 

1985). Moreover, following direct injury by stalk borers on sugarcane, microorganisms 

(especially Fusarium spp.) can easily attack the bored stalks and damage severity increase 

(Johnson et al., 1995). Hilal (1985) reported that in stalks bored by S. nonagrioides 10% of 

sucrose was inverted to dextran. 

Damage of stalk borer including: 1) Damage at tillering stage that cause dead heart. 2) Damage 

at forming of internodes stage that cause bored internodes. 3) Damage at ripening stage that 

cause reduction of sugar storage and sugar quality. 

Today’s one of the strategies for control borers in sugarcane fields in IPM system is usage of 

resistant cultivars. Resistant cultivars will reduce damage pest with least cost for farmers 

(Reagan et al. 1997). To investigate pest damage at the tillering stage, the influence of pest on 

the yield of millable cane was investigated using 12 sugarcane cultivars. This study was 

conducted in twelve cultivars of sugarcane to identify physical and biochemical characters of 

plant that cause resistance in different cultivars. 


Alkaloid and phenolic materials in two steps of tillering and harvesting times of stalks and 

millable cane with suksolet apparatus separated according with method of Dey and Harborne 

(1989) and after condensation, percentage was calculated. Percentage of POL (sugar solution 

particles) and Brix (sugar and nonsugar particles), juice and stalk fibre were measured 

according with standard of ICUMSA (1994). In order to measure mineral elements of stem 

including N, P, K, Si and Ca, first 0.2 g of dried stem with method of dry ashing, was placed in 

oven with 550 degree centigrade for two hours. Then obtained ash was solved in 2 M and 

solution volume reached to 100 ml. Then with Kajeldal methods (nitrogen), 

spectrophotometery (Phosphorus), Atomic absorption model AA55GL (Potassium, Calcium, 

Silicium), these elements were measured. Correlation of all measured factors with bored degree 

in plant was calculated using SPSS software. 

The methods and terminology used in the assessment of sugarcane quality are standard to the 

ICUMSA (1994). Experiments repeated 10 times for each cultivar. 

Data were analyzed using SPSS statistical software (Green et al., 2000). Comparisons of 

quality between groups of bored internodes in three cultivars were performed using Duncan’s 

multiple range test (p=0.05). Data reported as percentages (bored internodes, Pol, Brix and 

Juice purity) were transformed using the arcsine transformation before all statistical analyses 

but are presented as untransformed means at the table. Percentage of bored internodes and 

sugarcane cultivar were analyzed for their effects on sugarcane quality using a general linear 

model. Linear regressions were performed on sugarcane quality (dependent variables) as 

affected by percentage of bored internodes. 

131

RESULTS 

Data from different measured factors in twelve cultivars of sugarcane are shown in Table 1. 

Among the measured characters, phenolic compound in plant was inversely and significantly 

correlated with bored internodes (r=-0.53, P

Analyzing of data showed that there are significant difference between number of tillers at the 

tillering stage and number of millable canes at harvest time (F (8,15)= 28.01, P

134 

Figure 1. Regression curves percentage of bored internodes with weight of stalk on cvs 

CP69-1062 and CP48-103. 

DISCUSSION 

Based on results of this study, sugarcane has high potential for producing tiller and this level of 

injury and even more do not be influence number of millable canes. So, sugarcane can tolerate 

damage of Sesamia spp. borers at tillering stage due to producing too many tillers. There are 

reports that sugarcane tolerates damage by the borers Chilo infuscatellus (Rao Siva, 1962) and 

Scirpophaga exerptalis (Jepson, 1954) at tillering stage due to producing too many tillers. 

Our results showed that there is inverse correlation between phenolic compound in plant and 

infection because of negative effect of these materials on the pests. Studies of Godshal and 

Legendre (1988) showed that phenolic compounds in sugarcane cultivars are significantly

different. Also studies of Rutherford (1998) showed that flavonoids and chlorogenic acid 

(phenolic compound) in sugarcane shoot caused resistance to Eldana saccharina. High amount 

of calcium in sugarcane cultivars is increased infection of plant to stem borer, Diatrea 

saccharalis (Macedo, 1978). The findings of the present work are well consistent with other 

studies regarding stem borers, Sesamia spp. In regard to amount of Si, N, P and K elements in 

stem, significant difference is observed among resistance and susceptible cultivars of sugarcane 

to internode borer, Chilo sacchariphagus indicus (David, 1979) and the concentration of 

ionized salts in leaf cell extract is related to resistance to Sesamia excerptalis (Adlakha, 1964), 

but in this study relationship between these elements with infection to stem borers, Sesamia 

spp. was not observed. 

ACKNOWLEDGMENTS 

We thank the Sugarcane & By-Products Development Co. and conducted in Sugarcane 

Research Center/Khuzestan for financial support.. 

REFERENCES 

Adlakha P A (1964). Studies on the various factors responsible for resistance to top borer in the 

different varieties of sugarcane. Indian Journal of Sugarcane Research Development 8, 

343-344. 

Askarianzadeh A (2004). Mechanisms of resistance in sugarcane varieties to pink borers 

Sesamia spp. (Lepidoptera, Noctuidae). Ph.D. thesis, College of Agriculture, Tarbiat 

Modarres University, 129 pp. 

Danialy M (1985). Investigation of usage biological control, cultural and chemical methods 

against sugarcane borer in Hafttapeh/Khuzestan/Iran. M.Sc. thesis, Chamran university, 

Ahvaz, Iran, 114 pp. 

David H (1979). A critical evaluation of the factors associated with resistance to internode 

borer, C. sacchariphagus indicus (K.) in Slaccharum sp., allied genera and hybrid 

sugarcane. Ph.D. thesis, Calicut University, Calicut, 199 pp. 

Dey P M and Harborne J B (1989). Methods in plant biochemistry. Academic Press, London, 

UK. 552 pp. 

Godshall M A and Legendre B L (1988). Phenolic content of maturing sugarcane. 

International Sugar Journal 90(1), 16-19. 

Green S B; Salkind N J and Akey T M (2000). Using SPSS for Windows: Analyzing and 

Understanding Data. 2 nd Edition, Prentice Hall, USA. 

Hilal A (1985). Study of the technological alterations of sugarcane due to the attacks of 

Sesamia nonagrioides (Lef.)(Lep: Noctuidae). Actes de I, Institut Agronomique et 

Veterinaire Hassan-II 5(1-2), 37-42. 

ICUMSA (International Commission for uniform Methods of Sugar Analysis) (1998). Method 

GS5/7-1, the determination of pol (polarization), brix and fibre in cane and bagasse by 

the wet distegrator method official In: Methods book. British Sugar Technical Centre, 

England. 

135

Jepson W P (1954). A critical review of the world literature on the lepidopterous stalk borers of 

tropical graminaceous crops. Commonwealth Institute Entomology, London. 127 pp. 

Johnson J L; Zapata H and Heagler A (1995). Technical efficiency in sugarcane processing. 

Journal of Agribusiness 13, 28-33. 

Macedo N (1978). Varietal behavior, mechanism and inheritance of sugarcane resistance to the 

attack of Diatraea saccharalis. International Society of Sugarcane Technologist 

Entomology Newsletter 4, 687-688. 

Rao Siva D V (1962). Studies on the resistance of sugarcane to the early shoot borer, Chilo 

infuscatellus Snell. M.Sc. Thesis Andhra University, Waltair. 

Reagan T E; Ostheiner E A; Rodrigues L M; Woolwine A E and Schexnayder H P (1997). 

Assessment of varietals resistance to the sugarcane borer. Sugarcane Research, Annual 

Progress Report, 266 pp. 

136

Minaeimoghadam M, Askarianzadeh A: Comparison of feeding indexes of Sesamia nonagrioides Lef. (Lep., 

Noctuidae). In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 

137; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-19 Comparison of feeding indexes of Sesamia nonagrioides Lef. (Lep., 

Noctuidae) 

Minaeimoghadam M, Askarianzadeh A 

Department of Plant Protection, College of Agricultural Sciences, Shahed University, Tehran, 

Iran 

Email: minaeimoghadam@yahoo.com 

Abstract 

Stalk borers, Sesamia nonagrioides Lef. and Sesamia cretica Led. are important 

pests of sugarcane and cause considerable injury in the Khuzestan province of Iran 

annually. Today’s one of the strategies for control borers in sugarcane fields is 

usage of resistant cultivars. To study antibiosis resistance of sugarcane cultivars to 

Sesamia nonagrioides, feeding indexes on five cultivars (CP48-103 ،CP69-1062 ، 

CP57-614 ،NCO-310 and SP70-1143) were determined. Larvae (2 and 3 instars) 

feed on the cultivars for five days and then feeding indexes including: Consumption 

Index (CI), Approxymate Digestibility (AD), Efficiency of Conversion of Digested 

Food (ECD) and Efficiency of Conversion of Ingested food (ECI) were calculated. 

The trials were replicated six times. Data were analized by Kruskal-Wallis Test 

with SPSS 11.5 software. The results showed that CI, AD and ECD indexes were 

not significant among five cultivars but ECI index was significant. Comparison of 

means with Duncan's test showed that ECI index on NCO-310 and SP70-1143 

cultivars was more than CP69-1062, CP57-614 and CP48-103 cultivars. Therefore 

based on ECI index CP69-1062, CP57-614 and CP48-103 cultivars are resistance 

to Sesamia nonagrioides. 

137

Mansoor-ul-Hasan, Ashfaq M, Iqbal J, Sagheer M: Varietal resistance against Jassid, Amrasca biguttula biguttula 

(Ishida) on Okra under Faisalabad ecological conditions. In: Feldmann F, Alford D V, Furk C: Crop Plant 

Resistance to Biotic and Abiotic Factors (2009), 138-145; ISBN 978-3-941261-05-1; © Deutsche 


3-20 Varietal resistance against Jassid, Amrasca biguttula biguttula (Ishida) 

on Okra under Faisalabad ecological conditions 

Mansoor-ul-Hasan, Ashfaq M, Iqbal J, Sagheer M 

Department of Agri. Entomology, University of Agriculture, Faisalabad. Pakistan 

Email: mansoorsahi2000@yahoo.com 

138 

ABSTRACT 

The study was conducted as preliminary screening trials on 30 genotypes of okra 

for susceptibility/resistance against the jassid, Amrasca biguttula biguttula, during 

2006. From preliminary screening trials three genotypes showing comparatively 

susceptible responses (Pusa sawani, Dera local and Okra-3), three showing 

intermediate (Karam-5, Sabz pari and Clean spineless) and three showing resistant 

response (Makhmali, Punjab selection and Green wonder) to jassids were selected 

for final screening trials during 2007. Host plant susceptibility indices were also 

calculated to determine the contribution of each selected genotype towards 

susceptibility during 2006, 2007 and on average of 2006-07. Differences were 

found to be significant among genotypes of okra during both the study years 

regarding jassid numbers per leaf. The trend in selected genotypes towards 

susceptibility/resistance against jassid was found to be similar to those observed 

during preliminary screening trials. Pusa sawani showed maximum Host Plant 

Susceptibility Index (HPSI) (18% on average population of jassid per leaf recorded 

during 2006 and 2007). 

INTRODUCTION 

Okra, Abelmoschus esculentus is a member of family Malvaceae and is widely cultivated in 

tropics and sub tropics (Kochhar, 1986). Okra has gained considerable interest as an alternative 

to more traditional vegetables in many countries throughout the world. Okra is the most 

important traditional popular vegetable in Pakistan and is produced in different parts of the 

country. Increasing crop loss due to pest infestations is a major constraint in sustaining 

agricultural productivity and production. A. biguttula biguttula is one of the most serious pests 

of okra. It causes damage from early seedling stage to fruit-set, resulting losses of 50 % in

yield (Bindra and Mahal, 1981). Rawat and Sadu (1973) reported 49.8 % and 45.1 % 

reductions in the height and number of leaves respectively due to attacks of jassids. 

The increasing cost of pesticides has meant that they have become almost out of the reach of 

common farmers and a significant amount of government resources are exhausted every year 

on pesticide usage. The frequent use of systemic insecticides to manage insect pests leads to 

the destabilization of ecosystem, disrupting the delicate balance between the insect pests and 

their natural enemies and can lead to enhance insecticide resistance in pests (Ahmad et al. 

1999; Villegas et al. 2006), suggesting a clear need for alternatives. Varietal resistance 

occupies an important place in the Integrated Pest Management programme. It is one of the 

eco-friendly methods of pest control which besides being sustainable, reduces production costs 

and makes available to consumers good quality vegetables at accessible prices. It is therefore 

advisable to screen okra varieties/cultivars for possession resistance traits to jassids. 

Unfortunately little concentration has previously been paid to this aspect of control in Pakistan. 

A resistance variety can provide a base on which to construct an integrated control system and 

may be most fruitful when used in association with other methods of control. Host Plant 

Resistance (HPR) is seen to be a sustainable approach to pest management and varietal trials of 

different okra plants to jassids are essential. This work is an attempt to determine resistance/ 

susceptibility of different available genotypes of okra to jassids. 


Studies were carried out during 2006 and 2007 to screen okra genotypes based on leaf 

population density counts. Thirty genotypes of okra were sown in the experimental area of 

Post-graduate Agricultural Research Station, University of Agriculture, Faisalabad on March 

31, 2006 (Table 1). Based on leaf population density observations three genotypes, each 

showing resistant, susceptible and intermediate response for test insect was selected for further 

experiment. Thus, there were nine genotypes in total for testing insect response. The selected 

okra genotypes were sown on March 31, 2007. Experiments were laid out in a Randomized 

Complete Block Design (RCBD) with three replications. The row to row distance was 75 cm 

and plant to plant distance was 30 cm. The plot size was maintained at 15 m × 20 m during 

both study seasons. No plant protection measures were applied and the plant material was 

screened under natural insect pressure. All the recommended agronomic practices were 

adopted during the experiment. 

Jassid populations were recorded early in the morning twice in a week from 24 days after 

sowing. For counts of jassid population, 15 plants of each genotype in each replication were 

selected at random and tagged; one leaf at upper portion of the first plant, one leaf of the 

middle portion from the second plant and one leaf of the bottom portion from the third plant of 

each variety of similar age was taken to make insect counts. The data were analyzed 

statistically using M-Stat package. The means were compared by LSD test at P = 0.05. 

139


The results (Table 1) reveal that in 2006 the genotype Pusa sawani showed maximum jassid 

numbers (3.32 insects per leaf) followed by 3.24, 2.98 and 2.87 insects per leaf on Dera local, 

Okra-3 and Okra Sindh, respectively; counts which differed significantly with one an other 

and from those of observed on all other genotypes. The minimum jassid population (1.22 

insects per leaf) was recorded on Green wonder and showed non-significant difference with 

those of recorded on Punjab selection with 1.29 jassid per leaf. Full results are to be found in 

Table 1. The present findings are in agreement with those of Mahal and Singh (1979), 

Uthamasamy (1986), Singh (1988), Mahal et al., (1991), Mahal et al., (1993) who reported that 

Pusa swani was a susceptible genotype. In the present study, the genotype Pusa green was 

found to be moderately resistant with 1.84 jassids per leaf and these findings are in agreement 

with those of Shakeel et al., (2000). In the present study, the genotype Arka anamika appeared 

to be moderately resistant to jassids and Pusa swani was susceptible. Similar results were 

reported by Kumar and Singh (2002). 

Based on the data of jassid numbers during the study year 2006 in a preliminary screening trial, 

three genotypes (Pusa sawani, Dera local and Okra-3) showing the highest populations, three 

genotypes (Karam-5, Sabz pari and Clean spineless) having intermediate responses and three 

genotypes (Makhmali, Punjab selection and Green wonder) with the lowest population of 

jassid were selected for final screening trial during 2007. The objective of this study was to 

confirm the previous year’s results. Results (Table 1) reveal that maximum jassid numbers 

were recorded as 4.73 per leaf on genotype Dera local which did not differ significantly with 

those of observed on Pusa sawani with 4.65 jassids per leaf. The minimum jassid population 

observed was 1.25 and 1.26 per leaf on Punjab Selection and Green wonder respectively. Nonsignificant 

differences were found between Sabz pari and Karam-5 with 2.86 and 2.80 jassids 

per leaf respectively. The latter mentioned figure also showed non-significant difference with 

those of found on Clean spineless with 2.76 jassids per leaf. The genotypes Okra-3 and 

Makhmali differed significantly having 3.42 and 1.54 jassids per leaf respectively. From these 

results it was concluded that the genotype Dera local was the most susceptible genotype 

followed by Pusa swani. Punjab selection and Green wonder genotypes were considered 

comparatively resistant. During 2007, the selected genotypes of okra differed significantly 

regarding jassid population per leaf. The present findings can partially be compared with those 

of Khambete and Desai (1996) who studied the response of jassid on Pusa swani, MR10-1, 

MR-12 and IC-7194 genotypes and concluded that Pusa swani was a susceptible genotype. 

During 2006 the genotype Pusa sawani showed maximum HPSI (Host Plant Susceptibility 

Index) i.e. 17% followed by Dera local and Okra-3 with 16% and 15% HPSIs, respectively 

(Fig. 1). The minimum HPSI was observed to be 6% each in Green wonder and Punjab 

selection. The genotypes Karam-5, Sabz pari and Clean spineless each showed 11% HPSIs. 

The HPSIs based on jassid numbers on different okra genotypes during 2007 are shown in Fig. 

2. They are Dera local (19% HPSI),, Pusa sawani (18% HPSI) and the genotype Okra-3 (14% 

HPSI). 

140

Table 1. COMPARISON OF MEANS OF POPULATIONS OF JASSID ON 

VARIOUS GENOTYPES OF OKRA DURING 2006 and 2007. 

Genotypes Means ** 2006 Means ** 2007 

Pusa Swani 3.32 a 4.65 a 

Dera Local 3.24 b 4.73 a 

Okra-3 2.98 c 3.42 b 

Okra-Sindh 2.87 d 

COK-1418 2.78 e 

Ikra-2 2.76 ef 

COK-1396 2.69 fg 

Diksha 2.65 gh 

SPA-2001 2.65 gh 

PMS-55 2.61 hi 

Ikra-24 2.57 i 

Park-Karenti 2.49 j 

Ikra-1 2.27 k 

Karam-5 2.18 l 2.80 cd 

Sabz Pari 2.17 lm 2.86 c 

Clean Spineless 2.10 mn 2.76 d 

P-1999-31 2.06 no 

Ikra-3 2.02 op 

Ikra Anamika 2.00 op 

PMS-beauty 1.98 pq 

Namdahari 1.92 qr 

Lakshmi-24 1.86 rs 

Pusa Green 1.84 rs 

Arka Anamika 1.81 s 

Zeenat 1.73 t 

Green Star 1.59 u 

Super Star 1.51 v 

Makhmali 1.41 w 1.54 e 

Punjab Selection 1.29 x 1.25 f 

Green Wonder 1.22 x 1.26 f 

Means sharing similar letters are not significantly different by LSD Test at P = 0.05 

141

142 

Table 2. METEOROLOGICAL OBSERVATIONS AND JASSID NUMBERS 

RECORDED DURING 2006 and 2007 

Sr. No. Date Tempeature ºC Average 

Average 

Maximum 

Average 

Minimum Average 

Relative 

Humidity 

Average 

Rainfall 

(mm) 

Average 

Jassid 

Population 

1 24.04.06 37.8 21.0 29.4 32.0 0.0 0.38 

2 28.04.06 41.6 21.6 31.6 34.4 0.0 0.64 

3 01.05.06 40.7 23.7 32.2 35.7 0.0 1.11 

4 05.05.06 39.4 23.0 31.2 38.2 0.0 1.93 

5 08.05.06 44.4 23.6 34.0 29.2 0.0 2.88 

6 12.05.06 42.3 24.6 33.4 33.9 0.0 3.87 

7 15.05.06 45.5 26.5 36.0 29.7 0.0 5.23 

8 19.05.06 39.3 25.3 32.3 48.3 15.2 0.53 

9 22.05.06 38.5 25.2 31.9 49.3 0.0 0.97 

10 26.05.06 38.9 24.5 31.7 47.3 7.2 0.32 

11 29.05.06 43.1 27.0 35.1 39.2 0.0 0.67 

12 02.06.06 41.1 27.1 34.1 36.3 0.0 1.20 

13 05.06.06 39.8 25.5 32.7 37.0 0.0 1.59 

14 09.06.06 40.1 25.0 32.6 32.3 0.0 2.71 

15 12.06.06 42.2 23.6 32.9 27.8 0.0 4.16 

16 16.06.06 38.7 24.9 31.8 41.0 0.0 5.93 

17 19.06.06 34.9 21.0 28.0 60.8 4.0 4.66 

18 23.06.06 38.3 22.1 30.2 45.6 17.2 1.58 

19 26.06.06 40.9 27.5 34.2 48.5 0.0 2.62 

20 30.06.06 37.3 26.5 31.9 65.4 2.0 2.83 

21 03.07.06 38.0 24.8 31.5 61.8 23.0 0.80 

1 24.04.07 39.3 21.7 30.5 37.8 - 0.230 

2 28.04.07 39.8 19.1 29.4 32.4 - 0.32 

3 01.05.07 42.0 21.8 32.0 38.0 - 0.64 

4 05.05.07 40.7 23.4 32.1 39.4 11.2 0.21 

5 08.05.07 41.3 24.2 32.8 35.7 - 0.43 

6 12.05.07 38.5 22.8 30.7 45.7 4 0.38 

7 15.05.07 40.9 24.3 32.6 37.8 - 0.53 

8 19.05.07 41.3 27.2 34.3 40.4 - 0.69 

9 22.05.07 39.4 24.6 32.0 42.5 - 0.92 

10 26.05.07 39.6 23.9 31.7 33.6 - 1.50 

11 29.05.07 35.8 21.1 28.5 40.7 - 1.44 

12 02.06.07 40.1 22.5 31.3 35.0 - 3.27 

13 05.06.07 41.2 24.9 33.1 40.3 - 5.35 

14 09.06.07 42.7 26.6 34.6 35.6 - 9.58 

15 12.06.07 45.7 27.6 36.7 41.7 - 12.29 

16 16.06.07 40.6 29.3 35.0 55.1 20 1.43 

17 19.06.07 32.7 22.5 27.6 72.8 2.8 3.28 

18 23.06.07 36.2 24.2 30.2 56.4 7.1 4.17 

19 26.06.07 38.8 26.6 32.7 58.7 - 5.35 

20 30.06.07 36.7 26.2 31.5 67.9 0.5 5.86 

21 03.07.07 36.2 26.1 31.2 68.8 15.3 1.10

7% 

11% 

11% 

6% 6% 

11% 

17% 

15% 

16% 

Pusa Sw ani 

Dera Local 

Okra-3 

Karam-5 

Sabz Pari 

Clean Spineless 

Makhmali 

Punjab Selection 

Green Wonder 

Figure 1. Plant susceptibility indices (%) based on A. biguttula biguttula (Ishida) 

populations on various genotypes of Okra, Abelmoschus esculentus (L.) 

during 2006 

5% 

19% 

6% 

5% Dera Local 

Pusa Sw ani 

Okra-3 

11% 

11% 

11% 

14% 

18% 

Sabz Pari 

Karam-5 


Makhmali 

Green Wonder 



populations on various genotypes of Okra, Abelmoschus esculentus (L.) during 

2007 

11% 

7% 

11% 

5% 6% 

11% 

17% 

14% 

18% 

Dera Local 

Pusa Swani 

Okra-3 

Sabz Pari 

Karam-5 


Makhmali 

Green Wonder 



populations on various genotypes of Okra, Abelmoschus esculentus (L.) on 

cumulative basis during 2006-07 

143

These three genotypes considered to be comparatively susceptible to jassid according to their 

2007 HPSIs. The genotypes Sabz pari, Karam-5 and Clean spineless each showed 11% HPSIs 

and are considered to be intermediate. The minimum HPSIs was observed in genotypes Punjab 

selection and Green wonder, each having 5% HPSIs whereas Makhmali showed 6% HPSI and 

these genotypes are considered resistant. The results regarding HPSIs based on average 

population of jassid per leaf recorded during 2006 and 2007 are shown in Fig 3. It is evident 

from the results that Pusa sawani showed maximum HPSI (18%). The HPSIs for Dera local 

and Okra-3 were 17% and 14% respectively. The genotypes Sabz pari, Karam-5 and Clean 

spineless each showed 11% HPSI and are considered intermediate. The minimum HPSI was 

found for Green wonder (5%) while Makhmali and Punjab selection showed 6% and 7% HPSIs 

respectively and are therefore considered comparatively resistant. 

The data regarding jassid numbers recorded at different dates of observation during 2006 and 

2007 on various genotypes of okra are given in Table 2, respectively. In 2006 the lowest 

number of jassids per leaf was recorded as 0.377 on April 24 2006 and increasing trend was 

observed there after on the subsequent dates of observation. The population of jassid reached to 

a peak of 5.23 jassids per leaf on May 15. A tremendous decrease was observed thereafter on 

May 19 and after a slight increase on May 22 with population of 0.967 per leaf, the population 

again decreased and reached 0.322 insects per leaf. An increasing trend was observed 

thereafter on subsequent dates. The population reached its peak (5.925 per leaf) on June 16, 

2006, decreasing and increasing trends were observed thereafter with a peak of 4.827 jassid per 

leaf on June 30, 2006. From these results, it was observed that there were four peaks of jassid 

numbers during the study period with highest number of 5.925 jassid per leaf recorded on June 

16, 2006. In 2007 the lowest numbers of jassids (0.223 per leaf) were observed on April 24, 

2007. The population started to increase thereafter and reached to a peak of 0.635 per leaf on 

May 01. The population again decreased down to 0.21 per leaf and an increasing trend was 

observed thereafter on the subsequent dates of observation. The population reached a third 

peak on May 26 with 1.504 insects per leaf. A slight decrease was observed on May 29 with 

1.44 jassid per leaf and an increasing trend was again observed thereafter on the subsequent 

dates of observation and the population reached to the highest peak of 12.29 jassids per leaf on 

June 12. An increasing trend was again observed in jassid numbers after a tremendous 

decrease on June 16 with 1.432 jassids per leaf and reaching a peak of 5.862 jassids per leaf on 

June 30. The population had decreased to 1.096 per leaf by June 3. From these results it was 

observed that there had been five peaks of jassids on okra with the highest peak of 12.29 per 

leaf on June 12, 2007. The present findings do not agree with those of Preek et al. (1986), 

Patel et al. (1997), Gogoi and Dutta (2000) and Kumawal et al. (2000). They reported 

different periods of abundance to those of found in the present study probably because the 

sowing dates and ecological conditions were different. Similarly Mahmood et al, (1990) found 

that leafhopper populations started from June and remained active until the end of Okra crop, 

but in the present study, leafhopper populations started from the fourth week of April and 

remained present on the crop until July. 

144

REFERENCES 

Ahmad M, M I Arif, Z Ahmad (1999). Detection of Resistance to pyrethroids in Field 

Population of Cotton Jassid (Homoptera: Cicadellidae) from Pakistan. J. Econ. Ent., 

92(6): 1246-1250. 

Bindra O S, M S Mahal (1981). Varietal resistance in egg plant (brinjal) (Solanum melongena) 

to the cotton jassid (Amrasca biguttula). Phytoparasitica. 9: 119-131. 

Gogoi I, B C Dutta (2000). Seasonal abundance of cotton jassid, Amrasca biguttula biguttula 

(Ishidia) on okra. J. Agric. Sci. Soc. North East India, 13(1): 22-26. 

Khambete M S, B D Desai (1996). Studies on the varietal resistance of okra to jassid and shoot 

and fruit borer. TVIS-Newsletter, 1(2):18-19. 

Kochar S L, (1986). Tropical Crops. A text book of economic botany. Macmillan 1986. 

Indian Ltd., 263-264. 

Kumar M, A K Singh 2002. Varietal resistance of okra against cotton jassid, Amrasca 

biguttula biguttula under field conditions. Ann. Plant Prot. Sci., 10(2): 381-383. 

Kumawat R L, B L Pareek, B L Meena (2000). Seasonal incidence of jassid and whitefly on 

okra and their correlation with abiotic factors. Ann. Biology, 16(2): 167-169. 

Mahal M S, B Singh (1979). Population build-up of cotton jassid and index of its injury as a 

measure of resistance in okra. Indian J. Ecol., 6: 71-81. 

Mahal M S, H Lal, R Singh (1991). Standardisation of technique for screening okra 

germplasm for resistance against cotton jassid, Amrasca biguttula biguttula (Ishida) I. 

Development and survival of nymphs. J. Insect Sci., 4(2): 135-137. 

Mahal M S, H Lal, R Singh (1993). Standardization of a technique for screening of okra 

germlasm for resistance against cotton jassid, Amrasca biguttula (Ishida). II. 

Ovipositional preference of adults. J. Insect Sci., 6(2): 223-225. 

Mahmood T, K M Khokar, M Banaras, M Ashraf (1990). Effect of environmental factors on 

the density of leafhopper, Amrasca devastans (Distant) on okra. Trop Pest Manage., 36: 

279-284. 

Pareek B L, G K Sharma, K N Bhatnagar (1986). Seasonal incidence of major insect pests of 

okra in semiarid region of Rajastan. Ann. Arid Zone., 25: 222-224. 

Patel K I, J R Patel, D B Jayani, A M Shekh, N C Patel (1997). Effect of seasonal weather on 

incidence and development of major pests of okra (Abelmoschus esculentus). Indian J. 

Agric. Sci., 67(5): 181-183. 

Rawat R R, H R Sadu (1973). Estimation of losses in growth and yield of okra due to 

Empoasca devastans (dist.) and Erias spp. Indian J. Ent. 35: 252-254. 

Singh R (1988). Bases of resistance in okra (Abelmoschus esculentus) to Amrasca biguttula 

biguttula. Indian J. Agric. Sci., 58(1): 15-19. 

Shakeel M, K Ullah, M Zaman, S Ahmad, Z Hafeez (2000). Infestation of aphids, Aphis 

gossypii, Glov. (Homoptera: Aphididae) and jassid, Amrasca biguttula biguttula, 

Shir. (Homoptera: Jassidae) on different cultivars of okra at Mingora, Swat 

(Pakistan). Bal. J. Agic. Sci., 1(2): 34-37. 

Uthamasamy S, (1986). Studies on the resistance in okra, Abelmoschus esculentus (L) to the 

leafhopper, Amrasca devastans (Dist.). Trop. Pest Manag., 32(2): 146-147. 

Villegas R S, J L G Hernandez, B M Amador, A Tejas, J L M Carrillo (2006). Stability of 

insecticide resistance of silverleaf whitefly (Homoptera: Aleyrodidae) in the absence of 

selection pressure. Folia Entomol. Mex, 45 (1): 27-34. 

145

Iqbal J, Mansoor-ul-Hasan, Sagheer M: Seasonal abundance of the Cotton jassid Amrasca biguttula biguttula on 

Okra and their Correlation with Abiotic Factors. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to 

Biotic and Abiotic Factors (2009), 146-153; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 


3-21 Seasonal abundance of the Cotton jassid Amrasca biguttula biguttula on 

Okra and their Correlation with Abiotic Factors 

Iqbal J, Mansoor-ul-Hasan, Sagheer M 

Department of Agri. Entomology, University of Agriculture, Faisalabad. Pakistan 

Email: mansoorsahi2000@yahoo.com 

146 

ABSTRACT 

The data on jassid population per leaf obtained from varietals trials of 2006 and 

2007 at various dates of observation were correlated with the ambient weather 

conditions such as maximum temperature, minimum temperature, average 

temperature, relative humidity and rainfall. The coefficient of determination values 

were observed to determine the role of weather factors affecting population 

fluctuation of jassids on okra. Minimum temperature during 2007 and on 

cumulative basis of 2006 and 2007 showed significant and positive correlation with 

jassid populations. All the other factors showed non-significant correlations with 

jassid populations. Of the other factors, rainfall showed maximum contribution 

(12%) in population fluctuations of jassids during 2006 followed by maximum 

temperature, average temperature and relative humidity. During 2007 minimum 

temperature showed maximum contribution (20.5%) to population fluctuations 

followed by rainfall, relative humidity, maximum temperature and average 

temperature. On an average of two years data, rainfall was found to be the most 

important factor which contributed a maximum (13.4%) to population fluctuations 

of jassid. 

INTRODUCTION 

Insects are capable of surviving only within certain environmental limits, and when possible, 

individuals actively seek out favorable environments and therefore an understanding of their 

relative importance is an essential component of pest control . It is known that weather factors 

play important role in insect pest management. Prolonged periods of low or high temperatures, 

different level of humidity and rainfall can increase or reduce the population of certain insect 

pest species. The weather conditions prevailing in a season play a vital role in the incidence 

and subsequent upsurge of population of insect pests.

Okra, Abelmoschus esculentus, is one of the most common vegetables of Pakistan and is 

cultivated in tropics and sub-tropics on varying scales. Okra is susceptible to a large variety of 

pests that hamper its marketable fruit yield. Under tropical conditions, polyphagous insect pests 

like jassid, Amrasca biguttula biguttula (Homoptera: Cicadellidae), can attack several crops 

making intensive vegetable production difficult. This pest is especially important in the tropics 

and subtropics because environmental conditions are often favorable year round for growth and 

development of host and pest. This pest is among the most important sucking insects that 

attack okra (Singh et al. 1993; Dhandapani et al. 2003). Okra is most suitable host for A. 

bigattula bigattula in terms of number of eggs, and for nymph survival and feeding (Bernardo 

and Taylo, 1990; Sharma and Singh, 2002). Among various physical factors, temperature, 

humidity and rainfall are considered the most important causes of population fluctuation. The 

information available on the population fluctuation of jassid on okra in Pakistan is scanty. The 

present studies were therefore initiated to study the impact of abiotic factors on populations and 

seasonal abundance of jassids during 2006 and 2007. 


Thirty genotypes of okra were sown in the experimental area of the Post-graduate Agricultural 

Research Station, University of Agriculture, Faisalabad on March 31, 2006. Based on leaf 

population densities of jassids, nine okra genotypes were selected for further experiments; 

three genotypes, showing resistant, three showing susceptible and three intermediate responses 

to A. biguttula biguttula. These nine okra genotypes were sown on March 31, 2007. 

Experiments were laid out in a Randomized Complete Block Design (RCBD) with three 

replications. The row to row distance was kept at 75 cm and plant to plant was 30 cm. The plot 

size was maintained at 15 m × 20 m during both the study seasons. No plant protection 

measure was applied. All the recommended agronomic practices were adopted during the 

experiment. Jassid populations were recorded early in the morning twice in a week from 24 

days after sowing. For counts of jassid population, 15 plants of each genotype in each replicate 

were selected at random and tagged. One leaf of the upper portion of the first plant, one leaf of 

the middle portion from the second plant and one leaf from bottom portion from the third plant 

of each variety of similar age were used for observations. Metrological data of temperature, 

relative humidity and rainfall were recorded from the adjoining meteorological observatory of 

Physiology Section, Ayub Agricultural Research Institute, Faisalabad. The effect of abiotic 

factors on the adult and nymph population densities was determined by working out simple 

correlations (Steel et al. 1990). The combined effect of temperature, relative humidity and 

rainfall on the population of jassid for both study years was measured by using a Multiple 

Linear Regression Equation. 


The data regarding jassid population fluctuations during 2006 and 2007 were correlated with 

147

the weather factors both on a year and a cumulative basis. The impact of weather factors on 

population fluctuations were also determined by processing the data with a Multiple Linear 

Regression analysis. The results (Table 1) reveal that minimum temperatures both during 2007 

and on a cumulative basis showed a significant and positive correlation with the jassid 

population, whereas all other factors during both the study years individually as well as on 

cumulative basis resulted in non-significant correlations. 

148 

Table 1. CORRELATION COEFFICENTS (r) BETWEEN POPULATIONS OF 

JASSID ON OKRA AND VARIOUS WEATHER FACTORS 

(* = Significant at ≤ 0.05) 

Years 

Weather Factors 2006 2007 

Cumulative 

Maximum temperature (°C) 0.155 0.174 0.157 

Minimum temperature (°C) 0.073 0.484 * 0.342 * 

Average temperature (°C) 0.142 0.394 0.295 

Relative Humidity (%) – 0.130 0.242 0.110 

Rainfall (mm) – 0.313 – 0.189 – 0.231 

The multiple effects of weather factors on jassid population during 2006 (Table 2) reveals that 

the Rainfall contributed maximum (12%) in population fluctuations followed by maximum 

temperature with a 2.4% role in the population fluctuations. None of the regression equations 

was found to be fitted the best. The present findings are in conformity with those of Mahmood 

et al. (1990), Sharma and Sharma (1997), Prasad and Logiswaran (1997), Mahmood et al. 

(2002) and Arif et al. (2006) who also reported positive correlations of minimum temperature 

with density counts of leafhoppers. The present findings are not inconformity with those of 

Patel et al. (1997) who reported negative correlation between population of jassid and 

temperature. The present findings can partially be compared with those of Kumawat et al. 

(2000) who reported that maximum and minimum temperature showed positive and nonsignificant 

correlation with jassid population on okra. In the present study, all the other factors 

showed non-significant correlation with jassid populations. However, in multiple regression 

analysis rainfall showed a negative and non-significant impact on the populations of jassids. 

The present findings are in agreement with those of Kumawat et al. (2000) and Mahmood et al. 

(2002). The present findings can be compared with those of Srinivasan et al. (1981), who 

reported that rainfall reduced the mean density and increased aggregation among jassid on okra 

crop. Similar results were also reported by Lal et al. (1990) that continuous rainfall was 

unfavourable for the population build-up of jassids. The present findings are partially in 

agreement with those of Mahmood et al. (2002) who reported that rainfall had no significant 

contribution toward increasing or decreasing the leaf hopper numbers, whereas Prasad and 

Logiswaran (1997) found a negative association between jassid population and rainfall in

winter 1991 and during summer 1992. Similarly present findings cannot be compared with 

those of Sekhon and Singh (1985) and Lal et al. (1990) who reported significant and negative 

correlation between rainfall and jassid populations on cotton. 

Table 2. MULTIPLE LINEAR REGRESSION MODEL/S ALONG WITH 

COEFFICIENTS OF DETERMINATION (R²) REGARDING THE IMPACT 

OF WEATHER FACTORS ON POPULATIONS OF JASSID ON OKRA 

DURING 2006. 

REGRESSION EQUATION R² 100 R² 

Role of 

individual 

factor (%) 

Y = – 0.978658 + 0.40053 X1 0.024 2.4 2.4 

Y = – 1.105833 + 0.38283 X1 + 0.48055 X2 0.024 2.4 0.00 

Y = – 1.062264 + 22.788 X1 + 17.576 X2 – 40.231 X3 0.032 3.20 0.8 

Y = – 0.590469 + 21.377 X1 + 16.577 X2 – 37.847 X3 –22.206 X4 

Y = – 3.313567 + 54.699 X1 + 41.672 X2 – 96.648 X3 + 0.28898 X4 – 

0.033 3.3 0.1 

0.18644 X5 0.153 15.3 12 

Where: Y = Jassid Population per leaf; X1 = Maximum Temperature ( o C); X2 = Minimum Temperature ( o C); X3 = 

Average Temperature (°C); X4 = Average Relative Humidity (%); X5 = Rainfall (mm) 

Table 3. MULTIPLE LINEAR REGRESSION MODELS ALONG WITH 

COEFFICIENT OF DETERMINATION (R²) REGARDING THE IMPACT 

OF WEATHER FACTORS ON POPULATIONS OF JASSID DURING 2007 

ON OKRA. 

Role of 

REGRESSION EQUATION R² 100 R² 

individual 


Y = – 2.256243 + 0.61681 X1 0.030 3.00 3.00 

*Y = – 6.693761 + 0.11938 X1 + 1.5241 X2 0.235 23.5 20.5 

Y = – 6.514041 + 6.7429 X1 + 6.7152 X2 – 11.929 X3 0.236 23.6 0.1 

Y = – 16.217914 + 37.650 X1 + 27.978X2 – 63.880 X3 + 0.61813 X4 

*Y = – 16.840585 + 18.331 X1 + 13.578 X2 – 29.763 X3 + 0.72240 X4 – 

0.303 30.3 6.7 

0.32307 X5* 0.47 47 16.7 


Average Temperature (°C); X4 = Average Relative Humidity (%); X5 = Rainfall (mm); * = Significant at P ≤ 0.05 

The effect of weather factors on a cumulative basis during 2007 (Table 3) reveals that 

minimum temperature showed a maximum contribution (20.5%) to population fluctuations of 

jassid followed by rainfall (16.7%), relative humidity (6.7%) and average temperature (0.1%). 

The 100 R² value was 47 percent when the effect of all the factors on the population 

fluctuations were analyzed together. None of the regression equations were found to be best 

fitted. These findings can be compared with those of Prasad and Logiswaran (1997). Minimum 

149

temperatures during 2007 and on cumulative basis of 2006 and 2007 showed significant and 

positive correlation with the jassid populations on okra. In the present studies relative humidity 

showed a negative and non-significant correlation with the jassid populations during 2006 

while during 2007 and on cumulative basis this factor exerted positive and non-significant 

effects, showing that this factor was unimportant during the study. These findings are in 

agreement with those of Mahmood et al. (1990) who reported that relative humidity made no 

significant contribution towards increasing or decreasing the leaf hopper numbers. However 

these findings are contradicted by those of Bishnoi et al. (1996) who reported a significant 

relationship between population and relative humidity. Furthermore the present findings cannot 

be compared with those of Sharma and Sharma (1997) who found positive and non-significant 

correlation between relative humidity and jassid population densities on cotton crops. 

Similarly Prasad and Logiswaran (1997) found a positive association between jassid 

populations and relative humidity on brinjal. The present findings are partially in accordance 

with those of Kumawat et al. (2000), Mahmood et al. (2002) and Arif et al. (2006) who 

reported negative and non-significant correlation between relative humidity and jassid 

populations on okra. 

The multiple effect of weather factors for both year’s study (Table 4) reveals that rainfall 

showed significant and maximum contribution (13.4%) towards population fluctuation of the 

jassid on okra. Results of the other factors were non-significant; minimum temperature (9.5%), 

maximum temperature (2.5%), relative humidity (2.5%) and average temperature (0.1%). The 

100 R² value was calculated to be 28 when the effect of all the factors was computed together. 

Furthermore, none of the equations were found to be best fitted. 

150 

Table 4. MULTIPLE LINEAR REGRESSION MODELS ALONG WITH 

COEFFICIENT OF DETERMINATION (R²) REGARDING THE IMPACT 

OF WEATHER FACTORS ON POPULATIONS OF JASSID ON OKRA 

DURING 2006-2007. 

REGRESSION EQUATION R² 100R² 

Role of 

individual 


Y = – 1.540054 + 0.49604 X1 0.025 2.5 2.5 

Y = – 4.261055 + 0.16040 X1 + 0.97328 X2 0.120 12 9.5 

Y = – 4.117482 + 7.5551 X1 + 6.7666 X2 – 13.307 X3 0.121 12.1 0.1 

Y = – 8.977431 + 21.697 X1 + 16.551 X2 – 37.034 X3 + 0.26796 X4* 

Y = – 11.485062 + 29.113 X1 + 21.827 X2 – 49.709 X3 + 0.54537 X4* – 

0.146 14.6 2.5 

23.286 X5** 

0.280 28 13.4 


Average Temperature (°C); X4 = Average Relative Humidity (%); X5 = Rainfall (mm); * = Significant at P ≤ 0.05

70.000 

60.000 

50.000 

40.000 

30.000 

20.000 

10.000 

80 

70 

60 

50 

40 

30 

20 

10 

0.000 

0 

24.04.06 

01.05.06 

08.05.06 

15.05.06 

22.05.06 

29.05.06 

05.06.06 

12.06.06 

19.06.06 

26.06.06 

03.07.06 

7.000 

6.000 

5.000 

4.000 

3.000 

2.000 

1.000 

0.000 

Jassid Population 

Tempeature C 

Average Maximum 

Tempeature C 

AveragMinimum 

Tempeature C 

Average 

Average Relative 

Humidity (%) 

Rainfall (mm) 

FIGURE 1. SHOWING JASSID POPULATION PER LEAF VERSUS WEATHER 

FACTORS DURING 2006 

24.04.07 

01.05.07 

08.05.07 

15.05.07 

22.05.07 

29.05.07 

05.06.07 

12.286 

12.06.07 

19.06.07 

26.06.07 

03.07.07 

14.000 

12.000 

10.000 

8.000 

6.000 

4.000 

2.000 

0.000 

Average Jassid 

Population 

Tempeature C 

Average Maximum 

Tempeature C 

AveragMinimum 

Tempeature C 

Average 

Relative Humidity 

(%) Average 

Rainfall (mm) 

FIGURE 2. SHOWING JASSID POPULATION PER LEAF VERSUS WEATHER 

FACTORS DURING 2007 

151

The data regarding jassid population per leaf versus weather factors during 2006 and 2007 are 

depicted graphically in Figs 1 and 2. Variations were found to be significant in population 

fluctuations of jassid recorded on different dates during 2006 and 2007. Four peaks of jassid 

on okra were recorded during 2006, whereas five peaks were observed during 2007. The 

highest peak was observed on June 16, 2006 and on June 12, 2007 with 5.92 and 12.29 jassid 

per leaf, respectively. From these results it was observed that jassid population was highest 

during the study year of 2007 as compared to 2006. Furthermore the second week of June in 

both the study years was found to be favorable for the development of jassid on okra. The 

present findings disagree with those of Preek et al. (1986), Sharma and Sharma (1997), Patel et 

al. (1997), Gogoi and Dutta (2000), Kumawal et al. (2000) and Lokesh and Singh (2005) 

because they reported different periods of abundance to those found in the present study. 

Reasons for the observed differences could be that the sowing dates and ecological conditions 

were different in the other studies. Similarly Mahmood et al, (1990) found that leafhopper 

populations started from June and remained active till the end of Okra crop, but in the present 

study, the leafhopper infestations started from fourth week of April and remained present on 

the crop until July. 


Sincere thanks are to the Higher Education Commission, Islamabad for granting the Ph.D. 

scholarship under Indigenous Ph.D. program. 

REFERENCES 

Arif M J; Gogi MD; Mirza M; Zia K; Hafeez F (2006). Influence of Plant Spacing and Abiotic 

Factors on Population Dynamics of Sucking Insect Pests of Cotton. Pak. J. Biol. Sci., 

9(7): 1364-1369. 

Bernado EN; Taylo LD (1990). Preference of the cotton leaf hopper, Amrasca biguttula 

(Ishida) for okra, Abelmoschus esculentus (Linn.), and eggplant, Solanum melongena 

Linn. Philippine Agric., 73 (2): 165-177. 

Bishnoi OP; Singh M; Rao VUM; Niwas R; Sharman PD (1996). Population dynamics of 

cotton pests in relation to weather parameters. Indian J. Ent., 58(2): 103-107. 

Dhandapani N; Shelkar UR; Murugan M (2003). Bio-intensive pest management (BIPM) in 

major vegetable crops: an Indian perspective. Food, Agric. & Envir., 2: 333-339. 

Gogoi I; Dutta BC (2000). Seasonal abundance of cotton jassid, Amrasca biguttula biguttula 

(Ishidia) on okra. J. Agric. Sci. Soc. North East India, 13(1): 22-26. 

Kumawat RL; Pareek BL; Meena BL (2000). Seasonal incidence of jassid and whitefly on okra 

and their correlation with abiotic factors. Ann. Biology, 16(2): 167-169. 

Lal H; Mahal MS; Singh R; Singh B (1990). Influence of rainfall on population build-up of 

Amrasca biguttula biguttula (Ishida) on okra. J. insect sci., 3: 169-171. 

Lokesh; Singh R (2005). Influence of leaf vein morphology in okra genotypes (Malvaceae) on 

the oviposition of the leafhopper species Amrasca biguttula (Hemiptera: cicadellidae). 

Entom. Gen., 28(2): 103-114. 

152

Mahmood M; Hussain SI; Khokar KM; Jeelani G; Ahmad M (2002). Population Dynamic of 

Leaf Hopper (Amrasca biguttula biguttula) on Brinjal and Effects of Abiotic Factors on 

its Dynamics. Asian J. Plant Sci., 1(4): 403-404. 

Mahmood T; Khokar KM; Banaras M; Ashraf M (1990). Effect of environmental factors on 

the density of leafhopper, Amrasca devastans (Distant) on okra. Trop. Pest Manage., 

36: 279-284. 

Pareek BL; Sharma GK; Bhatnaga KN (1986). Seasonal incidence of major insect pests of okra 

in semiarid region of Rajastan. Ann. Arid Zone., 25: 222-224. 

Patel KI; Patel JR; Jayani DB; Shekh AM; Patel NC (1997). Effect of seasonal weather on 

incidence and development of major pests of okra (Abelmoschus esculentus). Indian J. 

Agric. Sci., 67(5): 181-183. 

Prasad SG; Logiswaran G (1997). Influence of weather factors on population fluctuation of 

insect pest of brinjal at Madurai, Tamilnadu. Indian J. Ent., 59(4): 385-388. 

Sekhon BS; Singh S (1985). Effect of temperature, relative humidity and rainfall on the 

population of Empoasca devastans Dist. on bhindi (Abelmoschus esculentus Moench.). 

Madras Agric. J., 5:90. 

Sharma A; Singh R (2002). Oviposition preference of cotton leafhopper in relation to leaf-vein 

morphology. J. Appl. Ent., 126, 538-544. 

Sharma GN; Sharma PD (1997). Population dynamics of cotton leafhopper, Amrasca biguttula 

biguttula (Ishida) on cotton and okra in relation to physical factors of environment in 

Haryna. Ann. Biol. Ludhiana, 13(1): 179-183. 

Singh J; Sohi AS; Dhaliwal ZS; Mann HS (1993). Comparative incidence of Helicoverpa 

armigera Hb. and other pests on okra and sunflower intercrops in cotton under Punjab 

conditions. J. Insect Sci., 6: 137-138. 

Srinivasan K; Krishnakumar NK; Ramachander PR; Krishnamurthy A (1981). Spatial 

distribution pattern of leafhopper Amrasca biguttula biguttula on okra Abelmoschus 

esculentus. Entomon, 6(3):261-264. 

Steel RGD; Torrie JH; Dickey DA (1990). Principles and Procedures of Statistics: A 

Biometrical Approach, 3 rd edition. WCB MeGraw Hill Companies, Inc., USA. 

153

Sagheer M, Ashfaq M, Hasan M-ul: Screening of various genotypes of rice against rice leaf folder, 

Cnaphalocrocis medinalis Guenee. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and 



3-22 Screening of various genotypes of rice against rice leaf folder, 

Cnaphalocrocis medinalis Guenee 

Sagheer M, Ashfaq M, Hasan M-ul 

Department of Agri. Entomology, University of Agriculture, Faisalabad, Pakistan 

Email: sagheersharif@yahoo.com 

154 

Abstract 

Sixteen advanced elite lines of coarse and fine rice including locally recommended 

varieties were screened for high, intermediate resistance and susceptible response 

based on percent leaves infestation caused by Cnaphalocrocis medinalis Guenee 

(Lepidoptera, Pyralidae) during 2005. The data for larval population for each 

line/variety were also recorded. Minimum leaf infestation was observed on Super 

Basmati (14.03 %) and was statistically at par with that of Basmati-370 (14.29 %) 

and Basmati-198 (15.29%) and differed significantly from genotypes PK-5261, 

99515, Basmati-2000, KS-133, IRRI-6, KS-282, Basmati-385 and 4-1-7909 which 

had 17.03, 18.68, 18.97, 19.81, 20.93, 21.06, 21.62 and 21.91 % leaves infestation 

caused by C. medinalis. The genotype 00518-2 was comparatively susceptible with 

maximum infestation (31.80%) and was statistically at par with genotypes 00515-1 

having 30.79% infestation and was statistically different with genotypes 7429-5-14- 

1-1, 48463 and 99518-2 which had 26.71, 24.56 and 22.82% leaves infestation due 

to rice leaf folder. The maximum infestation of C. medinalis was observed in 2nd 

week of September, 2005 and minimum observation was observed on first week of 

September. From these results, two genotypes, super basmati and Basmati-370 

having least infestation and four genotypes (Basmati-2000, KS-133, IRRI-6 and 

KS-282) showing intermediate leaf infestation (18.97, 19.92, 20.92 and 21.06%) 

and two genotypes (00518-2 and 00515-1) having maximum leaf infestation (31.80 

and 30.79% respectively) were selected for further screening during 2006 crop 

season. Minimum leaves infestation caused by C. medinalis was observed on Super 

Basmati (13.42 %) which was significantly different from that of Basmati-370, 

Basmati-2000, KS-133, IRRI-6 and KS-282 which showed intermediate response. 

Maximum leaves infestation was observed on genotype 00518-2 which was 30.84 

%. Overall results shows that fine varieties of rice are comparatively resistant as 

compared to coarse varieties.

Kazemi M H, Mashhadi Jafarloo M: Field assessment of antibiosis resistance of different wheat cultivars to the 

Russian Wheat Aphid, Diuraphis noxia (Hom.: Aphididae) at stem elongation growth stage. In: Feldmann F, 

Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 155-162; ISBN 978-3-941261- 


3-23-Field assessment of antibiosis resistance of different wheat cultivars to 

the Russian Wheat Aphid, Diuraphis noxia (Hom.: Aphididae) at stem 

elongation growth stage 

Kazemi M H 1 , Mashhadi Jafarloo M 2 

1. 

Department of Plant Protection, Faculty of Agriculture, Islamic Azad University, Tabriz 

branch, Tabriz, Iran 

2. 

Department of Plant Pests and Diseases, Agricultural and Natural Resource Research 

Center of East Azerbaijan, Tabriz, Iran 

ABSTRACT 

The Russian Wheat Aphid is one of the most important cereal pests in the world. 

Due to the economic importance of this aphid in Iran and also most parts of the 

world studies have been directed towards the introduction of resistant cereal 

varieties. In the present study, the resistance associated with antibiosis was sought 

at stem elongation growth stage in Alamoot, Alvand, Zarrin, Sabalan and Sardari, 

the most extensively planted wheat varieties in East Azarbaijan province of Iran. 

Antibiosis was determined by studying the percentage nymphal survival rate, their 

development time, fecundity of the first 10 and 15 days of their reproductive period, 

growth index and by calculating the relevant intrinsic rate of natural population 

increase (rm value). ANOVA of data indicated that, regarding the development time 

of nymphs, fecundity and rm values, there were significant differences between the 

varieties. The highest and lowest mean survival rates of nymphs were observed in 

rearings on Sabalan and Alvand with 77.78 and 66.67 percent respectively. 

Comparisons of means using Duncan’s multiple range test, showed significant 

differences (p

in the west (Kazemi et al. 2001a, Kazemi et al. 2001b; Blackman & Eastop, 1984; Stoetzel, 

1987). Its damage pattern differs from those of the other cereal aphids so that one can identify 

its occurrence by means of the resulting damage. White or yellow longitudinal bands appear on 

the leaves due to the feeding effects and injection of salivary toxins which, in colder climates, 

become red or pinkish due to the existing antocyanic pigments. The individual aphids feed on 

the upper surfaces of curled leaves. Young plants become stunted under heavy aphid attacks 

and pre-panicle infestations can result in curling of the flag leaves and panicle deformations 

(Jones et al. 1989; Kindler & Hammon, 1996; Kazemi et al. 2001a). 

In recent years, the Russian Wheat Aphid, has been included in the list of worldwide important 

pests of cereals, particularly wheat cultivars. Cereal losses in United States of America during 

years 1986-1989 were estimated at more than 650 million dollars (Kindler et al. 1992). The 

importance of this aphid in its native regions, especially in dry years, is high (Souza et al. 

1991), but in the opinion of Burd et al. (1993), this aphid can disturb the plant physiological 

patterns even in low populations. Archer and Bynum (1992) noted that the losses due to 

feeding damage of this pest on the crop in spring at growth stages GS 29-60 (Zadoks et al. 

1974) for one percent of plant contamination by the aphid, was evaluated as 0.46-0.48 percent. 

The Russian Wheat Aphid can also be damaging as a vector of plant pathogenic viruses 

including Barley Yellow Dwarf Virus (BYDV), Barley Stripe Mosaic Virus (BSMV) and 

Sugarcane Mosaic Virus (SCMV) (Damsteegt et al. 1992). It has also been reported that the 

susceptibility of the winter wheat to the cold weather increases due to feeding of this aphid and 

therefore leads to indirect crop losses. In recent years, due to the economic importance of this 

aphid in most parts of the world studies have been directed towards the introduction of resistant 

varieties (Du Toit, 1989; Kindler & Springer, 1989; Quick et al. 1991; Webster, 1990; Kindler 

et al. 1992; Robinson, 1993; Webster et al. 1993; Smith et al. 1992; Rafi et al. 1996 and 

Kazemi et al. 2001a, Kazemi et al. 2001b; Kazemi et al. 2007). Based on the observations 

made during these investigations, the highest level of aphid infestation has been observed in 

wheat fields of the Tabriz, Ahar and Kaleybar areas of East Azarbaijan province of Iran 

(Kazemi et al. 2001b; Kazemi et al. 2007). Thus, the present study was aimed at evaluating the 

existance of any resistance at the stem elongation growth stage of Alvand, Alamoot, Zarrin, 

Sabalan and Sardari wheat varieties (which had already shown some resistant and susceptible 

patterns to the aphid); these varieties being the most widely planted in the province. 


Plant and aphid culture 

The degrees of resistance of five wheat varieties (Alvand, Alamoot, Zarrin, Sabalan and 

Sardari) to the Russian Wheat Aphid, Diuraphis noxia, were evaluated at their stem elongation 

growth stage (GS 30-32). The seeds of the Sardari variety were obtained from the Institute for 

Dry Farming Studies and those of the remaining varieties from the Agricultural Organization 

of East Azarbaijan province. The aphid clones were collected from the Kaleybar wheat fields 

and transferred to the laboratory for morphological identification according to the relevant 

156

sources (Blackman & Eastop, 1984; Stoetzel, 1987). Stock cultures of aphids were reared 

under glasshouse conditions on Durum plants which are highly susceptible to the aphid 

(Formusoh et al. 1992) and kept in a germinator under 19-24 o C and 14: 10 (L: D) light regim. 

The seeds of each variety were sown in 200 m 2 plots at the Khosrov-shahr Agricultural 

Research Station wheat fields at the sowing rate of 180 Kg/ha. 

Plant infestation 

Aphids reared on the stock culture were individually confined in large clip cages on the upper 

leaves of experimental plants (Kazemi, 1988). Since the culture plant may influence the 

performance and preferences of the aphids, the aphids were reared on the experimental plants 

for at least one generation before the main experiments. For the main experiments, one adult 

apterous aphid from the appropriate culture was confined in a clip cage on the upper leaf of the 

experimental plant. After 24 hours the adult was removed, and one newly born nymph was 

allowed to develop to an adult and reproduce (Kazemi & van Emden, 1992). The position of 

the cages was changed once every three to four days to avoid local leaf damage. The 

experimental design was a completely randomized block design with five treatments (varieties) 

and each variety with 15 replicates using individual clip-on leaf cages as experimental units, 

set up on the last fully grown leaves of the main plants when the first node of the plant stem 

was visible from the beginning of May. In order to determine the maturation time and survival 

rate of encaged progeny, each individual nymph was allowed to develop into an adult. The 

fecundity of the resultant adults was determined by daily counts of their progeny between 9 

and 11 a.m. for periods of 10 and 15 days. All the progeny were removed from caged leaves 

after completion of the counts. To calculate the daily intrinsic rate of natural increase (rm 

value), nymphal survival on each variety (age specific survival rate: lx), developmental time 

and daily fecundity of individual aphids (age specific fecundity: mx) were used in the equation 

Σe -r m lxmx=1 (Birch, 1948), using van Emden’s STATSPAK version 8.00 based on Mallard 

Basic. Percentage of nymphal survival rate divided by the mean nymphal developmental time 

was used to calculate the Growth Index (GI) (Smith et al. 1994). 


Maturation time and survival rate of nymphs 

The data obtained during the developmental period indicated that there were significantal 

differences between treatment means. Comparisons made between treatment means using 

Duncan’s multiple range test showed significant differences (P ≤ 5%). The data presented in 

Table 1 show that the highest and lowest development time occurred on the Alvand and 

Sabalan varieties respectively. Also the highest and lowest nymphal survival rate was seen on 

Sabalan and Alvand varieties respectively. Combination of these two parameters, namely 

Growth Index (GI), demonstrates differences between the varieties, and due to a low GI on 

Alvand compared to the other varieties, Alvand is considered a resistant variety and Sabalan is 

157

susceptible one. The effect of aphid feeding on the resistant varieties leads to an increase in the 

nymphal maturation time and decrease in survival rate of the insects. 

Fecundity 

158 

Table1. Mean maturation time and survival rate of Russian Wheat Aphid nymphs f 

five wheat varieties under field conditions. 

Variety 

Mean maturation time 

(day) ( X ± SD) 

Survival rate (%) 

Growth 

Index 

Alamoot 13.13 ± 0.64 bc * 

70.37 5.36 

Alvand 13.73 ± 0.59 a 66.67 4.86 

Zarrin 13.47 ± 0.64 ab 

70.37 5.23 

Sabalan 12.67 ± 0.82 d 77.78 6.14 

Sardari 12.93 ± 0.70 cd 

74.07 5.73 

* Means followed by a similar letter are not significantly different at a level of 5% 

Comparisons made on mean fecundity (Table2) indicated significant differences 

(P ≤ 5%) in the mean fecundity of the aphid on five wheat varieties within the two 10 and 15 

day periods. 

Table2. Mean fecundity of adult apterae of Russian Wheat Aphid within 10 and 15 day 

periods of rearing on five wheat varieties. 

Variety 

10 day 

( X ± SD) 

15 day 

( X ± SD) 

Alamoot 23.67 ± 6.18 b * 

31.33 ± 9.61 ab 

Alvand 21.33 ± 6.00 c 30.20 ± 8.91 bc 

Zarrin 20.07 ± 5.50 c 28.73 ± 8.36 bc 

Sabalan 26.20 ± 6.95 a 33.33 ± 11.17 a 

Sardari 20.60 ± 5.94 c 28.60 ± 8.41 c 

* Means followed by a similar letter in each column are not significantly different at a 5% level 

The highest mean fecundity within the first 10 day periods of larviposition was recorded on 

Sabalan and the least progeny produced within the first 10 days of larviposition was observed 

on Zarrin, Sardari and Alvand.The trend of fecundity within the 15 day period of larviposition 

was more or less the same as within the first 10 days of reproduction.Trends in the aphid’s 

larviposition on five wheat varieties within 10 and 15 day periods have been shown as daily 

cumulative means in Figure 1. It is obvious that, from the beginning of the reproductive period, 

the rate of larviposition remained more or less the same on all varieties. However, there were 

remarkable deviations in fecundity on the Sabalan, Zarrin and Alamoot varieties which

continued until the end of the 15-day period, whilst changes in the larviposition rate on three 

other varieties (Alvand, Zarrin and Sardari) followed the same pattern. 

Accumulated maen nymphs No. 

40 

35 

30 

25 

20 

15 

10 

5 

0 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

Larviposition period (days) 

Alamoot 

Figure1. Daily cumulative means of larviposition within 10 and 15 day periods on five 

wheat varieties at stem elongation. 

However, at the end of the larviposition periods, the highest mean fecundity was observed on 

Sabalan and the lowest mean fecundity on Zarrin, Sardari and Alvand. The results of 

larviposition work indicate Sabalan suitability for aphid feeding due to its higher susceptibility 

to the aphid, and Sardari, because of the low larviposition of the aphid on it, to be a resistant 

wheat variety. The other varieties, especially at the end of 15 day periods of larviposition, 

showed no significant differences between them and were placed in one group. Kazemi et al. 

(2001b) studying the susceptibility of D. noxia at stem elongation stage under laboratory 

conditions on the same wheat varieties, had noted certain differences and same larviposition 

trends on the varieties. 

The intrinsic rate of natural population increase (rm value) 

Data indicated significant differences between rm values at P ≤ 5%. Based on the aphids’ 

intrinsic rate of increase within 10- and 15- day periods of rearing on test varieties, Sabalan had 

the highest rm value for both rearing periods and is therefore regarded as the most susceptible 

variety. Alvand and Zarrin had the lowest rm values and are considered to be resistant varieties. 

Sardari and Alamoot seem to be partially resistant (Table 3). 

Alvand 

Zarrin 

Sabalan 

Sardari 

159

160 

Table 3. Intrinsic rate of increase (rm values) of the Russian Wheat Aphid in rearing on 

five wheat varieties for 10 and 15 day periods under field conditions. 

Variety 

10- day period 

( X ± SD) 

15- day period 

( X ± SD) 

Alamoot 0.1536 ± 0.014 b * 

0.1586 ± 0.014 b 

Alvand 0.1377 ± 0.014 d 

0.1444 ± 0.014 c 

Zarrin 0.1407 ± 0.014 d 

0.1478 ± 0.014 c 

Sabalan 0.1712 ± 0.015 a 

0.1747 ± 0.015 a 

Sardari 0.1485 ± 0.015 c 0.1556 ± 0.013 b 

* The means followed by similar letter in each column are not significantly different at a 5% level. 

CONCLUSION 

The results and statistical analyses indicate that, at the stem elongation stage under field 

conditions, Sabalan appeared to be the variety most susceptible to the Russian wheat aphid, 

having the highest aphid fecundity and rm value. Alvand and Zarrin varieties appeared to be 

more resistant, having both the lowest aphid fecundity and rm values. The varieties, Alamoot 

and Sardari seem to be partially resistant. With the extension of the studies to the other 

phenological stages of the test varieties and using previously reported results, (Kazemi et al. 

2001a, Kazemi et al. 2001b; Kazemi et al. 2007) it is hoped that a probable "antibiosis" 

program would be a valuable tool towards lowering the damage potential of this aphid. 

REFERENCES 

Archer T L; Bynum JR E D (1992). Economic injury level for the Russian Wheat Aphid 

(Homoptera: Aphididae) on dryland winter wheat. Journal of Economic Entomology 

85(3), 987-992. 

Birch L C (1948). The intrinsic rate of natural increase of an insect population. Journal of 

Animal Ecology 17, 15-26. 

Blackman R L; Eastop V F (1984). Aphids on the world's crops an identification and 

information guide. John Wiley & Sons: Avon. 

Burd J D; Burton R L; Webster J A (1993). Evaluation of Russian Wheat Aphid (Homoptera: 

Aphididae) damage of resistant and susceptible hosts with comparisons of damage 

ratings to quantitative plant management. Journal of Economic Entomology 86(3), 974- 

980. 

Damsteegt V D; Gildow F E; Hewings A D; Caroll T W (1992). A clone of the Russian Wheat 

Aphid (Diuraphis noxia) a vector of the Barley Yellow Dwarf, Barley Stripe Mosaic 

and Brome Mosaic Viruses. Plant Diseases 76(11), 1155-1160. 

Du Toit F (1989). Components of resistance in three bread wheat lines to Russian Wheat Aphid 

(Homoptera: Aphididae) in South Africa. Journal of Economic Entomology 82(6), 

1779-1781.

Formusoh E S; Wilde G E; Hatchett J H; Collins R D (1992). Resistance to Russian Wheat 

Aphid(Homoptera: Aphididae) in Tunisian wheats. Journal of Economic Entomology 

85(6), 2505- 2509. 

Jones J W; Byers J R; Butts R A; Harris J L (1989). A new pest in Canada: Russian Wheat 

Aphid Diuraphis noxia (Mordvilko) (Homoptera: Aphididae). Canadian Entomology 

121(7), 623-624. 

Kazemi M H (1988). Identification and mechanisms of host plant resistance to cereal aphids in 

wheat. PhD Thesis, University of Reading: U.K. 

Kazemi M H; van Emden H F (1992). Partial antibiosis to Rhopalosiphum padi in wheat and 

some phytochemical correlations. Annals of Applied Biology 121, 1-9. 

Kazemi, M H; Talebi-Chaichi P; Shakiba M R; Mashhadi Jafarloo M (2001a). Biological 

responses of Russian Wheat Aphid, Diuraphis noxia (Mordvilko) (Homoptera: 

Aphididae) to different wheat varieties. Journal of Agricuitural Science and Technology 

3(4), 249-255. 

Kazemi, M H;Talebi-Chaichi P; Shakiba M R; Mashhadi Jafarloo M (2001b). Susceptibility of 

some wheat cultivars at stem elongation stage to the Russian Wheat Aphid, Diuraphis 

noxia (Mordvilko) (Homoptera: Aphididae) Journal of Agricultural Science 11(2), 103- 

111. 

Kazemi M H; Mashhadi Jafarloo M; Talebi-Chaichi P; Shakiba M R (2007). Biological 

responses of Russian Wheat Aphid, Diuraphis noxia (Mordvilko) to certain wheat 

cultivars at ear emergence stage. Journal of Agricultural Science-Islamic Azad 

University 12(4), 745- 753. 

Kindler S D; Hammon R W (1996). Comporisons of host suitibility of western wheat aphid 

with the Russian wheat aphid. Journal of Economic Entomology 89(6), 1621-1630. 

Kindler S D; Springer T L (1989). Alternate hosts of Russian Wheat Aphid. Journal of 

Economic Entomology 82(5), 1358-1362. 

Kindler S D; Greer L G; Springer T L (1992). Feeding behavior of Russian Wheat Aphid 

(Homoptera: Aphididae) on wheat and resistant and susceptible slender wheatgrass. 

Journal of Economic Entomology 85(5), 2012-2016. 

Quick J S; Nkongolo K K; Meyer W; Peairs F B; Weaver B (1991). Russian Wheat Aphid 

reaction and agronomic and quality traits of a resistant wheat. Crop Science 31(1), 50- 

53. 

Rafi M M; Zemerta R S; Quinesberry S S (1996). Interaction between Russian Wheat Aphid 

(Homoptera: Aphidida) and resistance and suceptible wheat genotypes. Journal of 


Robinson J (1993). Conditioning host plant affects antixenosis and antibiosis to Russian Wheat 

Aphid (Homoptera: Aphididae). Journal of Economic Entomology 86(2), 602-606. 

Smith C M; Schotzko D J; Zemetra R S; Souza E J (1992). Categories of resistance in plant 

introduction of wheat resistant to the Russian Wheat Aphid (Homoptera: Aphididae). 

Journal of Economic Entomology 85(4), 1480-1484. 

Smith C M; Khan Z R; Pathak M D (1994). Techniques for evaluating insect resistance in crop 

plants. CRC Press: Florida. 

Souza E; Smith C M; Schotzko D J; Zemetra R S (1991). Greenhouse evaluation of red winter 

wheats for resistance to the Russian Wheat Aphid (Diuraphis noxia Mordvilko). 

Euphytica 57(3), 221-225. 

161

Stoetzel M B (1987). Identification of Diuraphis noxia (Homoptera: Aphididae) and other 

Aphid species colonizing leaves of wheat and barley in the United States. Journal of 

Economic Entomology 80,696-704. 

Webster J A (1990). Resistance in Triticale to the Russian Wheat Aphid (Homoptera: 

Aphididae). Journal of Economic Entomology 83(3), 1091-1095. 

Webster J A; Porter D R; Baker C A; Mornhinweg D W (1993). Resistance to Russian Wheat 

Aphid (Homoptera: Aphididae) in barley: Effects on aphid feeding. Journal of 


Zadoks J C; Chang T T; Konzak C F (1974). A decimal code for the growth of cereal. Weed 

Research 14, 415-421. 

162

Zeinalzadeh Tabrizi H, Ghaffari M: Production of Sunflower Hybrids Based on New Cytoplasmic Male Sterility 

Sources. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 163; 


3-24 Production of Sunflower Hybrids Based on New Cytoplasmic Male 

Sterility Sources 

Zeinalzadeh Tabrizi H 1 , Ghaffari M 2 

1 Young Researchers Club, Islamic Azad University, Tabriz Branch, Tabriz, Iran 

2 Agricultural and Natural Resources Research Center, West Azerbaijan, Iran 

Email: hosseinzt@hotmail.com 

Abstract 

Since 1969, production of commercial sunflower hybrids has been based on a single 

cytoplasmic male sterility (CMS) source, PET1, discovered by Leclerq. Nowadays, 

development of new CMS sources of male sterility as well as fertility restorer 

systems are special interests of sunflower breeders for increasing genetic diversity 

and reducing the potential risk of vulnerability to different pathogens. In Recent 

years, more than 60 CMS sources reported in Helianthus germplasm, but instability 

and lack of appropriate maintainer and restorer lines have limited their use in 

hybridization programs. New CMS source, ANN5, seems to be different from 

Leclerq source and because of environmental stability, it can be used as a tester for 

discovering new genes for fertility and breeding sunflower hybrids in Iran. 

163

Heidari M, Ghanbari A: Relationship between Antioxidant activity and biochemical components of wheat and 

sorghum genotypes under salinity stress. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and 



3-25 Relationship between Antioxidant activity and biochemical components 

of wheat and sorghum genotypes under salinity stress 

Heidari M, Ghanbari A 

Department of Agronomy and Plant Breeding, Faculty of Agronomy, University of Zabol, 

Zabol, Iran 

Email: Haydari2005@yahoo.com 

164 

Abstract 

Seedling of two sorghum genotypes (Payam and Sistan) and four wheat genotypes 

(Bolani, Hirman, Star and Toss), were grown in Hogland nutrient solution 

containing 0, 100 and 200 mM NaCl in controlled environment. Antioxidant 

activity of catalase (CAT), ascorate perxidase (APX), guaiacol peroxidase (GPX) 

and osmolyte concentration, proline and carbohydrates, determined in the leaves 20 

days after salinity induced. Results showed that the activity of APX, GPX and CAT 

increased in both sorghum genotypes. Wheat genotypes showed significant 

differences during the experimental period. By increasing salinity levels from 0 to 

200 mM NaCl, the activity of APX and GPX decreased but among the antioxidative 

enzymes, the activity of CAT increased. At the 100 mM NaCl, the CAT activity in 

wheat genotypes were higher compared with that in 200 mM NaCl. Among the 

wheat genotypes, Toss and Hirman had the highest CAT activity. Total solube 

carbohydrates and proline increased in all wheat and sorghum genotypes with the 

increasing of salinity stress.

Ohadi S, Rahimian Mashhadi H, Tavakol Afshari R: Different response of intact siliques and naked seeds of 

turnipweed (Rapistrum rugosum) to light. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic 



3-26 Different response of intact siliques and naked seeds of turnipweed 

(Rapistrum rugosum) to light 

Ohadi S, Rahimian Mashhadi H, Tavakol Afshari R 

University of Tehran 

Email: sara_ohadi@hotmail.com 

Abstract 

Turnipweed is a problematic weed in different regions in Iran, especially in winter 

crops such as wheat. Experiment was conducted to investigate the effect of light, 

storage condition, duration of storing or burial and seed type (naked or intact 

silique) on germination and viability of turnipweed (Rapistrum rugosum). The seeds 

(siliques and naked) were kept under five different storage conditions including 

both indoor (room temperature (25±2 oC) and cold i.e. refrigerator (3±1oC)) and 

outdoor environments (soil depths of 10, 20 and 40 cm). All seeds were retrieved 

approximately every two months and tested for germination in light and darkness. 

At each time of exhumation, nongerminated seeds were treated with 

triphenyltetrazolium chloride to test their viability. The germination of seeds 

liberated from siliques (85%) was markedly greater than those in intact siliques 

(20%). The germination response of naked seeds and siliques to light varied 

between storage conditions and through time. Under indoor conditions (room and 

cold), both seed types had greater germination percentages in dark in most 

occasions. On the contrary, the germination of siliques buried at soil depths of 20 or 

40 cm was considerably simulated by light. Under indoor conditions, the percent 

viability of both seed types was only declined marginally, while those buried in soil 

showed high rate of mortality. Seeds in intact siliques persisted longer under either 

of indoor or outdoor conditions. The information on germination and viability of 

turnipweed seeds could be helpful in developing appropriate management strategies 

for the species. 

165

Mari J M: Seasonal abundance of Alfalfa Aphid (Therioaphis trifolli Monell) in Berseem field. In: Feldmann F, 

Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 166; ISBN 978-3-941261-05-1; 

© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

3-27 Seasonal Abundance of Alfalfa Aphid (Therioaphis trifolli Monell) in 

Berseem Field 

Mari J M 

Department of Entomology Sindh Agriculture University Tandojam Pakistan 

Email: janmarree@gmail.com 

166 

Abstract 

The study on population of alfalfa aphid on berseem crop assessed through two 

sampling methods, in situ plant count and yellow sticky trap from 15th December to 

15th March of both years (2005-2006 and 2006-2007). The two-year data in situ 

plant count method depicted that population was minimum (7.22 and 5.49) per 10 

tillers during 2nd week of December. The aphid population of each year increased 

gradually from December and reached to its peak (31.20 and 28.75) in 2nd week of 

February. Both regression equations showed that in the initial nine week the 

population growth was highly significant. There was a positive relationship between 

population growth and temperature which ranged (12 to 18°C) was linearly related 

at 17.75 to 722.02DD. It was highly significant with a slope of line 0.002DD and r 

=0.97. The aphid population started decreasing in March with a declining curve - 

0.208X r = 0.97. Aphid population decreased when temperature reached upper 

threshold limit 23 to 35°C. The regression analysis depicted that there was a 

significant negative correlation between cumulative degree-days (683 and 

1325.13DD) and aphid population with a slope of line- 0.002DD and r = 0.97.

Kumar S, Kaushik N, Proksch P: Seasonal abundance of Alfalfa Aphid (Therioaphis trifolli Monell) in Berseem 

field. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 167-174; 


3-28 Antifungal activity of endophytic fungi isolated from Tylophora indica 

in India 

Kumar S 1 , Kaushik N 2 , Proksch P 3 

1 

TERI University, Habitat center, Lodhi road, New Delhi-110003, India 

2 

The Energy and Resources Institute (TERI), Habitat center, Lodhi road, New Delhi-110003, 

India 

3 

Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität 

Düsseldorf-40225, Germany 

Email: kaushikn@teri.res.in 

Abstract 

Endophytic fungi were isolated from the medicinal plant Tylophora indica. 

Isolation was performed from disease- and pest-free, fresh plant material by surface 

sterilising with 70% ethanol for 2 min and then taking an extract of the sterilised 

plant part, chopping it and transferring it to a malt extract agar medium. Six 

endophytic fungi were isolated and identified as Chaetomium globosum, 

Cladosporium herbarum, Cladosporium cladosporides, Rhizocotonia sp and two 

isolates of Rhizoctonia solani. A seventh endophyte was isolated and tested but was 

not identified. All of them were tested by dual culture against Sclerotinia 

sclerotiorum, causal agent of root and stem rot of chickpea. Chaetomium globosum, 

Cladosporium cladosporides, and Cladosporium herbarum were found active in 

dual culture test and were mass multiplied and extracted with solvent and extracts 

were tested against Sclerotinia sclerotiorum. Methanol extract of Chaetomium 

globosum was most effective having 66.5% growth inhibition (GI) at 500 ppm 

followed by Cladosporium cladosporides showing 52.2% GI at 500 ppm. 

INTRODUCTION 

The natural and biological control of pests and diseases affecting cultivated plants has gained 

much attention in the past decades as a way of reducing the use of chemical products in 

agriculture. Endophytes are the micro-organisms that colonize interior of the plant parts, 

without causing any negative effect to the host (Arnold et al. 2003), rather helping the plant by 

167

imparting host plant resistance against biotic (Breen 1994; Schulz et al. 1999; Dingle & 

McGee 2003) and abiotic stresses (Siegel et al. 1990; West 1994). Every plant species 

examined to date harbours one or more endophytes (Strobel 2006). Endophytic fungi have been 

known to have wide range of activities against plant pathogens and phytophagous insects. Biomolecules 

of pharmaceutical and agricultural importance have been produced by most of the 

genera of endophytic fungi. Muscador albus, an endophytic fungus of rainforest plants, is 

known to produce volatile organic compound responsible for fumigant activity against plant 

pests (Strobel et al. 2001; Strobel 2006). Several antimicrobial metabolites such as colletotric 

acid (Zou et al. 2000), griseofulvin (Park et al. 2005) are reported from endophytic fungi. 

Metabolites of endophytic fungi responsible for pesticidal activity have been reviewed by 

Kumar et al. (2008). Nematicidal activity is also reported from the culture filtrate of Fusarium 

oxysporum, an endophytic fungus of tomato (Hallmann & Sikora 1996). The natural and 

biological control of pest and diseases affecting cultivated plants has gained much attention in 

the past decades as a way of reducing the use of chemical products in agriculture. Endophytic 

fungi are isolated from healthy plants and biopesticides developed from them have the 

potential to be environmentally safe and ecologically sound. 

Indiscriminate, non-judicious and unsafe use of chemical pesticides pose threats to human 

health and environment and thereby to biodiversity. Biological control of the crop pest is a 

good alternative to chemical, if they are not known to pathogenic against plant and animals, as 

endophytes are normally. Many of the bioactive metabolites reported from endophytic fungi 

act as plant defence activator and proved to be useful for novel drug discovery (Owen & 

Hundley 2004). Looking to the scope of endophytic fungi, present work was undertaken to 

explore the endophytic fungi of T. indica to find if they can be used as an alternative to 

chemical pesticide. T. indica, a medicinal plant of Asian origin, is known to host several 

endophytes having insecticidal and medicinal properties. The host plant is not known to be 

attacked by many plant pathogens and pests, so the endophytic micro-biota of the plant may be 

of use in protecting the plant.. 


Sample collection 

Leaf and stem samples of T. indica (Family: Ascalpediaceae) were collected from pot-grown 

plants at TERI, New Delhi. Immediately after the collection, plant parts were washed with tap 

water and processed for isolation of endophytic fungi. 

Media 

Malt extract agar medium [Malt extract (15 g/l); Agar (15 g/l), pH: 7.4-7.8] was used for 

isolation and purification of endophytic fungi. Antibiotic, Chloramphenicol @ 0.2 g/l of 

medium was used for isolation to avoid bacterial contamination. Wicherham medium [Malt 

extract (3g/l); Yeast extract (3 g/l); Peptone (5 g/l); Glucose (Qualigens)-10 g/l; pH-7.2-7.4] 

168

was used for small-scale multiplication of endophytic fungi being taken for extraction of 

metabolites. Potato dextrose agar (PDA) was used for dual culture bioassay. All the media 

chemicals and antibiotics were purchased from Himedia, India. 

Isolation of endophytic fungi 

Endophytic fungi were isolated from healthy plants of T. indica. The plant parts were surface 

sterilised with 70% ethanol for 2 min followed by 1% sodium hypochlorite for 3 min. Surface 

sterilized plant parts were dried on sterile blotting sheets and then chopped and transferred to 

malt agar plates, after taking an imprint of dried sterile plant part as suggested by Wang et al. 

2006. Plates were incubated at 24 o C for 3-7 days. Hyphal tips of the developing fungal 

colonies were transferred to fresh malt agar plates. After purifying by repeated sub-culturing, 

isolates were identified, dual-culture bioassayed followed by small-scale multiplication and 

extraction. 

Identification of endophytic fungi 

Identification was done by observing the microscopic slides of endophytic fungi prepared by 

mounting them on polyvinyl lacto-glycerol. Prof. K G Mukherji identified the endophytic 

fungi. 

Dual culture bioassay of endophytic fungi 

Dual culture bioassay was done against the plant pathogenic fungi, Rhizoctonia solani, 

Sclerotinia sclerotiorum and Fusarium oxysporum. The cultures were obtained from Indian 

Type Culture collection, Indian Agricultural Institute, New Delhi. Plant pathogenic fungi and 

endophytic fungi were inoculated on PDA plate at periphery, opposite to each other. After 

incubation at 24 o C for 3-7 days plates were observed and antagonism was expressed by 

presence of inhibition zones at the point of interaction. 

Extraction of fungal broth with organic solvent 

Endophytic fungi showing antagonistic activity against plant pathogenic fungi were inoculated 

in wickerham medium (300 ml in 1 litre conical flask) and incubated at 24 o C for 3-4 weeks. 

After attaining full growth each culture flasks were immersed in 250 ml of Ethyl acetate 

(Rankem, India) for 24 hrs, ground with a hand blender (INALSA Tech., India) and then 

filtered (Wicklow et al, 1998). Filtrate was extracted thrice with ethyl acetate, which was 

followed by Butanol (Qualigens, India) and then dried with vacuum rotary evaporator 

(Heidolph Inc, Germany). Ethyl acetate fraction was further partitioned between 90% methanol 

(Qualigens, India) and n-Hexane (Qualigens, India). 

169

Activity detection of extracts 

Extracts were tested against Sclerotinia sclerotiorum only. Thirty mg of dried extract was 

dissolved in 800 µl of methanol. From this solution 200 and 400 µl were mixed in 30 ml PDA 

media for 250 and 500 ppm concentrations respectively. Intoxicated media (30 ml) was poured 

to 3 plates, and upon solidification of the media, the plant pathogenic fungus S. sclerotiorum 

was inoculated at the centre of the plate and radial growth was measured until the check plate 

attained the full growth. To check if the growth inhibition is due to methanol 400 µl of it was 

added to 30 ml media and poured to three plates. Percent growth inhibition of the extract was 

calculated with respect the growth in methanol-containing plates. 

RESULTS AND DISCUSSIONS 

Isolation of endophytic fungi 

Isolation of endophytic fungi was performed during October 2006 to June 2007 at six different 

times. Seven endophytic fungi were isolated from 192 tissue segments (68 from stem and 124 

from leaf) of T. indica, pure cultures of which were identified by microscopic examination as 

Chaetomium globosum, Cladosporium cladosporoides, Cladosporium herbarum, Rhizoctonia 

solani-I, Rhizoctonia solani-II Rhizoctonia sp.. C. herbarum and R. solani-I were isolated from 

leaves and C. globosum, C. cladosporoides, R. solani-II and Rhizoctonia sp. were isolated from 

stems. 

C. globosum has been reported as being endophytic from several host plants including 

Canvalia maritime (Seena & Sridhar 2004), Ipopmea pes-caprae, Launea sarmentosa and 

Polycarpaea corymbosa (Beena et al. 2000), and some medicinal plants namely Terminalia 

arjuna, Crataeva magna, Azadirachta indica, Holarrhena antidysentrica (Tejesvi et al. 2006) 

Bioassay of endophytic fungi against plant pathogenic fungi 

In dual culture test C. globosum, C. cladosporoides, C. herbarum and Rhizoctonia solani-I 

were found active against theplant pathogenic fungi S. sclerotiorum (Figure 1), while C. 

herbarum and Rhizoctonia sp. were effective against F. oxysporum (Figure 2). 

Bioassay of culture extracts of endophytic fungi against Sclerotinia sclerotiorum 

Methanol and butanol extracts of the endophytic fungi C. globosum and C. cladosporoides 

were tested at 250 ppm and 500 ppm against S. sclerotiorum. Methanol extract of C. globosum 

gave 66.5% mycelial growth inhibition (GI) at 500 ppm and a similar effect was observed in 

butanol extract resulting in 63.9% GI at 500 ppm. The methanol extract of C. cladosporides 

was less effective (52.2% GI at 500 ppm) than the butanol extract of it (63.3% GI at 500 ppm) 

and also less effective than either of the methanol or the butanol extract of C. globosum 

(Figure 3). 

170

Sl. 

No. 

Table 1: Activity of endophytic fungi against plant pathogenic fungi tested in dual 

culture bioassay 

Endophytic fungi 

Activity against plant pathogenic fungi 

Rhizoctonia 

solani 

Fusarium 

oxysporum 

Sclerotinia 

sclerotiorum 

1 Chaetomium globosum - - + 

2 Cladosporium cladosporoides - - + 

3 Cladosporium herbarum - + + 

4 Rhizoctonia solani-I - - + 

5 Rhizoctonia solani-II - - - 

6 Rhizoctonia sp., - + - 

7 Unidentified - - - 

Table 2: Activity of extracts of endophytic fungi against plant pathogenic fungi tested 

poisoned food technique 

Sl Endophytic fungi 

% Growth Inhibition 

no. 

Methanol extract Butanol extract 

250 ppm 500 ppm 250 ppm 500 ppm 

1 Chaetomium globosum 3.8 66.5 0 63.9 

2 Cladosporium cladosporoides 0 52.2 0 63.3 

(a) (b) 

(c) (d) 

Figure 1: Dual culture bioassay of (a) Cladosporium herbarum (b) Rhizoctonia solani-I 

(c) Cladosporium cladosporoides (d) Chaetomium globosum against 

Sclerotinia sclerotiorum 

171

172 

(a) (b) 

Figure 2: Dual culture bioassay of (a) Cladosporium herbarum (b) Rhizoctonia sp. 

against Fusarium oxysporum 

(a) (b) 

(c) (d) 

Figure 3: Bioassay of 500 ppm of (a) methanol (c) butanol extract of Cladosporium 

cladosporoides (b) methanol (d) butanol extract of Chaetomium globosum 

against Sclerotinia sclerotiorum 

The present work provides evidence that endophytic fungi per se and their culture filtrate can 

be utilised for successful management of Sclerotinia stem and root rot of chickpea. The role of 

C. globosum in biological control has been well documented and commercial formulations 

have also been developed (Soytong et al. 2001). Culture filtrate of C. globosum has 

successfully inhibited the mycelial growth of Pythium ultimum in in vitro and pot culture 

experiments (Di Pietro et al. 1992). Cell wall degradation activity is one of the possible modes 

of action of C. globosum against P. ultimum (Inglis & Kawchuk 2002). Hexane extract of C. 

globosum has been reported as showing antifungal properties against S. sclerotiorum and 

Botrytis cineria (Nakashima et al. 1991)

REFERENCES: 

Arnold A E; Mejia L C; Kyllo D; Rojas E I; Maynard Z; Robbins N; Herre E A (2003). Fungal 

endophyte limit pathogen damage in a tropical tree. Proceedings of national academy of 

science of USA 100:15649-15654. 

Beena K R; Ananda K; Sridhar K R (2000). Fungal endophytes of three sand dune plant 

species of west coast of India. Sydowia, 52: 1–9. 

Breen J P (1994). Acremonium endophyte interactions with enhanced plant resistance to 

insects. Ann Rev Entomol 39:401-423. 

Di-Pietro A; Gut-Rella M; Pachlatka J P; Schwinn F J (1992). Role of antibiotics produced by 

Chaetomium globosum in biocontrol of Pythium ultimum, a causal agent of damping 

off. Phytopathology 82: 131-135. 

Dingle J; McGee P A (2003). Some endophytic fungi reduce the density of pustules of 

Puccinia recondita f. sp. tritici in wheat. Mycol. Res. 107:310-316. 

Hallmann J; Sikora R A (1996). Toxicity of fungal endophyte secondary metabolites to plant 

parasitic nematodes and soil-borne plant pathogenic fungi. European Journal of Plant 

Pathology 102:155-162. 

Inglis G D; Kawchuk L M (2002). Comparative degradation of oomycete, ascomycete, and 

basidiomycete cell walls by mycoparasitic and biocontrol fungi. Can. J. Microbiol. 48: 

60-70. 

Nakashina N; Moromizato Z; Matsuyama N (1991). The Antifungal substance produced by 

Chaetomium trilaterale var. diporum RC-5 isolated from sclerotia of Sclerotinia 

sclerotiorum. Ann. Phytopath. Soc. Japan 57: 657-662. 

Kumar S; Kaushik N; Edrada-Ebel R; Ebel R; Proksch P (2008). Endophytic fungi for pest 

and disease management, In: Integrated Management of Diseases Caused by Fungi, 

Phytoplasma and Bacteria, eds. A. Ciancio & K. G. Mukerji pp365–387, Springer. 

Owen N L; Hundley N (2004). Endophytes-the chemical synthesizers inside plants. Science 

progress 87: 79-99. 

Park J H; Choi G J; Lee H B; Kim K M; Jung H S; Lee S W; Jang K S; Cho K Y; Kim J C 

(2005). Griseofulvin from Xylaria sp. Strain F0010, and endophytic fungus of Abies 

holophylla and its antifungal activity against plant pathogenic fungi. J Microbiol 

Biotechnol 15:112-117. 

Schulz B; Rommert A K; Dammann U; Aust H J; Strack D (1999). The endophyte-host 

interaction: a balanced antagonism? Mycological Research 103:1275–1283. 

Seena S; Sridhar K R (2004). Endophytic fungal diversity of 2 sand dune wild legumes from 

the southwest coast of India. Can. J. Microbiol. 50: 1015-1021. 

Siegel M R; Latch G C M; Bush L P; Fannin N F; Rowan D D; Tapper B A; Bacon C W; 

Johnson M C (1990). Fungal endophyte-infected grasses: alkaloid accumulation and 

aphid response. J Chem Ecol 16:3301–3315. 

Soytong K; Kanokmedhakul S; Kukongviriyapa V; Isobe M (2001). Application of 

Chaetomium sp (Ketomium®) as a new broad-spectrum biological fungicide for plant 

disease control: A review article. Fungal Diversity 7:1-15. 

Strobel G (2006). Muscodor albus and its biological promise. J Ind Microbiol Biotechnol 

33:514–522. 

Strobel G A; Dirkse E; Sears J; Markworth C (2001). Volatile antimicrobials from Muscador 

albus, a novel endophytic fungus. Microbiology 147:2943-2950. 

173

Tejesvi M V; Mahesh B; Nalini M S; Prakash H S; Kini K R; Shetty H S (2006). Fungal 

endophyte assemblages from ethanopharmaceutically important medicinal trees. Can. J. 

Microbiol. 52: 427-435. 

Wang S; Li X-Ming; Teuscher F; Li D-Li; Diesel A; Ebel R; Proksch P; Wang B-Gui (2006). 

Chaetopyranin, a Benzaldehyde Derivative, and Other Related Metabolites from 

Chaetomium globosum, an Endophytic Fungus Derived from the Marine Red Alga 

Polysiphonia urceolata, Journal of Natural Products, p EST3.3 A-D. 

West C P (1994). Physiology and drought tolerance of endophyte- infected grasses. In: 

Biotechnology of endophytic fungi of grasses, eds Bacon C W and White J F, pp 87–99, 

CRC Press, Boca Raton, FL. 

Wicklow D T; Joshi B K; Gamble W R; Gloer J B; Dowd P F (1998). Antifungal metabolites 

(Monorden, Monocillin IV, and Cerebrosides) from Humicola fuscoatra Traaen NRRL 

22980, a mycoparasite of Aspergillus flavus Sclerotia. Applied and Environmental 

Microbiology, p. 4482–4484. 

Zou W X; Meng J C; Lu H; Chen G X; Shi G X; Zhang T Y; Tan R X (2000). Metabolites of 

Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica. J Nat 

Prod 63:1529-30. 


Authors are thankful to Prof. K G Mukerji for helping in identification of endophytic fungi also 

to DST-DAAD for financial support. 

174

Jain A, Mohan J, Singh M: Management of disease complex caused by Meloidogyne incognita and Fusarium 

oxysporum f. sp. lycopersici using different combinations of Karanj oilseed cake and/ or VA Mycorrhiza, Glomus 

fasciculatum, on tomato. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 

Factors (2009), 175-184; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, 

Germany 

3-29 Management of disease complex caused by Meloidogyne incognita and 

Fusarium oxysporum f. sp. lycopersici using different combinations of 

Karanj oilseed cake and/ or VA Mycorrhiza, Glomus fasciculatum, on 

tomato 

Jain A, Mohan J, Singh M 

Department of Botany, Janta Vedic College, Baraut , Baghpat, Uttar Pradesh (India) 

RVRS University of Agriculture, Gwalior, Madhya Pradesh (India) 

email: anju_oswal@reddifmail.com 

Abstract 

A pot experiment was conducted to investigate the effect of Karanj oilseed cake 

(Pongamia glabra) and/ or VAM (Glomus fasciculatum) in different combinations 

on Meloidogyne incognita and/ or Fusarium oxysporum infecting tomato 

(Lycopersicon esculentum, Mill.) cv. Pusa Ruby. Both the management components 

were equally effective in reducing the disease complex caused by fungus and 

nematode. However, the plant growth appeared to be better in the plants treated 

with both Karanj cake and VAM than the ones treated with either of the 

components. The effectiveness of both the pathogens was found to be enhanced in 

respect to increase in plant growth, mycorrhizal colonization, VAM chlamydospore 

counts in soil and decrease in population of nematode and fungal colonies. The 

outstanding performance of both the components is attributed to the strong 

nematicidal value of karanj cake with VAM supplementing the fungicidal property 

of the cake. In addition its nutritional value improved the general plant vigour. 

INTRODUCTION 

The Meloidogyne incognita and Fusarium oxysporum interaction causes damage to a wide 

range of crops resulting in significant yield and economic losses worldwide (Sikora and 

Fernandez, 2005). Control of disease complex caused has been accomplished primarily through 

chemical nematicides and fungicides, crop rotation and resistant cultivars where available. 

Many nematicides have been removed from the market for toxicological reasons or are 

scheduled for phasing out. In view of the changing scenario of pest management strategies a 

175

gradual shift from chemical to non-chemical methods. Apart from the nematicidal properties, 

karanj cake is also considered to be an organic nitrogenous manure. It is widely accepted that 

VAM enhances plant minerals nutritional especially phosphorus. An attempt was made for the 

management of the disease complex on tomato through non-chemical methods viz. organic 

amendment (karanj oilseed cake) and VAM fungus (Glomus fasciculatum). 


Materials 

A pot experiment was carried out on the susceptible tomato cultivar Pusa Ruby using Karanj 

oilseed cake (Pongamia glabra) and VAM fungus (Glomus fasciculatum) in different 

combinations. For all the observation in the experiment an average of five replicates for each 

data is presented in tables. The treatments for this experiment were as follows:- 

1. Uninoculated check (C) 

2. Nematode alone (N @ 2J2 /gm soil) 

3. Fungus alone (F) @ 2gm mycelial mat/500 gm soil 

4. Karanj cake alone (K.C) @ 2% w/w. 

5. VAM alone (Glomus fasciculatum) @ 50 chlamydospores/ 500 g soil 

6. Nematode + Fungus (N + F) 

7. Nematode + VAM (N + VAM) 

8. Nematode + Karanj Cake (N + KC) 

9. Fungus + VAM (F + VAM) 

10. Fungus + Karanj Cake (F + KC) 

11. Karanj Cake + VAM (KC + VAM) 

12. Nematode + Fungus + VAM (N + F + VAM) 

13. Nematode + Fungus + Karanj Cake (N + F + KC) 

14. Nematode + Karanj Cake + VAM (N + KC + VAM) 

15. Fungus + Karanj Cake + VAM (F + KC + VAM) 

16. Nematode + Fungus + Karanj Cake + VAM (N + F + KC + VAM) 

17. Karanj Cake + Carbofuran (root dip) + Nematode + Fungus {K.C + C (root dip) + N + F} 

Methods 

Isolation and maintenance of fungal culture 

The fungus was isolated from roots and collar region of tomato plants showing wilting and 

stunting and rot browning symptoms. After sterilization for two minutes with 0.01% HgCl2. 

The fungus was identified as Fusarium oxysporum f. sp. Lycopersici, identified through Indian 

Type culture, Mycological Number 2128.95, Division of Mycology and Plant Pathology, IARI, 

176

New Delhi and maintained as pure culture on potato dextrose agar (PDA) and maintained on P 

D broth for pot experiment . For F. oxysporum inoculum, fungus was cultured on 25 ml of 

potato dextrose broth (PDB) autoclaved in 100 ml Erlenmeyer’s flask. Flasks were incubated at 

25 ± 2 0 C for two weeks. The fungal mat from each flask was collected and blended in distilled 

water for 15 seconds to obtain a concentrate suspension (Stock solution). Inoculations were 

made by pouring the required amount of fungal suspension over the exposed root system which 

was then covered by autoclaved soil. 

Isolation and maintenance of nematode culture 

Root-knot nematode (Meloidogyne incognita) was obtained from egg masses of infected roots 

of tomatoes showing galls. A pure culture of the nematode was maintained. for nematode 

inoculum, by picking up a single egg mass from perennial pattern of an adult female and 

transferred to tap water in a watch glass to allow hatching. Hatchings were collected and the 

species was identified as Meloidogyne incognita. Nematode inoculum used for this study was 

maintained by inoculating the second stage juveniles (J2) onto the roots of tomato seedlings 

grown in earthen pots containing sterilized sandy loam soil. Egg masses were collected from 

these roots and incubated at 25 ± 2 0 C for hatching. Inoculation was made by adding counted 

number of freshly hatched J2 over the surface with autoclaved soil. 

Isolation and maintenance of VAM culture 

The Vesicular Arbuscular Mycorrhiza (VAM) Glomus fasciculatum culture for inoculation was 

obtained from Division of Microbiology, IARI, was multiplied and maintained on tomato 

plants raised on 30 cm. earthern pots containing sterilised soil and sand in a proportion of 1:1. 

The plants were cut at soil level after mixing of roots with the soil. Fresh plants were 

transplanted into the pots. Mycorrhizal inoculations were made using a 2.5 g of sand: soil 

mixture containing 50 chlamydospores (obtained from the culture) and placed at about 5 cm 

below the soil surface. Chlamydospores were retrieved from the soil by wet sieving and 

decanting techniques (Gardemann and Nicholson, 1963) for counting. The number of spores in 

a millilitre of suspension was determined by taking the average of their numbers in five 

different 1 ml aliquots. 

Amendment of Soil 

Sterilized soil was amended with 2% (w/w) of finely powdered Karanj oilseed cake which was 

sieved in 1 kg capacity 20 cm diameter earthenware pots. These pots were left exposed for two 

weeks to allow decomposition of the oilcakes. The pots were regularly watered to facilitate 

decomposition process. 

177

Plant growth parameters 

These observations were recorded after 60 days of inoculation. Length and weight of root and 

shoots for fresh and dry forms were recorded. For dry weight the material was kept in a hot air 

oven at 60 0 C for 72 hours. 

Examination of roots and soil for nematode 

For populations of M. incognita in roots, the number of galls, number of eggs and number of 

egg masses per plant was encountered by dissolving the roots in 1.5% NaOCl solution for one 

minute followed by counting an average of five eggmasses picked from each root system. For 

nematode populations in soil, 500g of soil from each pot was soaked in water for about 4 

minutes in a bowl and processed by modified Cobb’s sieving and decanting technique. 

Fig. 1 Tomato roots showing variability in mycorrhizal 

colonization 

178 

Fig. 3 Tomato roots showing mycorrhizal colonization 

Figures 1-3 Mycorrhizal colonization of tomato roots 

Fig.2 Typical vesicles and arbuscules of mycorrhiza 

in tomato roots

Staining of roots and assessment of Mycorrhizal colonization 

The roots were stained in 0.05% trypan blue in lactophenol followed by transferring to a 1: 1 

mixture of lactic acid- glycerol and left for 48 hours (Phillips and Hyman, 1970). Mycorrhizal 

colonization % (MCP) was determined. For VAM chlamydospore counts, 50g of well mixed 

soil from each pot was processed as per wet sieving and decanting technique. 

Statistical Analysis of data 

Appropriate statistical procedure was adopted to interpret the data. Data of number of galls, 

eggmasses, eggs/eggmasses, nematode population, fungus and VAM were converted into 

square root transformation @ {sqrt (X+0.5)}. 


There was an improvement in plant growth characters viz. shoot length and weight (fresh and 

dry) plus fresh root weight in all the treatments receiving either Karanj cake (Kc) or VAM 

(Glomus fasciculatum G.f.), singly as well as in various combinations when compared to those 

inoculated with M. incognita and/or F. oxysporum. Maximum shoot length, weights (fresh and 

dry) and fresh root weights were recorded in the treatment with KC+VAM and minimum in 

nematode (N) and fungicide (F) (Table 1). 

The data on number of galls and egg masses in roots, eggs/egg masses as well as nematode 

populations in soil showed significant reductions in the treatments when either of the 

management component , KC or VAM were applied singly or together. (Table 2) 

The data on mycorrhizal colonization percentage (MCP), chlamydospores count and number of 

Fusarium colonies, showed highest MCP in the treatment with KC+VAM and minimum in 

N+F+VAM. It was further observed that KC+VAM and F+KC+VAM treatments were on a par 

and showed significantly higher MCP compared to other treatments (Table 3). The intensity of 

mycorrhizal colonization was also more in the treatments with both the management 

components viz. KC+VAM and F+KC+VAM indicating that the F. oxysporum f. sp. 

lycopersici played no role in affecting mycorrhizal colonization. Further, the examination of 

roots infected with both root-knot nematode and VAM revealed the VAM colonization in most 

of the roots was in the area just behind the root cap and also in the zone of elongation. 

Moreover, the galled tissues were not found to be colonized by VAM fungus. 

A similar trend as that of MCP was observed in respect of VAM chlamydospore counts in soil,. 

The results clearly established that when both Karanj cake and VAM were applied before the 

plants were infected with F. oxysporum f. sp. lycopersici, the MCP and VAM chlamydospores 

counts were unaffected. When only VAM was applied before the plants infected, either with 

M. incognita or both the pathogens, the MCP and VAM chlamydospores counts were 

significantly reduced compared with KC + VAM or Karanj cake-alone treatments indicating 

that root-knot nematode reduced the MCP as well as production of VAM chlamydospores. 

179

The number of Fusarium colonies in all the treatments with Karanj cake or VAM either singly 

or in combination were significantly reduced compared with treatments with N+F and F alone. 

The highest reduction was observed in the treatment with F+KC+VAM followed by 

N+F+KC+VAM. The number of Fusarium colonies in Karanj cake-amended soil was found to 

be reduced significantly as compared to the unamended soil (Table 3). Goswami and Meshram 

(1991) reported a near 50% reduction in penetration of M. incognita juveniles in tomato roots 

in karanj-amended soil. Plant growth also showed enhancement in amended soil when 

compared to non-amended one. 

180 

Table 1 Effect of karanj oilseed cake and VAM (Glomus fasciculatum) both alone and 

in combination on plant growth parameters of tomato infected with M. 

incognita and /or F. oxysporum f.sp. lycopersici 

Treatments 

Shoot Length Shoot weight (g) Fresh Root weight 

(cm) Fresh Dry (g) 

Uninoculated check 38.7 9.7 2.3 5.1 

Nematode alone (N) 25.1 6.4 1.7 4.2 

Fungus alone (F) 25.7 6.6 1.7 3.4 

Karanj cake alone 42.7 10.6 2.5 5.6 

VAM alone 40.9 10.1 2.4 5.4 

Nematode + Fungus 19.9 5.1 1.3 2.3 

N + VAM 30.1 7.6 1.9 4.2 

N + KC 30.7 7.8 1.9 4.2 

F+VAM 31.8 8.0 2.00 4.3 

F + KC 33.0 8.3 2.1 4.4 

KC + VAM 46.7 12.0 2.9 6.4 

N+F+VAM 26.8 6.7 1.7 3.3 

N + F + KC 28.0 6.9 1.8 3.4 

N + KC + VAM 36.1 9.0 2.1 4.7 

F + KC + VAM 37.0 9.3 2.3 4.9 

N + F+ KC + VAM 35.7 8.4 2.1 4.1 

KC + C (Root Dip) + N + F 34.8 8.0 2.0 3.7 

S. Em 1.5 0.7 0.1 0.5 

CD at 0.05 3.1 1.4 0.3 0.5 

N=Nematode; F=Fungus; Kc=Karanj cake; C= Carbofuran

The host infection by M. incognita in terms of galls and eggmasses in roots and nematode 

multiplication in terms of eggs per eggmass and soil population of nematodes were observed to 

be decreased significantly in Karanj cake-amended soil compared to those receiving N alone or 

N+F (Table 2). The possible reason for this may be attributed either to accumulation of toxic 

substances such as phenolic compounds in roots (Alam et al. 1977) thereby hindering the 

penetration of nematodes or to the release of certain fatty acids during decomposition of the 

amendment which are nematotoxic as reported by Sayre et al. (1965). (Table 3). 

Table 2 Effect of Karanj cake & VAM alone & in combination on host infection and 

nematode multiplication of M. incognita &/or F. oxysporum infected tomato 

Treatments 

No. of galls/ 

plant 

Nematode multiplication 

No. of egg 

masses/plant 

No. of eggs/ 

egg masses 

Soil 

population 

Uninoculated check 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

Nematode alone (N) 204.3 (14.3) 126.3 (11.3) 215.3 (14.7) 7844.4 (88.6) 

Fungus alone (F) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

Karanj cake alone 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

VAM alone 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

Nematode + Fungus 116.5 (10.8) 70.1 (8.4) 172.4 (13.1) 3686.3 (60.7) 

N + VAM 81.3 (9.0) 47.3 (6.9) 140.5 (11.9) 2188.4 (46.8) 

N + KC 77.9 (8.8) 47.9 (7.0) 135.7 (11.7) 2240.9 (47.3) 

F+VAM 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

F + KC 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

KC + VAM 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

N+F+VAM 90.9 (9.6) 48.1(7.0) 155.4 (12.5) 2441.7 (49.4) 

N + F + KC 85.9 (9.3) 54.5 (7.4) 150.5 (12.3) 2619.5 (51.2) 

N + KC + VAM 43.4 (6.6) 19.7 (4.5) 124.9 (11.2) 1677.5 (41.0) 

F + KC + VAM 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

N + F+ KC + VAM 45.3 (6.8) 17.5 (4.3) 121.1 (11.0) 1834.4 (42.8) 

KC + C (Root Dip) + N + F 22.5 (4.8) 13.0 (3.7) 135.0 (11.7) 1720.1 (41.5) 

S. Em 0.6 0.5 0.5 4.1 

C.D. at 0.05 1.3 1.0 1.0 8.3 

N=Nematode; F=Fungus; KC=Karanj cake; C= Carbofuran 

181

It has been widely accepted that vesicular-arbuscular mycorrhizal (VAM) fungi enhance 

mineral nutrition especially phosphorus. Their interaction with plant pathogens such as fungi 

and nematodes also reduced severity of disease (Hussey and Roncadori, 1982). Increase in 

growth of soybean plants dually inoculated with M. incognita and G. fasciculatum as compared 

to those with M. incognita alone was observed by Hussey and Roncadori (1982) and Saleh and 

Sikora (1984). 

182 

TABLE 3 Effect of Karanj cake & VAM both alone & in different combinations on 

MCP, VAM chlamydospores count and Fusarium colonies in root and 

rhizosphere of tomato infected with M .incognita and/or F. oxysporum 

Treatments MCP Percentage Chlamydospores 

count/ 50gm soil 

No. of Fusarium 

colonies/ gm soil 

Uninoculated check 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

Nematode alone 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

Fungus alone 0.0 (0.7) 0.0 (0.7) 6178.0 (78.6) 

Karanj cake alone 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

VAM alone 61.4 (7.9) 190.2 (13.8) 0.0 (0.7) 

Nematode + Fungus 0.0 (0.7) 0.0 (0.7) 6538.0 (80.9) 

N + VAM 48.2 (7.0) 155.4 (12.5) 0.00 (0.7) 

N + KC 0.0 (0.7) 0.0 (0.7) 0.0 (0.7) 

F+VAM 59.0 (7.7) 181.4 (13.5) 4038.0 (63.5) 

F + KC 0.0 (0.7) 0.0 (0.7) 4548.0 (67.4) 

KC + VAM 74.6 (8.7) 231.2 (15.2) 0.0 (0.7) 

N+F+VAM 46.8 (6.9) 150.6 (12.3) 4818.0 (69.4) 

N + F + KC 0.0 (0.7) 0.0 (0.7) 4358.0 (66.1) 

N + KC + VAM 61.2 (7.8) 188.0 (13.7) 0.0 (0.7) 

F + KC + VAM 72.2 (8.5) 224.4 (15.0) 2578.0 (50.8) 

N + F+ KC + VAM 58.6 (7.7) 173.6 (13.2) 2848.0 (53.4) 

KC + C (Root Dip)+ N + F 0.0 (0.7) 0.0 (0.7) 2468.0 (49.7) 

S. Em 0.4 0.6 2.1 

C.D. at 0.05 0.8 1.1 4.2 

N=Nematode; F=Fungus; KC=Karanj cake; MCP= Mycorrhizal colonization percentage; C= Carbofuran

We consider that the reduction in number of galls and eggmasses in roots of mycorrhizal plants 

as compared to non-mycorrhizal ones, as recorded in the present investigation, is proposed to 

be either due to non colonization of galled tissues by VAM fungus. It was clearly demonstrated 

in the present investigation that the presence of VAM fungus inhibited the formation of galls in 

roots. When roots were colonised by mycorrhiza, the number of galls were found to be less. 

Similar observations were also recorded by Mittal et al, (1991) & Sharma et al.(1994).The 

most significant observations were recorded in terms of improvement in plant vigour, MCP and 

number of chlamydospore counts along with reduction in galls and eggmasses in roots and 

pathogen populations, when both the management components i.e., Karanj cake and VAM 

fungus (Glomus fasciculatum) were applied together to the plants infected with M. incognita 

and/or F. oxysporum f. sp. lycopersici. The reason for recording improved results, in the 

treatments receiving Kc and VAM fungi, was the positive influence of organic amendments on 

the proliferation of G. fasciculatum with increased plant growth. It was also observed that 

organic amendments with a narrow C: N ratio had a greater influence on VA-mycorrhizal 

proliferation as compared to those with a wider C:N ratio. 

In the present investigation, as many VAM chlamydospores were recorded in the treatment Kc 

+ VAM +N +F as that of VAM alone. This was due to the fact that Karanj cake could mask the 

adverse effect of M. incognita on VAM spore production. Goswami et al., (2007) also 

observed a significant reduction in disease incidence and improvement in the plant health of 

pigeonpea caused by a disease complex caused by RKN, Meloidogyne incognita & root-rot 

fungus when VAM, karanj cake and FYM were applied together. 

Similar observations were also recorded by Lingaraju and Goswami (1995) in mustard cakeamended 

and R. reniformis inoculated cowpea plants. The results of the present study indicate 

the possibility of using Karanj cake to overcome the difficulty in mass multiplication of VAM 

fungi. 

ACKNOWLEDGEMENT 

Authors would like to thank Dr. P. N. Chaudhary, Division of Mycology and Plant Pathology, 

IARI, Pusa Campus, New Delhi for identification of fungus, Dr. C. S. Singh, Division of 

Microbiology, IARI, Pusa Campus, New Delhi for providing VAM culture. Also thanks to 

Khadi and village Industries Commission, Pune for providing Karanj oilseed cake. 

REFERENCES 

ALAM M M, SIDDIQUI S A, KHAN A M (1977). Mechanism of control of plant parasitic 

nematodes as a result of the application of organic amendments to the soil. III. Role of 

Phenols and amino acids in roots. Indian Journal of Nematology 7, 27-31. 

GARDEMANN J W, NICHOLSON T H (1963). Spores of mycorrhiza Endogone spp. 

extracted from soil by wet sieving and decanting. Trans. British Mycological Society 

46, 235-244. 

183

GOSWAMI B K, RAJESH KUMAR PANDEY, JAIDEEP GOSWAMI, TEWARI D D 

(2007). Management of disease complex caused by root-knot nematode and root wilt 

fungus on pigeonpea through soil organically enriched with vesicular arbuscular 

mycorrhiza, Karanj (Pongamia pinnata) oilseed cake and Farm yard manure. Journal of 

Environmental Science and Health, Part B 42, 1-6. 

GOSWAMI B K, MESHRAM N J (1991). Studies on comparative efficacy of mustard and 

karanj oil seed cakes with a nematicide carbofuran against root-knot nematode 

Meloidogyne incognita in tomato. Indian Journal of Nematology 21, 66-70. 

HUSSEY R S, RONCADORI R W (1982). Vesicular-arbuscular-mycorrhizae may limit 

nematode activity and improve plant growth. Plant Disease66, 9-14. 

LINGARAJU S, GOSWAMI B K (1995). Effect of organic amendments in the interaction of 

Glomus fasciculatum and Rotylenchus reniformis. Indian Journal of Nematology 25, 

33-37. 

MITTAL N, SHARMA M, SAXENA G, MUKHERJI K G (1991). Effect of VA-mycorrhiza 

on gall formation in tomato roots. Plant Cell Incompatible Newsletter 23, 39-43. 

PHILLIPS J M, HAYMAN D S (1970). Improved procedures for clearing and staining 

parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. 

Trans. Brit. Mycolo. 55, 158-161. 

SALEH H, SIKORA R A (1984). Relationship between Glomus fasciculatum root colonization 

of cotton and its effect on Meloidogyne incognita. Nematologica 30, 230-237. 

SAYRE R M, PATRICH Z A, THORPE H J (1965). Identification of a selective nematicidal 

component in extracts of plant residues in soil. Nematologica 11, 263-268. 

SHARMA M P, BHARGAVA S, VARMA M K, ADHOLEYA A (1994). Interaction between 

the endomycorrhizal fungus Glomus fasciculatum and the root-knot nematode, 

Meloidogyne incognita on tomato. Indian Journal of Nematology 24, 133-139. 

SIKORA R, FERNANDEZ E (2005). Nematode parasites of vegetables. In: Luc, M., Sikora, 

R.A. & Bridge, J. (Eds.). Plant parasitic nematodes in subtropical and tropical 

agriculture, 2nd Edition. Wallingford, UK, CABI Publishing pp. 319-392 

184

Selvanarayanan V: Insect Resistance in Tomato Accessions in Tamilnadu, South India. In: Feldmann F, Alford D 

V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 185; ISBN 978-3-941261-05-1; © 


3-30 Insect Resistance in Tomato Accessions in Tamilnadu, South India 

Selvanarayanan V 

Department of Entomology, Faculty of Agriculture, Annamalai University, 608 002, Tamil 

Nadu, India 

Email: selvaento@gmail.com 

Abstract 

Use of chemical insecticides for managing the major pests of tomato is discouraged 

in view of the mounting resistance in the target pests besides other environmental 

considerations. Realizing the value of host plant resistance as a viable alternative, 

an attempt was made to gather huge germplam of tomato and to identify and 

develop insect tolerant / resistant tomatoes at Annamalai University, Tamil Nadu, 

India during 1995 to 2005. An exhaustive germplasm comprising 321 tomato 

accessions including cultivars, wild lines, land races, tribal / native tomatoes was 

gathered from various sources and screened for resistance against fruit worm 

initially. In the field screening, larval population and fruit damage was evaluated 

while in the glasshouse, foliage and fruit damage was assessed and four promising 

accessions namely, Varushanadu Local, Seijima Jeisei, Ac 238 and Roma were 

selected and subjected to intercrossing by conventional hybridization, which yielded 

three viable hybrids. Subsequently, the resistance potentials of these hybrids as 

well as their parents were probed both in the field and glasshouse against fruit 

worm, H. armigera, leaf caterpillar, Spodoptera litura Fab., whitefly, Bemisia tabaci 

Genn. and serpentine leaf miner, Liriomyza trifolii. In the field screening, a wider 

variation was observed with regard to resistance against the above pests. In the 

laboratory studies, the hybrids exerted lesser feeding and ovipositional preference 

and higher antibiotic effects on insect stages. Among the biophysical factors, 

density of non-glandular and glandular trichomes on the foliage and among the 

biochemical factors, phenols and chlorogenic acid content in the foliage, lycopene 

and ascorbic acid content in the fruits had a significant role in conferring tolerance / 

resistance. 

185

Obeng-Ofori D 1 , Oduro Owusu E: Current situation of insecticide resistance of major agricultural insect pests in 

Ghana. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 186; 


3-31 Current situation of insecticide resistance of major agricultural insect 

pests in Ghana 

Obeng-Ofori D 1 , Oduro Owusu E 2 

1 

Department of Crop Science, University of Ghana, Legon, Accra, Ghana 

2 

Department of Zoology, University of Ghana, Legon, Accra, Ghana 

Email: dobeng@ug.edu.gh 

186 

Abstract 

One major constraint to the cultivation of vegetable crops in Ghana is the high 

incidence of arthropod pests and diseases. Some of the key insect pests of vegetable 

crops in Ghana are aphids, whiteflies, the Diamondback moth, mealybugs and 

thrips. For a long time, farmers had relied heavily on the use of synthetic pesticides 

to combat the numerous pests attack these crops. The indiscriminate application of 

insecticides has created several problems such as the pollution of the environment, 

toxic residues in fresh vegetables, destruction of indigenous natural enemies 

resulting in resurgence of secondary pests and the development of resistant strains 

of pests. A systematic insecticide resistance monitoring and detection programme 

using dose-response, biochemical classification, carboxylesterase, 

acetylcholinesterase and glutathione S-transferase reactions have been in progress 

since the year 2000 to assess the insecticide resistance situation in Ghana with 

respect to major vegetable pests. There is conclusive evidence that the intensive 

application of insecticides on agricultural crops has resulted in a gradual build up of 

resistance among major insects of vegetables, such as aphids, whiteflies and the 

Diamondback moth. Resistance is widespread throughout the country and is likely 

to spread to other insects including non-targeted ones. There is the urgent need for 

stringent application of pesticide registration laws (Act 528), proper regulation and 

monitoring of pesticides on the market to check influx of unauthorized pesticides 

through unapproved routes, establishment of well-resourced laboratory for pesticide 

evaluation and management and intensified sensitization of farmers and other endusers. 

National monitoring networks should be established to monitor insecticide 

resistance in major insect pests. Pesticide importers/manufacturers should recognize 

the associated risks of pesticides and contribute to research on problem-solving.

Kusi F, Obeng-Ofori D: The new sources of resistance of some cowpea genotypes to the cowpea aphid (Aphis 

craccivora Koch) in Ghana. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 


Germany 

3-32 The new sources of resistance of some cowpea genotypes to the cowpea 

aphid (Aphis craccivora Koch) in Ghana 

Kusi F 1 , Obeng-Ofori D 2 

1 

Savannah Agricultural Research Institute of CSIR, Tamale, Ghana 

2 

Department of Crop Science, University of Ghana, Legon, Accra, Ghana 

Email: dobeng@ug.edu.gh 

Abstract 

Twenty-two advanced breeding cowpea genotypes were evaluated for their 

responses to infestation of cowpea aphid, Aphis craccivora Koch, at the Savanna 

Agricultural Research Institute, Nyankpala in the Guinea savanna ecology of 

Ghana. The aim of the study was to identify cowpea genotype(s) resistant to A. 

craccivora. The genotypes consisted of 10 advanced breeding lines (F6) developed 

from Apagbaala × UCR 01-11-52 and six from UCR 01-15-127-2 × Marfo-Tuya. 

These genotypes have been selected as lines with the highest yield potential in 

northern Ghana. The adapted parents (Apagbaala and Marfo-Tuya), a local variety 

in northern Ghana (SARC-LO2), and three varieties developed by the International 

Institute of Tropical Agriculture (IITA) namely, IT97K-499-35, IT95K-193-2 and 

IT98K-506-1 were used as controls. Seedling screening technique, aphid growth 

and reproduction on each genotype and yield assessment were used to classify the 

genotypes into resistant and susceptible genotypes. The genotypes SARC 1-57-2 

and SARC 1-91-1 were found to be the most resistant genotypes; the moderately 

resistant genotypes were SARC 1-36-1, SARC 1-71-2 and SARC 3-74A-2. Five of 

the genotypes namely, Apagbaala, IT 97K-499-35, IT 98K-506-1, IT 95K-193-2 

and Marfo-Tuya were highly susceptible. The high susceptibility of the IITA lines 

must be a cause for concern, particularly the IT 97K-499-35 line which is known to 

be resistant to A. craccivora in Nigeria. This may suggest the existence of cowpea 

aphid biotype in northern Ghana which is more virulent than the biotypes in 

Nigeria. The results support earlier findings of the development of aphid biotypes 

that are more aggressive and are not controlled by the type of effective aphid 

resistance cowpea varieties developed by IITA for Nigeria. 

187

Ofuya T, Balogun A O: Evaluation of the resistance status of twenty varieties of maize to infestation and damage 

by Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). In: Feldmann F, Alford D V, Furk C: Crop Plant 

Resistance to Biotic and Abiotic Factors (2009), 188; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 


3-33 Evaluation of the resistance status of twenty varieties of maize to 

infestation and damage by Sitophilus zeamais Motschulsky (Coleoptera: 

Curculionidae) 

Ofuya T, Balogun A O 

The Federal University of Technology, P.M.B. 704, Akure, Nigeria 

Email: tomofuya@yahoo.com 

188 

Abstract 

Twenty varieties of maize, Zea mays L. were compared for susceptibility/resistance 

to infestation and damage by Sitophilus zeamais Motschulsky under ambient 

laboratory conditions in Akure, south west Nigeria. They were compared using 

adult survival, F1 adult emergence, grain holing, length and breadth of grain, seed 

weight loss, phytic acid and tannin contents as variables. Significant varietal 

differences were observed in the susceptibility of maize to infestation and damage 

by S. zeamais. TZMI 205 and TZL COMP 4C3 varieties of maize manifested the 

highest level of resistance. High post infestation adult mortality, zero F1 adult 

emergence and lack of beetle punctures on grain was observed for these two 

varieties. Length of grain ranging from 0.82 to 1.22 mm, and breadth from 0.59 to 

0.83 mm were significantly different between the maize varieties. Phytic acid and 

tannin contents ranging from 0.99 to 1.98% (CV = 20%) and 0.38 to 1.37% (CV = 

40%) respectively, also varied among the maize varieties. Phytic acid content was 

significantly positively correlated with S. zeamais F1 adult emergence which was 

significantly positively correlated with percentage weight loss in grain and number 

of grain punctures. F1 adult emergence was significantly negatively correlated with 

post infestation adult survival. There were no significant correlations between 

tannin content, length and breadth of grain with other variables.

Kabeil S S, Amer M A, Matar S M, El-Masry M H: In planta Biological Control of Potato Brown Rot Disease in 

Egypt. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 189; 


3-34 In planta Biological Control of Potato Brown Rot Disease in Egypt 

Kabeil S S 1 , Amer M A 2 , Matar S M 1 , El-Masry M H 3 

1 

Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific 

Research and Technology Applications, New Borg-Elarab, Alexandria, Egypt 

2. 

Agricultural Botany Department, Faculty of Agriculture (Saba-Basha), Alexandria 

University, P.O. Box 21531-Bolkley, Alexandria, Egypt 

3 

.Institute of Graduate Studies and Research, Alexandria University, Egypt 

Abstract 

Potato crop in Egypt occupies 20% of the total area devoted for vegetable 

plantations and any 

disturbance in its production affects severely its local and export impact. Brown rot 

disease on potato is the 

most cereous disease affecting potato exportation. Therefore, competing such 

disease is an obligate practice 

to control the pathogenic causal bacterium. Biological control became recently an 

effective strategy for fighting plant pathogens, where the antagonist have the ability 

to compete with the phytopathogens. The present study was carried out with a 

biocontrol bacterium and proved a potent antagonist against Ralstonia 

solanacearum. The in planta trials were carried out using healthy and infected 

tuber-seeds treated with the biocontrol agent Biocine S2HA either by soaking or 

powdering or both. The Biocine S2HA was produced in large-scale using controlled 

bioreactor to obtain the optimal amount and active Biocine S2HA agent. Treating 

the healthy or infected tuber-seeds prior to plantation with biocine S2HA as soaking 

or powdering increased the potato yield compared with the untreated tuber-seeds. 

However, using the treated healthy tuber-seeds was better than using the infected 

ones. In addition, the most effective practices were powdering the growing plants 

near the stem base. The effectiveness of consequence powdering treatment is due to 

the repeatable treatment of root area with the biocine S2HA carried on the talk 

powder either in the infested soil or even in the infected tuber-seeds. 

189

Kabeil S S, Amer M A, Matar S M, El-Masry M H: Production of Bio-Active protein from some soil bacteria and 

biological use in controlling Erwinia amylovora. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to 

Biotic and Abiotic Factors (2009), 190; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, 


3-35 Production of Bio-Active protein from some soil bacteria and 

biological use in controlling Erwinia amylovora 

Kabeil S S, Hafez E E, Daba A S, Botros W, El-Saadani M A 

Mubarak City for Scientific Research and Technology,Borg-Elarab,Alexandria 

Email: Sanaa5769@yahoo.com 

190 

Abstract 

Alternative forms of plant disease control methods are needed to comply with 

environmental issues confronting the use of chemical pesticides. One alternative to 

pesticides is biological control of plant diseases using microorganisms that are 

antagonistic to plant pathogens. Generally, for a particular disease, the development 

of a successful biological control agent(s) involves initial selection of a suitable bioantagonist 

(by laboratory and small-scale field testing), followed by the formulation 

of an effective strategy of application, including both timing and method of 

application with final large-scale field trial(s) to establish the biological and costeffectiveness 

of control under agricultural conditions. Microorganisms that can 

grow in the rhizosphere are ideal for use as biocontrol agents since the rhizosphere 

provides the front line defense for roots against attack by pathogens. The crops 

(Pear and Apple) are economically important to Egypt, and any disturbance in its 

production affects severely its export impact. Recently, these crops are infected 

with the (fire blight) disease producing a major problem, especially because its 

control has not established yet. 

Soil samples were collected from Monofia governorate; many bacterial were 

isolated from these samples. Three isolates were obtained and used for bioassay 

against Erwinia amylovora three isolates showed high activity against the 

pathogenic bacteria. The bacteria was cultivated on Peptone Beef Glucose ( PBG) 

medium and the culture filtrate was fractionated and the fraction were used to 

control E. amylovora One of these fractions showed high ability to control the 

pathogenic bacteria E. amylovora. The results showed that bacterial strains isolated 

from soil sample produce protein and enzymes against the bacterial Pathogen.

Yasser, M: Survival, proliferation of Trichoderma harzianum in Egyptian soil and the role of Trichoderma on the 

plant growth. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 


3-36 Survival, proliferation of Trichoderma harzianum in Egyptian soil and 

the role of Trichoderma on the plant growth 

Yasser, M 

Botany Department, Beni Suef University Egypt 

Email: manal_yaser2006@yahoo.com 

Abstract 

The present study is planned to evaluate the efficiency of potential antagonistic 

Trichoderma as biocontrol agent for biocontrol damping-off of tomato and root rot 

of kidney bean plants, by Optimization of cultural factors of Trichoderma in soil 

under laboratory conditions. Results revealed that introducing Trichoderma to the 

soil as mycelial preparations growing on rice husk, resulted in better survival and 

proliferation, than when grown on corn meal at concentration of 5 % with moisture 

content 30 % and optimum temperature 28°C. Also Studying some culture 

conditions of the bioagent on the antimicrobial activity, The incubation period of 

12 days, pH of 5.5, the optimum incubation temperature was 25°C, and 20°C and 

using chitin and sodium nitrate as a carbon and nitrogen sources gave high growth 

and the best antagonistic potential. However treatment with the bioagent only or 

treated with combination Trichoderma harzianum and Rizolex-T, resulted in an 

increase in chlorophyll content in comparison with the untreated plants 

191

Pós V, Hunyadi-Gulyás É, Manninger K, Szikriszt B, Rab E, Kabai M, Medzihradszky K, Lukács N: Wheat leafrust 

infection response – from apoplast proteomics to transcriptional aspects in near-isogenic lines of the 

’Thatcher’ cultivar. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 

(2009), 192-193; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, 

Germany 

3-37 Wheat leaf-rust infection response – from apoplast proteomics to 

transcriptional aspects in near-isogenic lines of the ’Thatcher’ cultivar 

Pós V 1 , Hunyadi-Gulyás É 2 , Manninger K 3 , Szikriszt B 1 , Rab E 1 , Kabai M 1 , Medzihradszky 

K 2 , Lukács N 1 

1 

Dept. of Plant Physiology and Plant Biochemistry, Corvinus Univ. of Budapest, Ménesi út 44, 

H-1118 Budapest, Hungary 

2 

BRC Proteomics Research Group of H.A.S., Temesvári krt 62, H-6701 Szeged, Hungary 

3 Plant Protection Institute of H.A.S., Herman Ottó út 15, H-1022 Budapest, Hungary 

Email: noemi.lukacs@uni-corvinus.hu 

192 

Abstract 

Besides the aim of increasing crop yield and grain quality of wheat, traditional 

cross-breeding as well as transgenic strategies exploiting the genetic variability of 

wild relatives have been developed to enhance abiotic or biotic resistance of 

Triticum aestivum. Despite of the emerging new cultivars, infection of the obligate 

biotrophic leaf-rust (Puccinia recondita fsp. tritici) still results in substantial yield 

losses or even epidemics in the Central-European region. About 30 resistance genes 

conferring resistance to individual races of leaf-rust were identified until now. To 

understand the role of a given resistance gene precisely and to estimate its actual 

usefulness, near-isogenic (NIL) leaf rust resistant lines were developed in wheat. 

This opened the possibility to explore the specific influence of resistance genes on 

individual proteins and genes involved in the defense process and probably 

contributing the resistance development. 

We analyzed the susceptible cv. ‘Thatcher’ and two of its near-isogenic lines, Lr1 

and Lr9 with respect to changes in the apoplastic protein pattern associated with 

defense response and/or seedling resistance. During 7 days p.i. a complex set of 

proteins from different stress-related plant families (e.g. extracellular PR1, PR2 

proteins, chitinases/chitin-binding proteins, several members of the Barwin-family 

and TLP-s or different peroxidases) were identified after 2D-PAGE separation 

followed by MS analysis (MALDI-TOF and LC-MS/MS). Some dominant, 

infection-induced proteins, such as a chitinase I (27,5 kDa), a beta-1,3-glucanase

(35,4 kDa) and at least two, closely related PR-1 proteins (16,7-17,6 kDa) were 

found in all three or in both Lr1 and Lr9 resistant lines, but their amount, activity 

and expression kinetics showed clear genotype-dependent differences. 

Considering that many members of the latter protein families are known defense 

factors of numerous resistant as well as of sensitive plants, but still can be major 

factors contributing to seedling resistance by their differential expression, we 

performed RT-PCR and RT-qPCR analyses of chitinase and glucanase mRNA with 

a double aim. First, to confirm the proteomic results and second, to find out whether 

a rise in the total amount of mRNA or possibly individual isoenzymes are 

responsible for the changes of enzyme activity in the apoplast of the individual NIL 

lines. Results of these analyses will be shown and compared with data from the 

literature. 

193

Mavrikou S, Flampouri K, Moschopoulou G, Michaelides A, Kintzios S: Assessment of Organophosphate and 

Carbamate Pesticide Residues with a Novel Cell Biosensor. In: Feldmann F, Alford D V, Furk C: Crop Plant 

Resistance to Biotic and Abiotic Factors (2009), 194; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische 


3-38 Assessment of Organophosphate and Carbamate Pesticide Residues 

with a Novel Cell Biosensor 

Mavrikou S, Flampouri K, Moschopoulou G, Michaelides A, Kintzios S 

Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece 

Email: skin@aua.gr 

194 

Abstract: 

The conventional analysis of pesticide residues in analytical commodities, such as 

tobacco and tobacco products is a labor intensive procedure, since it is necessary to 

cover a wide range of different chemicals, using a single procedure. Standard 

analysis methods include extensive sample pretreatment (with solvent extraction 

and partitioning phases) and determination by GC and HPLC to achieve the 

necessary selectivity and sensitivity for the different classes of compounds under 

detection. As a consequence, current methods of analysis provide a limited sample 

capacity. In the present study, we report on the development of a novel cell 

biosensor for detecting organophosphate and carbamate pesticide residues in 

tobacco. The sensor is based on neuroblastoma N2a cells and the measurement of 

changes of the cell membrane potential, according to the working principle of the 

Bioelectric Recognition Assay (BERA). The presence of pesticide residues is 

detected by the degree of inhibition of acetylcholine esterase (AChE). The sensor 

instantly responded to both the organophoshate pesticide chlorpyriphos and the 

carbamate carbaryl in a concentration-dependent pattern, being able to detect one 

part per billion (1 ppb). The observed response was quite reproducible, with an 

average variation of +5,6%. Fluorescence microscopy observations showed that 

treatment of the cells with either chlorpyrifos or carbaryl was associated with 

increased [Ca 2+ ]cyt . The novel biosensor offers fresh perspectives for ultra-rapid, 

sensitive and low-cost monitoring of pesticide residues in tobacco as well as other 

food and agricultural commodities.

Markoglou A N, Vitoratos A G, Doukas E G, Ziogas B N : Phytopathogenic and mycotoxigenic characterization 

of laboratory mutant strains of Fusarium verticillioides resistant to triazole fungicides. In: Feldmann F, Alford D 

V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 195; ISBN 978-3-941261-05-1; © 


3-39 Phytopathogenic and mycotoxigenic characterization of laboratory 

mutant strains of Fusarium verticillioides resistant to triazole fungicides 

Markoglou A N, Vitoratos A G, Doukas E G, Ziogas B N 

Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece 

Email: markan@aua.gr 

Abstract 

Mutants of Fusarium verticillioides (formerly called F. moniliforme) resistant to the 

triazole fungicides (Rf: 20-60, based on EC90s) were isolated at high mutation 

frequency (1.8 x 10-5) after UV-mutagenesis and selection on media containing 

epoxiconazole. Cross resistance studies with other fungicides showed that the 

mutation(s) for resistance to epoxiconazole also reduced the sensitivity of mutant 

strains to other C-14 demethylase inhibitors (DMIs), as flusilazole, difenoconazole, 

propiconazole, flutriafol and imazalil. No effect of epoxiconazole-resistant 

mutation(s) on fungitoxicity of fungicides which affect other cellular pathways or 

other steps of the sterol biosynthesis was observed. Study of saprophytic fitness 

determining parameters showed that the mutation(s) for resistance to epoxiconazole 

did not significantly affect the mycelial growth rate, sporulation and conidial 

germination. Pathogenicity tests on maize seedlings under greenhouse conditions 

showed that most mutant strains presented infection ability similar to the wild-type 

strain. Liquid chromatographic-mass spectrometric (LC-ESI/MS) analysis of 

mycelial extracts from the wild-type and mutant strains, that were grown on PDA 

medium, showed that all epoxiconazole-resistant isolates produced fumonisins 

(FB1, FB2) at similar or even higher (up to 6-fold) concentrations than the wildtype 

parent strain. In addition, in most of these mutant strains the mycotoxigenic 

ability was further increased (2 to 4-fold higher) when the mutants were grown on 

epoxiconazole-amended medium. Similar results were also found in tests with 

artificially inoculated corn seeds. The data of the present study indicate, for the first 

time, the potential risk of increased fumonisin contamination of cereals after 

intensive use of triazole fungicides. 

195

Hamolka L, Krut'ko N, Gutkovskaya N: New Lignin-Phenolic Compounds for Plant Protection. In: Feldmann F, 



3-40 New Lignin-Phenolic Compounds for Plant Protection 

Hamolka L 1 , Krut'ko N 1 , Gutkovskaya N 2 

1 

The Institute of general and inorganic chemistry of the National Academy of Sciences of 

Belarus, 9, Surganava Str. Minsk BY-220072, Republic of Belarus 

2 

RUP The Plant Protection Institute, 2, Mira Str. 220011 Priluki, Minsk region, Republic of 

Belarus 

Email: l.hamolka@mail.ru 

196 

Abstract 

One of the mechanisms of induced crop resistance to biotic and abiotic stress 

factors is lignification, i.e. lignin biosynthesis and cell wall strengthening due to 

deposition of lignin as well as phenolic components. Natural lignins are complex 

three-dimensional phenolic polymers. In plant cell walls they serve as physical 

and/or chemical barriers against the penetration of pathogens and abiotic stress 

impact. Materials for crop protection synthetically derived from natural lignins are 

of high biological activity; they enhance defense reactions. We developed new 

improved formulations of lignin chemicals and tested and evaluated them against 

some fungal pathogens and under adverse soil conditions. New materials have a 

potential to diminish use of synthetic pesticides and to decrease environmental load.

Ben Slimane R, Bancal P, Bancal M-O: Regulation of Grain N Accumulation in Wheat. In: Feldmann F, Alford D 



3-42 Regulation of Grain N Accumulation in Wheat 

Ben Slimane R, Bancal P, Bancal M-O 

INRA, UMR 1091 INRA/INAPG Environnement et Grandes Cultures, F-78850 Thiverval 

Grignon, France 

Email: bancal@grignon.inra.fr 

198 

ABSTRACT 

Grain filling regulation is disturbed when late crop management or pest attacks 

occur, because of source/sink ratio variation. In order to strongly modify fluxes of 

remobilized nitrogen (N) during grain filling, leaf blades 2 and 3 were either 

wrapped with aluminium foil or cut one week after anthesis. The evolution of green 

area, dry matter (DM), and N in all organs were followed from heading to full 

maturity in wheat crops managed under two levels of N fertilization in field. 

Both leaf treatments resulted in an immediate two third loss of green area, without 

affecting either the green area of untreated leaves (flag leaf and lower leaves) or 

their green duration. Net assimilation rate was also reduced similarly, and grain DM 

filling rate declined immediately to the same extent. 

N uptake was very low in control and not affected by leaf treatments, which 

differed in the fate of remobilized N from leaf blades 2 and 3. This N was 15% of 

final grain N in control; it was lost in cutting treatment, whereas N from wrapped 

blades was suddenly released and accumulated in stems and sheaths. Conversely 

these organs didn’t compensate for the lack of remobilized N from cut leaves. 

Therefore grain N filling rate remained unchanged after wrapping treatment as 

compared to control, while it was immediately reduced after cutting treatment. It is 

concluded that grain filling by remobilized N was up-regulated by stem and sheaths, 

without on-line control by grains. 

INTRODUCTION 

Grain protein content (GPC) is one of the main determinants of wheat international market 

price. Late nitrogen (N) fertilization is the common practice used to produce a high GPC. 

However this goal can be achieved only by ensuring crop protection through high levels of 

pesticides. A major challenge of modern agriculture is to limit the excessive use of chemicals 

and at the same time to improve grain quality without affecting yield. Attention should thus be

paid to N remobilization, the process by which nutrients are translocated from vegetative 

organs to grains during the post-anthesis period. In wheat, up to 60-90% of grain nitrogen is 

provided by N remobilization during grain filling, while a lesser fraction comes from postanthesis 

N absorption (Kichey et al. 2007). Thus, it is clear that N remobilization is highly 

involved in grain filling, but it is disturbed when late crop management or pest attacks occur 

(Bancal et al. 2008). 

Late foliar diseases primarily induce early green surface inactivation, but perhaps also an 

accelerated senescence of surrounding tissues, thus temporarily increasing N availability and 

severely affecting source/sink ratio in crops. Late fertilization effects are not straighter, as 

vegetative organs are not only sources, but also temporary sinks. Indeed 15 N labeling studies 

indicate that N absorbed after anthesis is competitively incorporated in vegetative parts 

(Oscarson 1996; Kichey et al. 2007). Late fertilization commonly results in a delayed (Martre 

et al. 2006) or reduced N remobilization (Gooding et al. 2007). To improve plant N economy it 

is thus needed to clarify the relative roles of vegetative organs as both source for N 

remobilization and temporary sink for N either derived from senescing tissues or from late N 

uptake. However, N fluxes changing with time should be studied, as well as N balances from 

anthesis to maturity. 

In many previous works that dealt with manipulating sinks or sources, only the source/sink 

ratio was considered, assuming that a reduction of source availability was equivalent to an 

increase of sink demand and the converse. However, according to Bancal & Soltani (2002), the 

response curve relating sink and source activities is non-linear, and consequently the 

source/sink ratio could be a misleading index. Therefore sinks and sources should be 

manipulated independently. This paper studied the regulation of grain N filling avoiding any 

variation in post-anthesis absorption while varying N availability through manipulating N 

remobilization of vegetative plant organs. This was achieved by wrapping or cutting both 

second and third leaf blades at the start of rapid grain filling. These two leaves were important 

sources for both dry matter (through photosynthesis) and N (through remobilization) during 

grain filling. Leaf cutting resulted in the sudden loss of these sources, whereas leaf wrapping 

induced an accelerated senescence of tissues, and thus a temporarily exaggeration of these 

sources. However, other vegetative plant parts, such as the first untreated leaf, the stem and the 

chaff might counteract the induced variation in N availability, the extent of which was 

estimated in this study. 


Field experiment 

Winter wheat (Triticum aestivum) cv. Cap Horn was sown on October 26 th , 2006 in an 

experimental field of INRA at Thiverval-Grignon, France at a density of 250 seeds /m². Two 

levels of nitrogen fertilization were applied before flowering thus leading to a nitrogen 

nutrition index (NNI) of 0.5 and 0.8 respectively around anthesis. Six treatments were 

199

conducted, where a treatment is a combination of two factors: nitrogen fertilization and abiotic 

stress applied post-anthesis (leaf wrapping, partial defoliation or control). 

Leaves were numbered downward from 1 to 4. On May, 23 th , 2007 (121 °Cd after anthesis), 

300 shoots per treatment were tagged based on their ear length close from the main shoot 

average, as previously measured in plots. Treatments were then applied: leaf blades 2 and 3 

were either wrapped in aluminium foil, or cut at the ligule, or saved as control. 

Plant sampling and assessments 

Fifteen plants were sampled weekly from heading (-79 °Cd) to full grain maturity (949 °Cd) 

and shared into three replicates, from which organs were separated into leaf blades, stems plus 

surrounding sheaths, and ears. The leaf blades, when still green, were scanned and then total 

and green leaf areas were measured by image analysis. The leaf blades were recovered and 

lyophilized. Ears were also lyophilized, whereas the stems were oven dried. Dry matter (DM) 

was weighed from all organs. Once ears had been lyophilized, chaff and grains were separated 

and weighed, and grain numbers determined. Finally, all organs were lyophilized again and 

ground to a fine powder in preparation for subsequent analyses of the total N content, using the 

Dumas combustion method. 

RESULTS 

Evolution of green leaf area as well as DM and N amount in all organs were assessed from -79 

°Cd till 949 °Cd. Two way ANOVA was calculated for each organ at each sampling date. The 

effect of crop NNI was commonly very significant, while the effect of leaf treatments appeared 

only in some cases (never before 138 °Cd, one day after they were done), and interactions 

between crop NNI and leaf treatments almost never occurred. 

Green leaf area evolution 

At anthesis, the green blade areas were 81 ± 5 cm² per shoot in the NNI 0.5 plot vs. 105 ± 4 

cm² per shoot in the NNI 0.8 plot, from which blades 2+3 contributed one half in both cases. 

ANOVA (Table 1) indicated that until 337 °Cd green leaf area remained significantly higher in 

the NNI 0.8 plot than in the NNI 0.5 plot. At 138 °Cd, one day after treatment started, the 

wrapped leaves not yet differed from the control. One week later, wrapped leaves had a 

yellow-green color which was assessed as green by the image analysis; therefore no significant 

difference to control appeared (Figure 1). Later, green area in wrapped leaves decreased 

sharply until 337°Cd, while it continued to decline smoothly until 720 °Cd in untreated leaves. 

Therefore green area was significantly lower (P < 0.1%) in wrapped leaves 2+3 from 337 °Cd 

to 599 °Cd (table 1). In the other, untreated, leaf blades (flag leaf and lower leaves) leaf 

treatments of blades 2+3 had no significant effect (P > 1%) at any sampling date. Thus the 

green duration of untreated blades was neither accelerated nor slowed down by both leaf 

treatments. 

200

Table 1. Effect of NNI and leaf treatments on the evolution of green areas. 

NNI Effect 

Treatment Effect Interaction N×T 

Sampling 

Date (°Cd) 

–––––––––––––––––––– 

Green area Green area 

of blades of other 

–––––––––––––––––––– 



–––––––––––––––––––– 



2+3 blades 2+3 blades 2+3 blades 

-79 *** *** NS NS NS ** 

12 ** *** NS NS NS NS 

138 *** *** NS NS NS NS 

227 * ** NS NS NS NS 

337 * * *** NS NS NS 

470 NS NS *** NS NS NS 

599 NS *** ** NS NS NS 

720 NS NS NS NS NS NS 

840 nd nd nd nd nd nd 


Multiple factor ANOVA was carried out at each sampling date fully crossing crop NNI (N) and leaf treatment (T) 

effects. First order interaction between factors was examined (N×T). A probability lower than 1% denotes a 

significant effect of a treatment or an interaction, with *: 0.1% 

The DM of above ground parts was 2.9 ± 0.2 g·culm -1 at heading and increased later, becoming 

higher in the NNI 0.8 plot than in the NNI 0.5 plot from 337 °Cd (Table 2). DM was also 

higher in control than in leaf treated plants from 599 °Cd, without interaction between NNI and 

leaf treatment. No significant differences were recorded between wrapping and cutting 

treatment. In the NNI 0.8 plot, DM increased quite linearly from 138 °Cd to 720 °Cd and a 

mean rate for net assimilation was calculated. This rate was statistically higher (P < 1%) in 

control than in either wrapping or cutting treatment, which did not differ together (2.4 ± 0.3 vs. 

1.8 ± 0.2 mg·culm -1 ·°Cd -1 , respectively). 

202 

Table 2. Two way ANOVA for the effect of NNI and leaf treatments on above ground 

DM and N at the varying sampling dates. 

NNI Effect 

Treatment Effect Interaction N×T 

Sampling 

Date (°Cd) 

–––––––––––––––––––– 

Above Above 

ground ground 

–––––––––––––––––––– 

Above Above 

ground ground 

–––––––––––––––––––– 

Above Above 

ground ground 

DM N DM N DM N 

-79 ** *** NS NS NS NS 

12 NS *** NS NS NS NS 



337 * *** NS NS NS NS 


599 ** *** * NS NS NS 

720 ** *** ** NS NS NS 

840 *** *** * NS NS NS 

949 * *** * NS NS NS 

At heading, the N amount in above ground parts was 29 ± 1 mg·culm -1 in the NNI 0.5 plot. It 

was very significantly higher at 40 ± 1 mg·culm -1 in the NNI 0.8 plot (P < 0.01%), and this 

difference was maintained until 720°Cd, when N in above ground parts stabilized. Conversely, 

leaf treatments never had an effect (P > 1%), and no interaction was shown. Regression lines 

of N with time were obtained from 138 °Cd until 720 °Cd, and statistical comparison of their 

slopes did not indicate any significant effect of leaf treatments (P > 10%). The average rate of 

net N uptake was therefore 8 ± 1 µg·culm -1 ·°Cd -1 in the NNI 0.5 plot vs. 10 ± 1 µg·culm -1 ·°Cd -1 

in the NNI 0.8 plot.

Nitrogen amount evolution in vegetative parts 

Within control plants, leaf blades 2+3 lost N quite linearly from 138°Cd to full maturity 

(Figure 2A). The mean rate for remobilization was -6 ± 0.2 µg·culm -1 ·°Cd -1 in the NNI 0.5 plot 

vs. -11 ± 1 µg·culm -1 ·°Cd -1 in the NNI 0.8 plot. The direct N lack by cutting treatment on grain 

filling was thus not negligible, representing 10 to15% of the final grain N. The N amount 

decreased abruptly in wrapped leaves 2+3 from the start of treatment leading to a very 

significant effect of wrapping (P < 0.01%) by ANOVA after 237 °Cd (table 3). Interactions 

with NNI of the crop were observed only after 599 °Cd, when leaves were N-depleted. The 

maximum rates of N remobilization occurred during the first week after wrapping; it reached - 

26 ± 3 µg·culm -1 ·°Cd -1 in the NNI 0.5 plot vs. -41 ± 2 µg·culm -1 ·°Cd -1 in the NNI 0.8 plot, 

around four fold more (P < 0.01%) than in control. Therefore wrapping treatment resulted in an 

earlier N release from leaves 2+3, thus increasing N availability in other plant parts. But this 

enhancement was transient (figure 2A), as N amount stabilized rapidly in wrapped leaves 2+3, 

while it still declined at a constant rate in control. After 599 °Cd, control leaves were slightly 

more N-depleted that wrapped leaves. Since the difference was significant only in the NNI 0.8 

plot, interactions were observed between NNI and leaf treatment (table 3). 

Table 3. Two way ANOVA for the effect of NNI and leaf treatments on the evolution 

of N in leaf blades 2+3, in mixed stem and sheaths, and in other vegetative 

parts (VP). 

Date NNI Effect 

Treatment Effect 

Interaction N×T 

(°Cd –––––––––––––––––––– –––––––––––––––––––– –––––––––––––––––––– 

) Blades2+ Stem + Othe Blades2+ Stem + Othe Blades2+ Stem + Othe 

3 sheath r 3 sheath r 3 sheath r 

N s N V.P. N s N V.P. N s N V.P. 

N 

N 

N 

-79 *** *** *** NS NS NS * NS NS 

12 *** *** *** NS NS NS NS NS NS 

138 *** *** *** NS NS NS NS NS NS 

227 ** *** *** *** ** NS NS NS NS 

337 *** *** *** *** ** NS NS NS NS 

470 *** *** *** *** * NS * NS NS 

599 *** *** *** *** ** * NS NS * 

720 *** ** *** ** NS NS *** NS NS 

840 *** *** *** *** NS * NS NS * 

949 *** *** * *** NS NS ** NS NS 

203

204 

N in leaf bl 2+3 (mg.culm-1 N in leaf bl 2+3 (mg.culm ) -1 N in leaf bl 2+3 (mg.culm ) -1 ) 

10 

8 

6 

4 

2 

0 

A 

-200 0 200 400 600 800 1000 

Time from anthesis (°Cd) 

N in stem+sheaths (mg.culm-1 N in stem+sheaths (mg.culm ) -1 N in stem+sheaths (mg.culm ) -1 ) 

20 

15 

10 

5 

0 

B 

-200 0 200 400 600 800 1000 

Time from anthesis (°Cd) 

Figure 2. Evolution of Nitrogen in leaf blades 2+3 (A), and in stem + sheaths (B) within 

the NNI 0.8 plot. Symbols are as in figure 1. No open circles could be plotted 

on figure 2A after the cutting treatment was done. 

In stem and sheaths N was remobilized less rapidly after leaf wrapping (figure 2B), and its 

amount was higher (P < 0.1%) than that of control from 227 until 599 °Cd. The N amount then 

declined rapidly in stem and sheaths of wrapped leaf treatment, lastly stabilizing at the same 

level as in the other treatments. Stem and sheaths could then be regarded as temporary 

reservoirs for N released from wrapped leaves 2+3. Conversely stem and sheaths did not 

compensate for the lack of remobilized N from cut leaves 2+3, as no difference was found to 

control on any sampling date (table 3). The other vegetative parts (upper and lower untreated 

leaf blades and chaff), were not significantly affected by treatments (P > 1%). 

Grains 

The effect of NNI on grain N was significant at each sampling date, while the effect of leaf 

treatment appeared at 337 °Cd without interactions with NNI (table 4). Final grain N yield in 

control was 28 ± 2 mg·culm -1 in the NNI 0.5 plot vs. 38 ± 2 mg·culm -1 in the NNI 0.8 plot, 

respectively. N accumulated quite linearly in grains until 720 °Cd, and the mean grain N filling 

rate was not different (P >10%) in control and wrapping treatment, at 36 ± 1 µg·culm -1 ·°Cd -1 

within NNI 0.5 plot vs. 51 ± 1 µg·culm -1 ·°Cd -1 within NNI 0.8 plot. In cut leave treatment, 

grain N filling rate was significantly lower than in control (P < 0.01%) at 28 ± 1 

µg·culm -1 ·°Cd -1 in the NNI 0.5 plot vs. 41 ± 1 µg·culm -1 ·°Cd -1 in the NNI 0.8 plot. Thus cutting 

leaves 2+3 likely reduced immediately the grain N filling rate whereas wrapping those leaves 

did not have a significant effect on the grain N at any time. 

Unlike observed for N, no difference was identified throughout in grain DM between wrapped 

and cut leave treatments. At 470 °Cd and later, the DM of grains from leaf-treated plants was

very highly significantly (P < 0.01%) lower than that of control plants. The difference at 

maturity reached 0.2 ± 0.1 g·culm -1 in the NNI 0.5 plot vs. 0.3 ± 0.1 g·culm -1 in the NNI 0.8 

plot, respectively. In the 0.8 NNI plot, grain DM increased quite linearly from 138 °Cd until 

720 °Cd, and grain DM filling rate was very significantly (P < 0.1%) higher in control than in 

either cutting or wrapping treatment (3.8 ± 0.1 vs. 3.2 ± 0.1 mg·culm -1 ·°Cd -1 ). Thus the 

decrease in rate of grain DM filling was the same as the decrease in net assimilation rate, what 

was suggested by the absence of effect of leaf treatments on the DM of vegetative parts. 

Actually apart from the grain, only the treated leaves 2+3 were significantly affected in their 

DM by treatments. But DM variation in those leaves amounted to less than 3% of final grain 

DM, and therefore their effect on grain filling was not detectable. 

Table 4. Two way ANOVA for the effect of NNI and leaf treatments on grain DM and 

N at the varying sampling dates. 

Sampling 

Date (°Cd) 

NNI Effect 

–––––––––––––––––––– 

Grain DM Grain N 

Treatment Effect 

–––––––––––––––––––– 


Interaction N×T 

–––––––––––––––––––– 


-79 nd nd nd nd nd nd 


138 NS * NS NS ** *** 

227 NS * NS NS NS NS 

337 * *** * * NS NS 

470 * *** NS * NS NS 

599 *** *** *** *** ** * 

720 *** *** *** *** NS NS 

840 *** *** *** ** NS NS 

949 ** *** ** * NS NS 

“nd” refers to data not available (grains earlier than 138 °Cd after anthesis). 

DISCUSSION 

In wheat, after anthesis, literature identified one main N sink (grain filling), and two main N 

sources (N remobilization and post-anthesis N uptake), but there are some indications that 

vegetative organs may also be temporary N sinks, thus competing with grain filling. In this 

paper, a more clear understanding of the way grain N filling is regulated was investigated by 

altering only N remobilization through either leaf wrapping or leaf cutting. The experiment was 

moreover carried out at low crop N nutrition in order to minimize post anthesis N uptake. Two 

levels of crop nutrition, with NNI at 0.8 and 0.5 respectively, were studied to look on their 

possible interaction with treatments, i.e. to assess if N shortage induced qualitative changes in 

N partitioning. The ANOVA indicated that the extent of N shortage did not change 

qualitatively the results of leaf treatments. However, as 0.8 NNI crops could remobilize more 

N than 0.5 NNI crops, the trends in data were clearer in the first case. 

205

Effects of leaf treatments on DM fluxes 

The two treatments (leaf cutting or wrapping) were not different from a DM point of view. 

Carbon net assimilation was reduced following both treatments by one third as compared to 

control. The reduction of net assimilation was not compensated by DM remobilization from 

vegetative organs. Consequently grain DM filling rate declined immediately after leaf 

treatments were applied, and to the same extent as net assimilation decrease. No compensatory 

change in grain filling duration was observed, and final grain DM was then significantly lower 

than that of the control, which was commonly observed in both defoliation or shading 

experiments (Martinez-Carasco & Thorne 1979; Guitman et al. 1991; Ma et al. 1996). 

Effects of leaf treatments on N fluxes 

In our experiment, N uptake was very low, amounting less than 20% of final grain N, 

approximately the same level as N remobilization from the roots (Andersson & Johansson 

2006). The part of true N absorption in N uptake was thus likely at zero because of the low N 

availability in the soil. Therefore the path of N remobilized from organs was not hidden by any 

variation in N absorption, which may explain paper discrepancies with previous studies. For 

instance, N uptake was neither accelerated nor slowed down by our leaf treatments whereas N 

absorption is frequently reduced when flag leaf is removed (Guitman et al. 1991). 

In defoliation experiments, N from excised leaves is lost, and obviously final grain N is always 

lowered. Literature suggests that moreover N remobilization in remaining organs could be 

impaired. Guitman et al. (1991) reported that both N and soluble protein decreased later in 

remaining leaves following flag leaf excision. Ma et al. (1996) indicated that following a 

complete defoliation, the nitrogen concentration at grain maturity was higher in chaff and stem. 

In spite of this, when leaves 2 and 3 were cut in our experiment, neither remaining leaves nor 

stem or chaff exhibited either lower or delayed N remobilization. However, N remobilization 

from vegetative tissues is actually the balance between N input and output from N uptake. In 

defoliation studies where N absorption was not nil, the part of N uptake available for removed 

leaves could be re-allocated to the remaining organs, thus delaying the time for net N decrease 

in these receiving vegetative organs. In our study, the weakness of N uptake showed that 

vegetative organs did not react to defoliation by any change in their N output. As a result, the 

rate of grain N filling immediately declined after leaf cutting. Moreover it did it at the same 

rate as N remobilization from control leaves 2 and 3, which clearly advocated against a sink 

regulation of grain N filling. 

In wrapping treatment, N from darkened leaves 2 and 3 was suddenly released, thus 

temporarily increasing the fluxes of remobilized N. However this increased N availability did 

not result in any change in grain N filling. Released N was instead entrapped for a while in 

stem or in sheaths. This N was further remobilized from stem plus sheaths, so that the final 

grain N was the same as in control. It might suggest a sink regulation of grain N filling rate in 

opposition to defoliation results. Data could however be interpreted in the frame of source 

206

egulation, providing “source” would be segmented: stem and sheaths were downstream as 

compared to wrapped leaves (actually blades). Following wrapping treatment, stem and sheaths 

received enhanced N inputs, and as commonly observed following late fertilization, they were 

able to temporarily incorporate them, resulting in a delayed net remobilization pattern. 

However, late fertilizations also result in an enhanced rate of grain N filling, which we did not 

observe, even for a while. It could be hypothesized that in our study the fluxes of remobilized 

N were small enough to be fully entrapped by stem and sheaths, which would be overflowed 

under high fertilization. Indeed, the extra-flux of remobilized N amounted 30 µg °Cd -1 during 

the first week after wrapping in the 0.8 NNI plot, far below N uptakes reached under high 

fertilization (around 100 µg °Cd -1 ). 

Conclusion 

Under our experimental conditions, without post anthesis N uptake, N released by leaf 

senescence seemed delivered to grains with up-regulation by stem or sheaths. Lowering of N 

availability resulted in lower grain filling rate, whereas increase of N availability was leveled 

through temporary storage. We suggest that any variation in N remobilization by diseased 

leaves would not modify the senescence of other leaves. Variation in DM yield could thus be 

obtained from decrease in green surface. As this experiment thus mimicked part of fungusinduced 

disorders (i.e. induced senescence), although in an accelerated way and without pure 

pathogenic effects, further work is needed to refine variations in N yield, concerning possible 

accelerated remobilization from infected organs, and also the impact of disease on post anthesis 

absorption. 


We thank Mrs. J Jean-Jacques and M. P Belluomo for technical assistance in the trials work 

undertaken. 

REFERENCES 

Andersson A; Johansson E (2006). Nitrogen partitioning in entire plants of differing spring 

wheat cultivars. Journal of Agronomy and Crop Science 192, 121-131. 

Bancal M O; Roche R; Bancal P (2008). Late foliar diseases in wheat crops decrease nitrogen 

yield through N uptake rather than through variations in N remobilization. Annals of 

Botany 102, 579-590. 

Bancal P; Soltani F (2002). Source-sink partitioning. Do we need Münch? Journal of 

Experimental Botany 53, 1919-1928. 

Gooding M J; Gregory P J; Ford K E; Ruske R E (2007). Recovery of nitrogen from different 

sources following applications to winter wheat at and after anthesis. Field Crop 

Research 100, 143-154. 

207

Guitman M R; Arnozis P A; Barneix A J (1991). Effect of source-sink relations and nitrogen 

nutrition on senescence and N remobilization in the flag leaf of wheat. Physiologia 

Plantarum 82, 278-284. 

Kichey T; Hirel B; Heumez E; Dubois F; Le Gouis J (2007). In winter wheat (Triticum 

aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlates 

with agronomic and nitrogen physiological markers. Field Crop Research 102, 22-32. 

Ma Y Z; MacKown C T; Van Sanford D A (1996). Differential effects of partial spikelet 

removal and defoliation on kernel growth and assimilate partitioning among wheat 

cultivars. Field Crop Research 47, 201-209. 

Martinez-Carrasco R; Thorne G N (1979). Physiological factors limiting grain size in wheat. 

Journal of Experimental Botany 30, 669-679. 

Martre P; Jamieson P D; Semenov M A; Zyskowski R F; Porter J R; Triboï E (2006). 

Modelling protein content and composition in relation to crop nitrogen dynamics for 

wheat. European Journal of Agronomy 25, 138-154. 

Oscarson P (1996). Transport of recently assimilated 15 N nitrogen to individual spikelets in 

spring wheat grown in culture solution. Annals of Botany 78, 479-488. 

208

Chand S, Rishi M, Preeti M, Anjali C, Saurabh K: Characterization of novel endophytic Bacillus licheniformis 

strain CRP-6 from apple seedlings displaying multiple plant growth promoting activities. In: Feldmann F, Alford 

D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 209; ISBN 978-3-941261-05-1; © 


3-43 Characterization of novel endophytic Bacillus licheniformis strain 

CRP-6 from apple seedlings displaying multiple plant growth 

promoting activities 

Chand S, Rishi M, Preeti M, Anjali C, Saurabh K 

Department of Basic Sciences, Dr. Y.S. Parmar University of Horticulture and Forestry, 

Nauni, Solan, INDIA 

Email: shirkotcp@yahoo.co.in 

Abstract 

Phylogenetic characterization of carbendazim tolerant endophytic rhizobacterial 

isolate CRP-6, based on sequence homology of a partial 846-bp fragment of 16s 

rDNA amplicon, with the ribosomal database sequences 

(http://www.ncbi.nlm.nih.gov) validated the strain as Bacillus licheniformis. The 

strain CRP-6 produced a substantial amount of soluble phosphate from insoluble tricalcium 

phosphate in Pikovskaya’s (PVK) medium. The rate of P-solubilization 

increased with concomitant decrease in pH of the medium. The strain CRP-6 also 

produced high amount of indole-3-acetic acid (IAA) in tryptophan amended 

medium. Besides, the strain also exhibited significant production of the siderophore 

on Chrome- azurol-S- (CAS) medium both by plate assay and liquid assay methods, 

respectively. Significant growth inhibition of phytopathogenic fungi occurred in the 

order as Dematophora necatrix > Fusarium oxysporum f.sp. lycopersici > 

Rhizoctonia solani >Sclerotinia sclerotorium upon incubation with strain CRP-6 

cells in dual culture. The data revealed 100% inhibition of mycelial growth of 

Dematophora necatrix at 5% (v/v) of cell free supernatant. Seed treatment with 

strain CRP-6 resulted in 100% disease control of white root rot of apple caused by 

D. necatrix in one year old apple seedlings under net house conditions. The data 

revealed significant per cent increase in shoot and root parameters over untreated 

control. Thus, the secondary metabolites producing new Bacillus licheniformis 

strain CRP-6 exhibited innate potential of biocontrol and plant growth promotion 

activities under in vitro as well as field conditions. 

209

Grbic M, Grbic V: Dissection of plant resistance to pest using a genomic approach: Arabidopsis-Two Spotted 

Spider Mite Tetranychus urticae, a novel model for plant-herbivore interactions. In: Feldmann F, Alford D V, 

Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 210; ISBN 978-3-941261-05-1; © Deutsche 


3-44 Dissection of plant resistance to pest using a genomic approach: 

Arabidopsis-Two Spotted Spider Mite Tetranychus urticae, a novel 

model for plant-herbivore interactions 

Grbic M, Grbic V 

Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 

Email: mgrbic@uwo.ca 

210 

Abstract 

In response to herbivore attack, plants have evolved a variety of mechanisms to 

deter herbivore from feeding. The fundamental mechanisms of plant resistance to 

pest are the basis for breeding of pest-resistant crops. Currently, the system for 

dissecting the genetics of the plant-pest interaction is lacking. Although Arabidopsis 

makes an excellent plant genetic model for such studies, a genetically defined planteating 

arthropod is lacking. The two-spotted spider mite Tetranychus urticae is a 

generalist herbivore and major agricultural pest. It feeds on more then 1500 plant 

species (about 150 of which are of economic value). Chemical pesticides are the 

predominant method of controlling spider mites, but mites have rapidly evolved 

resistance to all major pesticide groups. Conveniently, T. urticae develops on the 

model plant Arabidopsis, allowing utilization of the plethora of genomic tools 

available in this plant model species to dissect plant-pest interactions. 

Our effort to sequence the genome of T. urticae sequencing project (USA 

Department of Energy, Joint Genome Institute http://www.jgi.doe.gov 

/sequencing/why/CSP2007/spidermite.html) that will open new perspectives for 

genomic analysis of plant-herbivore interactions and plant resistance to pests. We 

characterized the differential resistance among natural Arabidopsis accessions to 

spider mite damage and isolated Arabidopsis accessions resistant and susceptible to 

T. urticae. In addition, we profiled the transcriptome of naturally resistant and 

susceptible Arabidopsis accessions upon spider mite feeding using ATH1 

Arabidopsis microarray. We isolated more that 500 genes induced by spider mite 

feeding in susceptible and resistant Arabidopsis ecotypes including potential 

candidate genes for plant resistance to spider mites. Our screen for natural variation 

of plant resistance and transcriptome profiling represents the first systematic step 

toward uncovering plant genes for breeding/biotechnological modification of crop 

plants for resistance against major pest in agriculture.

Denzer H W: FieldClimate.Com offering Weather data based Plant Disease Information for Growers and 

Advisors. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 211- 


3-45 FieldClimate.Com offering Weather data based Plant Disease 

Information for Growers and Advisors 

Denzer H W 

Pessl Instruments GmbH, Werksweg 107, A-8160 Weiz, Austria 

OVERVIEW 

GSM Telephone networks offers GPRS technology to connect to the internet from everywhere. 

This technology is used by modern agricultural electronic weather stations of Pessl Instruments 

GmbH. Wheater data from the field are collected on FieldClimate.Com. On FieldClimate.Com 

the data can be displayed in graphs or tables. The data can be downloaded into other 

applications or into other databases. 

Most of our clients are using the services of FieldClimate.Com to calculate reference 

evapotranspiration and plant disease models for different crops to use the climate data to 

improve their plant protection praxis. Plant disease models for the following crops are 

available: 

• Viticulture: Grape Vine Downy mildew and Powdery Mildew, B. cinerea, Black rot 

o This models are available since 2005 

o 46% of our clients are using this models 

• Apple: Apple Scab, Fire Blight 


o 40% of our clients using this models 

• Pear: Pear Scab, Brown Spot, Fabraea Leaf Spot, Fire Blight 

o This models are available since 2007 and 2009 

o 15% of our clients are using this model 

• Stone Fruit Models: Leaf Curl, Shut Hole, Powdery Mildew, Monilia 



• Strawberry: Botrytis cinerea, Powdery Mildew 



• Citrus: Alternaria, Post Bloom Fruit Drop 



• Onion: Perosnospora destructor, Botrytis squamosa 



211

212 

• Potato: NoBlight, Smith Periods, NegFry, TomCast 



• Asparagus: Puccina aparagi, Botrytis cinerea, Stemphylium versicarium 



• Rice: Sheath Blight, Rice Blast 



• Wheat: Fusarium, Septoria, Puccinia ssp. 



• Tomato: Leveillula taurica, Botrytis cinerea, Septoria lycopersici, Colletotrichum 

gloeosporioides, Cladosporium fulvum, Phytophtora infestans, /Phytophtora capsic/, 

Alternaria alternata, Sclerotium rolfscii 



• Carrots and Red Beets: TomCast, Sclerotinia 

o This model is available since 2006 


• Turf Gras: Dollar Spot, Brown Patch, Pythium Blight, Snow Mould 

o This model is available since 2005 


In total 2.500 weather stations are reporting their data to FieldClimate.Com and 3.500 Users 

are registered to use its services. 577 users have actually registered to plant disease models on 

FieldClimate.com. A high number of this user accounts is used by user groups or institutional 

users. This data are from February 2009. Registration for the use of the disease models is 

needed since September 2008. Most clients from northern hemisphere will register themselves 

during May. 

FUSARIUM HEAD BLIGHT MODEL USED IN FIELDCLIMATE.COM 

Fusarium Head Blight Infections will only take place in periods of very high relative humidity 

or the availability of free moisture (LINEMANN 2003, MARIN et al (1995), WOLF et al 

(2008). In dependence of temperature infection will take longer or shorter to be finished. This 

gives us the possibility to describe the conditions for infection and to calculate if this 

conditions have been fulfilled in the ongoing climate situation. 

From this the fusarium head blight model on FieldClimate.Com is first at all an infection 

model like we are used to this by apple scab (Venturia inaequalis) or Grape vine Downy 

Mildew (Plasmopara viticola).

Fusarium head blight infections from primary inoculumcan take place in stage 61 to 69. 

Infection in this stage during extended moist periods will lead to the total damage of parts of 

the head. Ongoing infections from already infected heas can go in to stage 85. This late 

ongoing infections will need extended mosit periods and they will increase the mycotoxin 

contends in the corn. 

The model evaluating the mycotoxin risk by fusarium head blight is accumulating the possible 

infection periods from stage 61 up to stage 85. in dependence of field history (non tillage corn 

or wheat before, tillage corn or wheat before, no corn or wheat before) an accumulated leaf 

wetness periods needed for 2, 4 or 6 fulfilled infection periods will lead to the maximum 

accepted risk. 

213

Rajkovic S, Tabakovic–Tosic M: Sustainable Development – Good Agriculture Practice Eriterion in Crop 

Production System. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

3-46 Sustainable Development – Good Agriculture Practice Eriterion in 

Crop Production System 

Rajkovic S, Tabakovic – Tosic M 

Institute of Forestry, Kneza Viseslava 3, 11030 Belgrade, Serbia 

Email: izbisfu@betel.yu 

214 

Abstract 

Agriculture has been dramatically changing since the Second World War. New 

technologies in for example farm machinery, increased use of chemicals and 

specialization have found a use in food production, controlled by government 

support for maximizing production. These changes have enabled a small number of 

farmers to produce larger quantities of food with limited resources. 

Although, these changes have had positive effect and have reduced many risks in 

agriculture, a high price has been paid. The most frequent risks are soil pollution, 

ground water contamination, family farms bankruptcy, low living and working 

conditions of the farm workers, increased production costs, and disintegration of 

economic and social conditions in rural communities. During the production 

process there was an occurrence of pathogen resistance to applied pesticides, thus 

making production more difficult. 

Over the last two decades the role of agriculture in promoting the practical solutions 

to these problems has been developed. Today, this movement for sustainable 

agriculture contributes to the increased support and acceptance even in organic 

agriculture. Sustainable agriculture does not solely refer to numerous and social 

concerns, it also offers innovative and economical possibilities for farmers, 

workers, consumers, policy makers and many others in the food production chain. 

This paper summarizes the ideas, practices and policies which contribute to our 

concept of sustainable agriculture. This includes overcoming plant resistance 

problems in the production process as well as the avoidance of plant diseases 

resulting from pathogen resistance to pesticides.

INTRODUCTION 

The agricultural production has been under the attack of media because of the use of natural 

resources faster than they are restoring, while we are at the same time witnessing the abrupt 

and disproportional rise of world population having the effect on increasing food demand. The 

idea and call for sustainable agriculture development are imposed in this situation. In times 

when the world is experiencing crises through the lack of food, when we witness the non – 

ethical work conditions, when the pollution has become an existential problem; we can reach a 

conclusion that the need for sustainable agriculture development is even more pronounced. 

It is important to explain the sustainable agriculture development itself. The point of 

sustainable agriculture is how to grow enough of accessible good quality food, and at the same 

time keep the capacity for future and constant production. The application of sustainable 

agriculture is to improve natural resources management, protect the environment, develop 

cooperation with partners and contribute to the well – being of the local community and others. 

The sustainable development enables an even approach in order to satisfy our community’s 

current and future needs for food, while at the same time we take care of ecosystem 

preservation which in fact is the foundation for sustainable agriculture development. 

SUSTAINABLE DEVELOPMENT 

According to the present understanding of the concept the sustainable development comprises 

the following: 

− Balanced and righteous economic development which can last for a longer period; 

− Poverty reduction through strengthening the poor and providing better access to 

indispensable services and assets; 

− Participation of all interested parties in decision – making process (central and local 

authorities, non – governmental organizations (NGOs), private business sector, 

professional organizations, unions), through dialog and trust forming, with social 

ownership development; 

− Careful management and preservation (to the fullest extent possible) of non – restorable 

resources; 

− Rational use of power and natural resources (water, soil, forests, etc.); 

− Waste reduction, effective prevention and control of pollution, and ecology risks 

minimization; 

− Education and health system improvement as well as improvement in the equality of 

sexes; 

− Protection of cultural identities. 

There are four principles of the sustainable development representing guidelines to the 

sustainable agriculture development: 

215

− Restorable resources may be used up to the level permitted by their restorability degree; 

− Raw material sources which are threatened by destruction can be used in agriculture only 

if they can be replaced materially and functionally by restorable raw material and their 

application guaranties higher productivity; 

− Ecological pollution must not exceed the limit and hazardous material decomposition 

capacities offered by main ecologic media – water, air, and soil; 

− The time equivalent must exist between periods of supplemental fertilizing and soil 

damage on the one hand and natural time period of soil restoration on the other hand 

(Rajkovic 2007). 

These sustainable development concept imperatives have very strong ecologic dimension 

connected with the fact that the sustainable development discussion was from the beginning 

based on ecological updating issues and tightly connected to novelties in environment 

protection policy. 

SUSTAINABLE AGRICULTURE 

Sustainable agriculture is ecologically sustainable, economically capable for maintaining, 

socially responsible, preserving natural wealth from complete annihilation or eradication and 

serves as the foundation for future generations. 

Sustainable agriculture: 

− Uses methods and work procedures maximizing the land productivity, while at the same 

time minimizes harmful effects on soil, water, air and health of both farmers and 

consumers; 

− Places production methods and procedure maintaining natural resources in the centre of 

its interest; 

− Makes an effort to reduce the use of non – degradable matter and chemicals made on oil 

basis, to replace them and on long-term notice completely stops using them. These 

chemicals should be replaced by those made from degradable materials; 

− Uses work methods and procedures adjusted to work conditions in the localities in 

question; 

− Is based on the knowledge and capabilities of farmers and cattle breeders making an 

effort to include them completely in the production process (Deutscher 2002). 

However, we must not forget to pay considerable attention to both economical and social 

functions. These last two factors require respect of certain game rules concerning equal 

intergeneration division of immaterial and material resources. The economic component 

requires special attention, because connecting and regulating of economic interests is of 

extreme importance for sustainable development. Economic profit, achieved today during a 

very short time frame through damaging environment or thanks to social injustice, cannot be 

tolerated on the further route of sustainable development. 

216

If we apply the aforementioned principles to the agricultural policy, we will conclude that we 

must shape the sustainable agriculture policy in order to support the agriculture which: 

− Is economically speaking marked by productive trading, not dependant of the subsidies 

thus being competitive itself. The employed in agriculture do not earn solely by 

producing healthy food, by processing it and placing it on the market, but by including 

other possibilities for profit to their work, e.g. tourism sector, production of usable raw 

material and bio – mass energy. Besides, there are other possibilities for profit, through 

state fees instigating nature and environment protection; 

− When ecologic dimension is in question, natural resources soil, waters and air are used in 

order to prevent long – term negative influence on them. Meaning the minimum use 

possible of fertilizers and pesticides in order not to pollute surrounding soil and water 

areas. This correlation should protect natural wealth; maintain nature and genetic 

potential of plant and animal species; 

− Socially speaking it provides jobs in area of agriculture; 

− When ethics is in question, it provides protection for animals which are reared, provided 

with food and not subjected to torture; 

− Consumer protection represents a new policy paradigm. Historical compromise was 

made after the Second World War, according to which the imperative was to secure 

enough food stocks for the employed in industry and for the state needs; it has served its 

purpose. This compromise has now brought itself in question due to social structure 

changes. The skepticism more and more rises toward permanent subsidizing of certain 

products, independently of their quality and consequences caused by their production. 

This is changed by forming of the new social milieu which places higher quality and 

other demands. 

“Healthy, nourishing products made under ecologic and animal protection aspects are 

demanded. Consumer statements and demands to the farmers contribute to the attitude change 

from lack of confidence to utter confidence between farmers and consumers. Every consumer 

is under the obligation to reward sustainable agrarian policy by his behavior and to break the 

closed circle of domination of competitive production homesteads.” (Meyer 2002). 

However, we can all contribute to it when: 

− We decide to buy products from homesteads improving sustainable agriculture 

development; 

− We decide to buy products produced in our immediate surroundings that have not been 

transported a long way to our shelves; 

− We use products we produced ourselves, various cultures can be raised in the garden or 

on the balcony; 

− We properly dispose garbage which can be used for fertilizing; 

− We use fertilizers in small amounts or not at all; 

217

− We do not use pesticides; 

− We decide to sow in our garden plants which thrive in our climate. 

ENVIRONMENT PROTECTION 

The most important environment problems consist of: 

− Old and polluting industries (technologies) – pollution of certain locations and poor 

ecological performance of main polluters; 

− The high pollution degree from urban sources – solid waste, waste water; undeveloped 

utility infrastructure also represents an obstacle to economic (and especially tourist) 

development; 

− Unsustainable use / over exploitation of resources like: energy, water and forest; 

− The increasing influence of traffic – road traffic (dissatisfying fuel quality, old vehicles, 

traffic jams in large cities and the bad public transport system, the need for building new 

roads), the non – existence of measures for solving pollution problems caused by boats / 

vessels; 

− Biodiversity threats (because habitats of certain species are disappearing, as well as the 

over – use of commercial species); 

− Problems with soil use and building without plans, which is especially pronounced in 

localities suitable for tourism development. 

The environment system is weak and without capacities to solve these problems in an effective 

way. The most important problems comprise weak / low institute capacity, fragmented and 

sometimes contradictory laws and not abiding by them in practice, dissatisfying finance (less 

than 0. 1% of GDP is spent on environment protection from public sources). Monitoring and 

informing systems do not provide good information basis for decision – making, while 

ecologic indicators are not available and / or not comparable. There is a large space for 

improvement connected to information approach and participation of the public in the decision 

– making process on environment issues (UNFCCC – Framework of United Nation 

Convention on Climate Changes, CBD – Convention on Biological Diversity). 

ORGANIC AGRICULTURE 

All over the world more and more farmers are turning to “biodynamic” or “organic” 

production. Meaning? First and foremost, it means that they are using techniques and work 

methods that are not based on chemical and genetic substance and technologies of agro – 

industry, but are based on ecological knowledge. Without harmful “counter effects” they can 

increase their crop yield, control the pests and keep the soil fertile. It is important to mention 

the giving up on mono – cultures, which was the common practice during the colonial period. 

218

Various cultures are planted, and according to the principle of changing of planting places, so 

that the insects which gather around one culture disappear during the next planting period. 

They know it is not wise to completely eliminate the pests, because that would mean disturbing 

the balance of a healthy ecosystem. Instead of artificial fertilizers these farmers fertilize the soil 

with plant remains, thus returning the organic matter without disrupting natural biologic 

circling of the matter (Capra 2002). 

The organic production is sustainable because it is in accordance with ecologic principles. This 

production means the taking of the total complexity of ecosystem in which is found and of 

which it lives on and participates in other natural cycles. This way of thinking and acting is the 

constituent part of every production aspiring to be sustainable. 

Bio – farmers know that the fertile soil is alive, filled in its every cubic centimeter with billions 

of live organisms. That it is a complex eco – composition whence all substances are cyclically 

changing from plants to animals, fertilizers and bio – bacteria. The sun energy is a natural 

material for burning which instigates those cycles. 

The motto of the sustainable agriculture could be: Learn from Nature, and do not try to 

overcome or manipulate with Nature! 

It is often emphasized that sustainable way of production cannot provide the satisfying 

amounts of the food needed for the world population which is constantly growing. This attitude 

can be refuted with arguments obtained from experience so far and conducted researches over 

the years. Frijof Capra has summarized a few interesting results in one exposition given on 

International Conference on Sustainable Agriculture Development, held in Bellagio, an Italian 

town in 1999: 

In Bellagio the scientists have handed in a report saying” that the use of experimental 

techniques in certain parts of the world – change of crop planting places, the use of natural 

fertilizers and compost, production on balconies and water areas – different zones up until then 

considered inadequate for production of food surplus have yielded spectacular results. 

To illustrate, agro – ecological projects participated by 730, 000 families in Africa have 

assisted the production increase from 50 to 100 per cent, while the production costs were 

drastically reduced ensuring production expansion. During the conducting of these projects it 

showed over and over again that organic production not only increases producing and offers a 

large number of ecologic advantages, but it is also profitable for farmers.” (Capra 2002). 

The person buying merchandize and products made in the process of fair trade takes on a 

global responsibility. The one buying products coming from organic agricultural production 

protects us and our environment. The individual buying products from his surrounding, cares 

about relieving traffic, ensures jobs and instigates the economic development in rural regions 

(Duesterhaus 1990). 

219

SUSTAINABLE AGRICULTURE AND RURAL DEVELOPMENT 

The potential for agricultural development is not sufficiently used, and represents a significant 

possibility for developing, especially concerning the health food production. Agriculture 

development would, also, help in slowing down the negative trends like leaving the village and 

people migrations and increasing employment. However, significant stimulation and living 

conditions improvement are needed to achieve more balanced development of rural areas. 

Tourism is also an area which could contribute to the rural area development. 

Priority activities should be taken toward more integrated, diversified and participating forms 

of rural development, in the framework of agricultural development policy integrating 

ecological aspects and promoting synergy between agriculture, tourism, industry and service, 

to provide sustainable management of vital resources (soil, biodiversity, water), limit the risk 

factors (fires, floods, pollution), offer a way out of rural poverty and reduce village leaving 

(urban areas, the coastal region emigration). 

A significant growth in institutional abilities is necessary as well as considerable financial 

funds in overcoming challenges and achieving progress in realization of goals for sustainable 

development. Thus it is necessary: 

− To build capacities and rise the conscience level on sustainable development concept. 

Building capacities and rising the conscience levels among all parties interested, 

including short – term measures and systematic, long – term process (e.g. through 

education system reform); 

− To mobilize all relevant interested parties not only on the national, but on the regional 

and global level and form partnerships. Mobilization of the parties interested is especially 

important as a mean of providing good acceptance of the strategy and for building trust 

among different participants, firstly in national context; 

− To set real goals and priorities and coordinate competitive priorities; 

− To mobilize the necessary finance funds from all the sources available; and 

− To make efforts to improve efficiency of international help for implementing the 

sustainable development concept. 

CONCLUSION 

It could be said in conclusion that: 

− Sustainable development implies a continuous process which should be conducted 

without rigidity; 

− Flexibly and via institutional resources which are to be adjusted to ever changing 

circumstances; 

− And which should be managed by improvements and dialogues between all sides 

included in the process. 

220

− The current financial and institutional funds should in that sense be expanded, increased 

and efficiently used; 

− At the same time create and use resources and instruments for acting. 

− We should identify and implement monitoring mechanisms and sustainable development 

indicators, which are in accordance with those elaborated on by the Mediterranean 

commission on sustainable development (MCSD) in order to evaluate the results of the 

priority of actions undertaken. 

REFERENCES 

Capra F (2002). Verborgene Zusammenhänge. Vernetzt denken und handeln - in Wirtschaft, 

Politik, Wissenschaft und Gesellschaft, Scherz: Bern. 

Duesterhaus R (1990). Sustainability's Promise. Journal of Soil and Water Conservation 

45(1), 4. 

Meyer H; Gaum W (2002). 10 Jahre nach Rio – Wie nachhaltig ist die Agrarpolitik? Aus 

Politik und Zeitgeschichte 31-32, 28-29. 

Rajković S; Dražić D; Rakonjac Lj; Veselinović M; Ratknić M (2007). Održivi razvoj i 

poljoprivreda. Ecologica 14, 159-164. 

221

Srivastava M P: Role of Phytomedcine and Plant Health Clinic in Plant Health Security. In: Feldmann F, Alford D 



3-47 Role of Phytomedcine and Plant Health Clinic in Plant Health Security 

Srivastava M P 

Haryana Agricultural University, F-44 Tulip Garden, Sushant Lok-II, Gurgaon, India 

E-mail: mpsrivastava@indiatimes.com 

INTRODUCTION 

Food is the basic requirement of man without which his survival is at stake. Ever growing 

population and more importantly unprecedented losses due to plant diseases and other pests 

pose serious threat to food security. In nutshell, food security implies availability of food to 

every individual. The Food and Agriculture Organization of the United Nations (FAO) defines 

“food security” as a state of affairs where all people at all times have access to safe and 

nutritious food to maintain a healthy and active life. This implies that in order to enjoy food 

security, there must be on the one hand a provision of adequate safe and nutritious food and, on 

the other, rich and poor, male and female, old and young must have access to it. Managing 

burgeoning population appears to be an uphill task as it involves social and political 

commitment. However, devastating losses due to plant diseases and other pests could be 

prevented to a larger extent by providing stringent health security to plants and thereby 

assuring food security to ever growing population. But this is not as simple as it appears. It 

requires careful short- and long-term planning. Besides we have to produce more food from 

continuously reducing arable land by genetic manipulation, biotechnology, or organic farming, 

but most importantly providing health security in all eventualities to plants through 

phytomedicines (Srivastava 1998c) and relentless support of plant health clinics (Srivastava 

1998b, 2003, 2005a,b, 2008). I shall deliberate on these issues as to how food security can be 

achieved by strengthening plant health clinic worldwide and promoting rational use of 

phytomedicines. 

POPULATION AND FOOD REQUIREMENT 

Ever rising population poses myriad problems, the most important being food security. The 

world population was more than doubled in the last half century and reached 6 billion in 1999. 

Each year it is adding approximately 73 million people – a population nearly the size of 

Vietnam. By 2030, it is projected to reach 8 billion, and nearly all that increase is expected to 

occur in developing nations, which are also expected to see rapid urbanization and consequent 

222

eduction in arable land. At the same time world’s hungry and chronically malnourished 

remain at about 840 million people, despite global pledges and national effort to improve food 

security 

POPULATION SCENARIO IN DIFFERENT CONTINENTS 

According to US Census Bureau (2009) as of March 2009, the world population is estimated to 

be 6.8 billion and is expected to reach 9 billion by 2040. Today Asia accounts for over 60% of 

the world population with almost 3800 million people. The People Republic of China and India 

alone comprise 20% and 17% respectively. Africa follows with 840 million, 12% of the world 

population. Europe 716 million people make up 11% of the world population. North America 

has a population of 514 million with US population of 305 million while S. America is home to 

371 million, 5.3% of the world population, and Australia 21 million. Food grain requirement of 

different regions of the world are provided in Table 1, which hints for higher food requirement 

in Asia and Africa in view of ballooning population. Looking at the world demographic 

scenario, Europe has the distinction of more or less stable population, while Asia, Africa and 

South America have witnessed continuous increase in population. Therefore growing 

population may not pose as serious a problem towards food security in Europe but higher per 

capita consumption cannot be ignored. 

Table 1. Food grain requirement of different regions of the world in 2025 (Sinha et al 1988) 

Regions Population Average per capita Food Requirement 

(Billions) consumption (kg) (MT) 

Africa 1.62 257 416 

South America 0.78 296 231 

Asia 4.54. 300 1362 

North America 0.35 885 310 

Europe 0.52 700 364 

USSR 0.37 983 364 

Oceania 0.04 578 23 

World 8.22 373 3070 

The phenomenal increase in population has lead to faster urbanization an industrialization 

affecting land resources. Against this backdrop, the continuing shrinkage of arable land poses 

serious threat to food security resulting in reduction in arable land. Therefore increase in arable 

land would be required to meet the food demand of the growing population (WECD 1987). 

How far this actually happens remains to be judged but the fact cannot be denied that reduction 

in arable land will be on going, we may have to strive hard to grow more from the limited area 

adopting even strategies like biotechnology & organic agriculture, and more importantly 

protecting the crops from onslaught of diseases and other pests. 

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BIOTECHNOLGY 

Biotechnology is believed to play an increasingly important role for food and health security. 

David King, Britain’s Chief Scientific Advisor while stepping down from his post in 2007 has 

called GM technology as the only “sophisticated” solution to feed future billions of hungry 

mouth. There are, however, widespread concern about the potential adverse impact of GMOs 

on human health, biodiversity and environment. Therefore to strengthen this area and to benefit 

mankind, more strenuous research covering health and safety aspects is required besides 

mandatory labeling regulation worldwide. 

The technology has also helped in producing genetically engineered plants resistant to insect 

pests and diseases (Dempsy et al. 1998). Use of transgenics has been helpful in reducing pest 

attack and investments in pesticides. However, there are instances where pesticide umbrella 

was provided to transgenics as was applied to non-transgenics. How long resistance lasts is yet 

to be seen and therefore use of pesticides may be required in long run to protect crop produced 

involving biotech. 

ORGANIC FARMING 

Promoted by environmentalists, organic farming is aimed at producing food without using 

chemicals and pesticides. Although produce with organic tags/certificates may fetch higher 

price, it cannot feed an ever-increasing population more so in Asia and Africa. It appears more 

to be illusion. It may, however be practiced in western world where the population has 

stabilized, but certainly not in India or in China, which dominate the demographic scenario 

(Srivastava 2003). 

Dr. Norman Borlaug, the renowned agricultural scientist and Nobel Laureate, who was 

instrumental in ushering in the Indian green revolution in the 1970s, said: “The idea that 

organic farming is better for the environment is ridiculous, because organic farming produces 

lower yields and, therefore, requires more land under cultivation, to produce the same amount 

of food. Thanks to synthetic fertilizers, global cereal production tripled between 1950 and 

2000, but the area of land used increased by only 10 per cent. According to a report from 

Murthy (2008) in spite of wide media support, the area under organic farming in India is about 

2 per cent of the total cultivated area while balance 98 per cent is cultivated by the farmers to 

produce food for the billions at affordable price. It is worth quoting Dr Jacques Diouf, Director 

General, FAO: “Organic agriculture can contribute to fighting hunger but can not feed over six 

billion people today and nine billion by 2050 without judicious use of chemical and fertilizers” 

In view of the above considerations, we have to lay greater emphasis on security of crops from 

plant diseases and pests which may result is 40 per cent increase in productivity world wide, 

which is otherwise lost due to various pests. 

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PLANT DISEASES 

Plants diseases are known to cause unprecedented losses throughout the world. Intensive and 

extensive cropping with a view to produce more from limited land has led to an increase in 

disease and pest problem. Globally various pests cause 40% yield reduction in principal food 

and cash crop every year of which plant diseases account for 20% (Oerke et al. 1994). In India, 

annual loss due to various pests amount to INR 30 000 crores (approx. $ US 60 billion) of 

which diseases account for 28% costing approximately INR 8 400 crores ($ US 16.8 billion; 

Srivastava 2001). As per recent estimates, India is loosing annually INR 1,40,000 crores worth 

of crops to various insect-pests, diseases and weeds (Kumarasamy 2008) Thus an everincreasing 

population on one-hand and plant diseases and pests on the other continuously 

threaten food security. Since increasing arable land to produce additional food to meet 

requirement of the growing population is not an easy proposition, the option lies in providing 

health security to our crops and protecting the losses from pests and diseases. 

PLANT HEALTH SECURITY 

Plant health security implies providing health care to plants from the very beginning by taking 

holistic or integrated approach. This involves availability well proven socio-economically 

viable technology from knowledge resource centers (Srivastava 1998a, 2003) besides 

uninterrupted 7x24 hrs access to such centers, and more importantly diagnostic and advisory 

support from plant health clinic, on use of pesticides or phytomedicines besides other 

preventive measures and reliance on host resistance. 

THE PLANT HEALTH CLINIC 

Failure in timely diagnosis of diseases and other pests has often been responsible for 

devastating losses. However, the losses can be avoided with timely diagnostic support of plant 

health clinic. These clinics exist only some of the countries and there too they are not widely 

prevalent. Such clinics operate as unit of Plant Pathology Department in India. Under 

Horticulture Mission more plant health clinics are in the offing. In USA and Canada too plant 

health clinics are operating with the Department of Plant Pathology of various state 

universities. Global Plant Clinic of CABI is operational in UK and Afro-Asian countries. 

Unfortunately we don’t have organized plant clinic with its independent identity like the ones 

for humans and animals. Plant clinic is likely to revolutionize plant protection. As such we 

need well-organized clinic/polyclinic for comprehensive diagnosis of all pests through skilled 

professionals equipped with diagnostic tools, monitor-aided microscope, audio, video, internet 

facility and toll free telecommunication. Most of the countries therefore are required to create 

plant clinic. Srivastava (2008b) in his opening remarks in ‘Plant Health Clinic’ Session, 

organized by him during ICPP 2008 at Torino, Italy called upon the nations to join hands in 

‘Mission Plant Health Clinic’ and work towards creation of more and more plant health clinics 

so that world may witness boom of plant clinic as global phenomenon. Mobile plant clinic 

225

deserve equal attention as they come to the rescue of the growers by providing needed health 

care during epiphytotic outbreaks. Mobile plant clinics with modest diagnostic tools and 

trained professionals may provide on-the-spot diagnosis in field condition during disease 

outbreak. Such clinics have helped in averting epiphytotics in India (Srivastava 2008). Plant 

clinics may also organize camps on plant health, judicious use of pesticides, fungicide 

resistance management and promoting IPM besides issuing pest alerts like Plant Disease 

Warning issued by the author in the past. Plant clinics thus can play an important role of Savior 

of plants from pests and diseases world-wide. Therefore plant clinics must be created in 

nations, which lack it, and revamp them wherever they exist to make them more growersfriendly. 

While diagnosis is one of the major roles of plant clinics, advisory does not end by providing 

needed prescription with regard to use of phytomedicines at that moment of time. The clinics 

additionally have to lay greater emphasis on preventive measures, which often prove better 

than cure. Depending upon the disease scenario, it may exclusively seed treatment in case of 

loose smut of wheat and barley – being internally seed-borne, or seed-, soil treatment as in 

damping off, or prophylactic spray in late blight, or crop rotation in some soil borne diseases, 

or burning of refuge to minimize the inoculum load for the next season or providing suitable 

IPM module for future guidance for retrieving the situation. Since host resistance offers one of 

the best means of controlling the diseases and insect-pests, the clinics are required to impress 

upon the growers invariably to use resistant or tolerant cultivars so as to minimize the use of 

phytomedicines, aimed at minimizing the cost of cultivation and protecting environment and 

ecosystem from hazards arising from their excessive use. Therefore the role of plant health 

clinic needs to be redefined in view of foregoing mandate, and shall not remained confined to 

diagnostic and advisory but much more as explicitly provided in the foregoing paragraphs. 

PHYTOMEDICINES IN PLANT HEALTH SECURITY 

Pesticides also rightly referred to as phytomedicines are undoubtedly the medicines for treating 

plant ailments. Plant clinics can appropriately recommend the right phytomedicine for 

prevention and treatment of plant ailments. They are the best arsenals, which offer utmost 

security security to plants from ravages of plant diseases and other pests. The losses can be 

prevented to a larger extent by rational and timely use of pesticides. It is, however, unfortunate 

that many of us really lack insight to so called ‘Materia Medica’ of plant diseases. The notion 

regarding ill effect of fungicides or for that purpose pesticides or phytomedicines such as 

environmental pollution, accumulation of residues in food, feed, soil and water, and 

development of resistance are uncalled for. The issue has been highlighted very often by 

environmental lobby, which believe that pesticides have done more harm than good while 

ignoring the fact that it is user who is responsible and not the pesticides. Pesticides are 

poisonous entity and they need to be used with utmost care at the recommended dose as per 

need and not as per will. The role of pesticides in pest control is incredible; the ill effects have 

resulted due to poor knowledge, misuse and abuse of pesticides and therefore some ascribe 

226

pesticides as evil in plant protection. But considering their immense contribution in pest 

control, such evils are necessary in plant protection (Srivastava 1999). Pesticides, if used with 

caution, hazards to humans, environment and ecosystem can be avoided. IPM propounded by 

Stern et al. (1959) long ago is one of the means of overcoming such problems. Srivastava 

(2003) has asserted that plant pathology cannot be practiced without phytomedicines or 

fungicides, which can only provide respite during a serious disease outbreak. 

Fungicides and plant diseases 

Fungicides are the Phytomedicines that offer security to plants from various plant pathogens. 

Scarcity of food due to outbreaks of epiphytotics led to development of wide range of 

fungicides viz., Bordeaux mixture, copper oxychloride, Sulphur, dithiocarbamates, dinocap. 

The first systemic fungicides carboxin and oxycarboxin made their debut in 1966 followed by 

carbendazim 2 years later (Table 2). Discovery of phenylamides and fosetyl-Al revolutionized 

control of Oomycetous fungi. The process of discovery gained momentum and more and more 

effective safe fungicides such as SBIs, MBIs (tricyclazole, pyroquilon etc.), strobilurins were 

developed in spite of outbursts against pesticide by Rachel Carson in 1962. Today with the 

availability of safer and effective fungicides of 4 th generation including novel fungicides, most 

of the diseases can be effectively controlled and crop yields can be improved. While 

phenylamides and fosetyl-Al have revolutionized control of downy mildews and 

phytophthoras, SBIs have offered control of diverse group of fungi, MBIs to rice blast and 

strobilurins unusually wide array of crop diseases from all four classes of plant pathogens, 

namely the Ascomycetes, Basidiomycetes, Deuteromycetes and Oomycetes. 

Therefore judicious use of fungicides may provide considerable security from onslaught of 

diseases and ensure adequate food security by preventing the losses. However, failure is not 

ruled out if wisdom is not applied in their use. We have not to be misled by environmentalist 

lobby and we have to adopt realistic approach towards use of Phytomedicines in order to 

ensure crop security and consequently food security. 

Measure to ensure optimum gain from fungicides 

Often use of outdated and dubious pesticides have resulted in poor or no control of pests and 

diseases and a double loss to the users. The worst hit are often poor, gullible farmers. 

According to Kumarasamy (2008) considering cost benefit ratio of 1:5 for genuine pesticides, 

and on an estimated sale of INR 1200 crores of spurious pesticides, farmers lose about INR 

600 crores hence users must exercise utmost caution to ensure genuineness of pesticides and 

also that they are not outdated. 

Another matter of concern is repeated use of systemic fungicides leading to resistance 

development and consequently poor control of diseases and ultimately setback to growers. 

Development of resistance has assumed significant importance with benzimidazoles, 

phenylamides, and antibiotics. Strobilurins – the novel fungicides, which offer security to 

227

plants from invasion of fungi from oomycetes, ascomycetes, basidiomycetes and 

deuteromycetes have to be used with caution as development of resistance is an innate problem 

with this group of fungicides. It is therefore imperative to stringently follow the guidelines of 

Fungicide Resistance Action Committee (FRAC). Unfortunately many people are unaware or 

are least concerned and hence face the music later. 

228 

Table 2. Milestones in the discovery of fungicides in the last 50 years of the 20 th 

century 

Fungicide group Common name Year of Discovery/launch 

Aromatics Dinocap, PCNB & 

chlorothalonil 

1946, 1930, 1964 

Oxathiins Carboxins and oxycarboxins 1966 

Benzimidazoles Carbendazim, benomyl 1968 

Organophosphorus Edifenphos, IBP, tolcophos 

methyl (Rhizolex) 

1970s 

Phenylamides Acylalanines, butyrolactones & 

oxazolidinones 

Late 1970s, 80s 

Carbamates Prothiocarb & propamocarb 1974, 1981 

Alkylphosphonate (Fosetyl Al) 1977 

Cyanoacetamide oxime 

(cymoxanil) 

1976 

Cinnamic acid derivative 

(dimethomorph) 

1988 

Triazole Tridemefon, tridimenol, 

propiconazole 

1973, 1978, 1979 

Penconazole, tebuconazole, 

hexaconazole 

1983, 1986 

Melanin Biosynthesis Inhibitor Pyroquilon, tricyclazole, KTU 

3616 

1980s 

Protein Synthesis Inhibitor Blasticidin S, kasugamycin 1995, 1965 

Phosphatidylinositol Syn. 

Inhibitor 

Validacin (Validamycin) 1970 

Natural products/ Novel 

fungicides 

Phenyl pyrroles 1980 

Strobilurin [Kresoxim methyl & 

Azoxystrobin] 

1990 

(Source: Srivastava 2003) 

It has also been observed that very often a cocktail of pesticides have been used. Not 

necessarily the combination of the two pesticides may be compatible or even synergistic; 

conversely incompatible or antagonistic. Often situation may not warrant use of fungicides and 

insecticide simultaneously but used on the behest of pesticide dealer. This should be avoided, 

as it only increases the cost of operation without any substantial gain. In certain situation it 

may cause even adverse effect on crops or poor management of the pests. Therefore 

compatibility of two or more pesticide in question must be ensured before mixing.

EPILOGUE 

Reduction in arable land due to an ever-increasing population and unprecedented losses due to 

plant diseases and other pests poses serious threat to food security. This can, however, be 

averted by increasing productivity by resorting to improved and sophisticated technology on 

one hand and more importantly providing health security to plants on the other and thereby 

preventing losses from pests and diseases. This can be achieved by timely diagnostic support of 

plant health clinics. Such clinics with diagnostic facility for plant diseases and other pests need 

to be created word wide and revamped to meet the aspiration of the growers. Simultaneously 

we must endeavour to promote mobile plant health clinic, which can come to the rescue of 

growers during epiphytotic outbreaks. Recommendation emanating from plant health clinic 

will go a long way in managing the problem. Have a deep insight to Materia Medica of plant 

diseases before selecting or using fungicides. Pesticide hazards, environmental pollution, 

resistance development can be avoided following guidelines of Pesticide Action Network 

(PAN) and Fungicide Resistance Action Committee (FRAC), and significant improvement in 

crop yield can be obtained. The use of pesticides are likely to increase productivity by 40 per 

cent which is otherwise lost to pests and diseases world wide. Let’s strengthen plant health 

clinic and promote phytomedicines to ensure food security without undermining preventive 

measures including deployment of host resistance. 

REFERENCES 

Carson R (1962). Silent Spring. Houghton Miffin: Boston, USA. 

Dempsey D A; Silva H; Klessig D F (1998). Engineering diseases and pest resistance in Plants. 

Trends in Microbiology 6, 54-61. 

Kumarasamy S (2008). Spurious pesticides, a growing menace, ruining farmers’ economy. 

Pestology 32(6), 15. 

Murthy K S R K (2008) Organic farming and food security (2008). Hindu Business Line, 

Friday, July 11, 2008. 

Oerke E C; Dehne H W; Schonbeck F; Webber A (1994). Crop Protection and Crop 

Production. Elsivier: Amsterdam. 

Sinha S K; Rao N H; Swaminathan M S (1988). Food security in the changing global climate. 

In: Conference proceeding for The Changing Atmosphere: Implications for Global 

Security, 27-30 June, 1988, Toronto, Canada, pp. 167-192. WMO- No. 170. Geneva: 

World Meteorological Organiztion 

Srivastava M P (1998). Information technology flow: an Asian perspective. 7 th International 

Congress of Plant Pathology (Abstr. 4.3.9), Edinburgh, 9-16 August, 1998. 

Srivastava M P (1998a). Plant clinic – a technological marvel in plant disease control. 

Pestology 23 (7), 70-71. 

Srivastava M P (1998b). Pesticides: The necessary evil in plant protection. In: Research 

progress in plant protection and plant nutrition, eds H Fuzeng & L Kexin, pp 239-243. 

China Agriculture Press: Beijing, China. 

229

Srivastava M P (2001). Pragmatic approach in plant protection in the third millennium. In: 

Plant diseases and their control – the selected papers of the First Asian Conference of 

Plant Pathology, eds Z Shimai, Z Guanghe & L Huifang, pp 4-7. China Agricultural 

Scientech Press: Beijing, China. 

Srivastava M P (2003). Transfer of plant pathology knowledge for rural prosperity – an Asian 

perspective. Presented as a Keynote Address at the 8 th International Congress of Plant 

Pathology, Christchurch, New Zealand, 2-7 February 2003. Australasian Plant 

Pathology 32, 187-194. 

Srivastava M P (2005a). Plant clinic in diagnosis and control of plant diseases – an Asian 

perspective. 2 nd Asian Conference of Plant pathology (Abstr p 3) Singapore, 25-28 

June 2005. 

Srivastava M P (2005b). Redefining the role and modalities of plant clinic. Natinal Symposium 

on Plant Disease Diagnosis, Epidemiology and Management (Abstr p 2), CCS Haryana 

Agricultural University, Hisar, 21-22 December, 2005. 

Srivastava M P (2008a). Plant health clinic – a global perspective. 8 th International Congress of 

Plant Pathology, Torino, Italy, 24-29 August 2008. Abstr: Journal of Plant Pathology, 

90 (2, Supplement), p S2.278 

Srivastava M P (2008b). Plant health clinic: challenges and opportunities – a global vision. An 

opening address in Evening Session 17: Plant Health Clinic, organized by Dr. M. P. 

Srivastava at 9 th International Congress of Plant Pathology, Torino, Italy, 27 August, 

2008. 

Stern V M; Smith R F; Bosch R v d; Hagen K (1959) The integrated control concept. 

Hilgardia 29, 81-101. 

US Census Bureau: US and World Population Clock Projection. http://www.census.gov/www/ 

popclockworld.html, accessed 15/02/2009 

WECD (1987). Food 2000: Global policies for sustainable agriculture. Report to the World 

Commission on Environment and Development. Zed Books Ltd: London. 

230

Sharma H C: Potential for Exploiting Host Plant Resistance to Insects for Food Security Under Subsistence 

Farming Conditions in the Semi-Arid Tropics. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to 



3-48 Potential for Exploiting Host Plant Resistance to Insects for Food 

Security Under Subsistence Farming Conditions in the Semi-Arid 

Tropics 

Sharma H C 

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 

324, Andhra Pradesh, India. 

Email: H.Sharma@cgiar.org 

Abstract 

Large scale application of pesticides to reduce the losses due to insect pests has not 

only led to serious environmental hazards, but has also resulted in development of 

resistance in pest populations. It is in this context that varieties capable of resisting 

pest damage will play a vital role in pest management, particularly under the harsh 

environments in the semi-arid tropics (SAT) in Asia and Africa, where insecticide 

use is either uneconomical or beyond the reach of resource poor farmers. 

Considerable progress has been made in identification of sources of resistance to the 

insect pests in crops such as sorghum, pearl millet, groundnut, chickpea, and 

pigeonpea – the principle cereal and grain legume crops in SAT.. However, there is 

a need to transfer the resistance genes into high-yielding cultivars with adaptation to 

different agro-ecosystems. Cultivars with stable resistance to midge (Stenodiplosis 

sorghicola) and shoot fly (Atherigona soccata) in sorghum, and pod fly 

(Melanagromyza obtusa) in pigeonpea have been developed and released for 

cultivation by the farmers; while genes from the wild relatives of sorghum, 

pigeonpea chickpea, and groundnut can be introgressed into varieties with low to 

moderate levels of resistance to stem borers (Chilo partellus, Busseola fusca, and 

Sesamia inferens) and shoot fly (A. soccata) in sorghum, and pod borers 

(Helicoverpa armigera and Maruca vitrata) in pigeonpea and H. armigera in 

chickpea to make host plant resistance an effective weapon in pest management. In 

addition, marker assisted selection and genetic engineering are being used to 

develop cultivars with resistance to stem borers and shoot fly in sorghum and pod 

borer, H. armigera in chickpea and pigeonpea. Development of insect-resistant 

varieties will not only cause a major reduction in pesticide use, but also lead to 

increased activity of beneficial organisms, and a safer environment to live. 

231

232 

PLENARY SESSIONS 4 - 12

Wege C: Mixed Infections of Geminiviruses and Unrelated RNA Viruses or Viroids in Tomato: A Multitude of 

Effects with a Highly Probable Impact on Epidemiology and Agriculture. In: Feldmann F, Alford D V, Furk C: 



4-1 Mixed Infections of Geminiviruses and Unrelated RNA Viruses or 

Viroids in Tomato: A Multitude of Effects with a Highly Probable 

Impact on Epidemiology and Agriculture 

Wege C 

Department of Molecular Biology and Virology of Plants, Universität Stuttgart, Institute of 

Biology, Pfaffenwaldring 57, D-70550 Stuttgart, Germany; 

Email: christina.wege@bio.uni-stuttgart.de; www.uni-stuttgart.de/bio/bioinst/molbio/ 

ABSTRACT 

Geminiviruses have become a major threat for tomato and pepper cultivation all 

over the world. Rising numbers of epidemics are caused by different mono- or 

bipartite members, or by multi-component disease complexes, in the genus 

Begomovirus. International transport of infected plant material has contributed to an 

unprecedented dissemination of tomato yellow leaf curl disease-causing 

geminiviruses into new environments, which gives rise to novel mixed infections 

with unrelated RNA viruses or viroids. The minireview summarises a set of case 

studies analysing the molecular effects of double-infections with ssDNA-containing 

begomoviruses and RNA-based pathogens under controlled experimental 

conditions. Several pathogen combinations resulted in a synergistic increase in 

symptoms, which, however, did not necessarily correlate with the molecular 

observations: Despite enhanced pathogenicity, co-invading RNA agents supported, 

did not affect at all, or even interfered negatively with two distinct begomoviruses 

analyzed in the studies, respectively. The interplay between antiviral silencing 

exerted by the host, and counteracting silencing suppression by both co-infecting 

vir(oid)al agents seemed to play a major role for the resulting infection. Situations 

with increased virus titres and enhanced tissue infiltration occurred, which might be 

of high ecologic and economic relevance. Notably, striking differences existed 

between the molecular responses of distinct tomato-infecting begomoviruses to one 

and the same co-infecting RNA agent, rendering any prediction for the outcome of 

mixed infections nearly impossible. 

233

MINIREVIEW 

The circular ssDNA-containing geminiviruses are amongst the agronomically most relevant 

viruses worldwide, having gained major importance for completely different types of crop 

plants during the past three decades (for a recent review on geminiviruses, refer to Jeske 2009). 

These range from vegetables such as tomato, pepper, cucurbits or Leguminosae via essential 

carbohydrate plants (potato and sweet potato, cassava, maize, main cereals of the temperate 

zones, sugarcane, beet), drugs and spices (tobacco, Lamiaceae, e.g. mint) up to fibre source 

plants, especially cotton. Geminiviruses in the whitefly-transmitted genus Begomovirus 

(Stanley et al. 2005; Fauquet et al. 2008) have been involved in the majority of recent 

economically relevant epidemics. Most of the respective diseases are caused by either bipartite 

begomoviruses, or by viral disease complexes consisting of usually a single helper virus 

component in association with one or more dependent, typically subviral molecules of different 

types (DNAs-β and DNAs-1; Briddon et al. 2003; Briddon et al. 2004; Briddon & Stanley 

2006; Mansoor et al. 2006; Briddon et al. 2008). However, the strictly monopartite Old World 

tomato yellow leaf curl virus (TYLCV), originating from the Middle East, is currently 

attracting special attention: This virus has been accidentally distributed internationally to an 

unprecedented extent. It threatens tomato and pepper cultivations of incalculable economic 

value - many of them only recently established - in almost any warm to temperate climate, 

between Venezuela, the U.S., European and African Mediterranean and sub-Saharan countries, 

China, Japan and Australia (Moriones & Navas-Castillo 2000; Ueda et al. 2004; Morales 2006; 

Delatte et al. 2007; Duffy & Holmes 2007; Garcia-Andres et al. 2007a; Ssekyewa et al. 2007; 

Walker 2007; Fernandes et al. 2008; Yongping et al. 2008). Due to its high prevalence in 

internationally transported plant material, including tomato fruit bunches from which the virus 

can be easily accessed and further transmitted by whiteflies (Delatte et al. 2003), TYLCV has 

become one of the first geminiviral examples of a plant virus spread nearly worldwide through 

human activity. In turn, it may be expected that bipartite New World begomoviruses or 

Eurasian begomoviral disease complexes of similar host range will be internationally 

distributed soon. Climate change further promotes the dissemination of whitefly vectors and, 

concomitantly, the vectored geminiviruses into newly accessed geographic regions (Jiu et al. 

2007; Liu et al. 2007; Morales 2007). The different viral travellers thereby get into competition 

and contact with each other more frequently than ever before. 

Symptoms, temporal, and spatial characteristics of the systemic spread, as well as the strict 

phloem limitation of TYLCV or related monopartite tomato-infecting viruses (such as tomato 

yellow Sardinia virus, TYLCSV) are very similar to those of several bipartite ones (e.g. Horns 

& Jeske 1991; Wege et al. 2000; Saunders et al. 2001; Wege et al. 2001; Morilla et al. 2004; 

Wege 2007; Fernandes et al. 2008). In double infections, distinct begomoviruses were shown 

to frequently replicate within the same accessible phloem nuclei (Morilla et al. 2004), which 

promotes intermolecular recombination via recombination-dependent replication (Jeske et al. 

2001; Preiss & Jeske 2003) and thus the occurrence of new, and - following selection - in 

several cases more pathogenic virus variants (as shown e.g. by Padidam et al. 1999; Fondong 

234

et al. 2000; Sanz et al. 2000; Pita et al. 2001; Monci et al. 2002; Kitamura et al. 2004; Legg & 

Fauquet 2004; Garcia-Andres et al. 2006; Garcia-Andres et al. 2007b; Duffy & Holmes 2008). 

Upon their dissemination into new habitats, begomoviruses also meet to an increasing extent 

unrelated RNA viruses and viroids, both of which are common pathogens of Solanaceous 

crops, too. 

A set of model experiments, using the bipartite tomato-infecting abutilon mosaic virus 

(AbMV; Wege et al. 2000, and references therein), has recently revealed that the outcome of 

mixed infections with begomo- and non-related viral or viroidal infectious agents is essentially 

non-predictable. Phenotypically, mixed infections of AbMV with unrelated RNA tobamo- or 

cucumoviruses or with viroids (potato spindle tuber viroid, PSTVd) led to striking synergistic 

symptom enhancement (Boschert, Kadri & Wege unpublished data; Pohl & Wege 2007; Wege 

& Siegmund 2007). Unexpectedly, however, molecular analyses showed that viroids and 

AbMV did not influence each other's titres, and tobamoviruses even exerted a negative effect 

not only on AbMV titre, but also on its infectivity. Both these infectious RNA-based agents did 

not alter the geminiviral tissue distribution (Boschert, Kadri & Wege unpublished; Pohl & 

Wege 2007). By contrast, the presence of cucumoviruses strongly raised the level of AbMV 

DNA in tomato as well as in further hosts (Wege & Siegmund 2007). The use of transgenic 

plants identified an important role of the cucumoviral 2b silencing suppressor in enhancing 

AbMV titres and numbers of invaded cells. Furthermore, the findings yielded the first evidence 

that AbMV can replicate in nonvascular cells: Upon co-infection with cucumber mosaic virus 

(CMV; subgroup I strains Le or Fny; family Bromoviridae; Palukaitis & García-Arenal 2003), 

the geminiviral phloem-limitation was broken at several sites of heavily affected leaves (Wege 

& Siegmund 2007). AbMV DNA accumulated in non-vascular mesophyll cells in spongy or 

palisade parenchyma, entering numerous cells far distant from the bundle sheath. This study 

represented the first molecular analysis on a true synergism of an RNA/ssDNA virus 

combination in plants. It suggests a significant impact of mixed infections involving 

begomoviruses and RNA pathogens on virus epidemiology, and thus ecology and agriculture: 

Raised virus titres have been shown to enhance insect transmission efficiencies (Rochow 

1972), and delocalization from phloem tissues has even been detected to confer mechanical 

transmissibility to an otherwise non-transmissible luteovirus (Ryabov et al. 2001). Similar 

results were recently obtained with co-infecting Poty- as well as Tombusviridae, which, 

however, despite of their positive effects on AbMV accumulation, did not induce any obvious 

increase in pathogenicity (Sardo, Tavazza, Accotto, Noris & Wege unpublished data). 

Concomitant experiments based on transgenic plants expressing potyviral (HC-Pro), or 

tombusviral (P19) silencing suppressors verified a supportive effect also of these RNA viral 

suppressor proteins on the DNA virus AbMV (unpublished). 

To analyse a putative additional role of the viral transport machineries in determining tissue 

infiltration and symptom severity, a number of studies was carried out using different plant 

lines expressing functional movement proteins of begomo- as well as of RNA viruses. 

Nicotiana benthamiana plants were established which expressed both movement-associated 

AbMV genes, BV1 and BC1, from DNA B component replicons released in every AbMV 

235

DNA A-multiplying cell (Wege & Pohl 2007). Their susceptibility for mechanical virus 

inoculation was strongly enhanced, verifying that AbMV proteins BV1 and BC1 were fully 

functional not only inside the phloem, but also in non-vascular parenchyma upon 

complementing viral transport from epidermal into the conductive tissues. In the opposite 

direction, however, AbMV was not able to traverse the vascular boundary and thus remained 

phloem-limited in systemically invaded transgenic tissues. Hence, some deficiency of the 

movement-associated proteins of AbMV in trafficking the viral transport complexes through 

asymmetric bundle sheath plasmodesmata cannot be ruled out (Wege & Pohl 2007). Bundle 

sheath cells are involved in the control of phloem unloading and signal routing (Ding et al. 

2003; Waigmann et al. 2004; Lough & Lucas 2006) and thus may represent a major barrier for 

the passage of viruses moving systemically via ternary complexes of nucleic acid and two 

types of movement-associated proteins, as it has been deduced from a number of different 

experiments for AbMV (Wege & Jeske 1998; Zhang et al. 2001; Aberle et al. 2002; 

Frischmuth et al. 2004; Hehnle et al. 2004; Frischmuth et al.a 2007; Kleinow et al. 2008). 

Transgenic plants constitutively expressing RNA (tobamo- or cucumo-) viral movement 

proteins known to increase plasmodesmatal size exclusion limits and to complement cell-tocell 

movement of non-related viruses, though, did not mediate AbMV egress from the phloem 

(Pohl & Wege 2007; Wege & Siegmund 2007). 

Taken together, these results indicate that in single infections, the limited AbMV accumulation 

as well as its strict phloem restriction involve tissue-specific antiviral silencing by the host, 

which may be specifically counteracted by suppressor proteins of the cucumoviral (2b), the 

potyviral (HC-Pro), or the tombusviral (P19) type, respectively, but not by tobamoviral (126K 

or 130K), viroidal, or any AbMV function (details including information on silencing 

suppressors which have been involved in synergistic effects are reviewed by Palukaitis & 

MacFarlane 2006; for an update on plant viral silencing suppressors, refer e.g. to Levy et al. 

2008). Additional deficiencies of AbMV movement-associated protein competences, namely at 

the bundle sheath boundary, remain possible. Bundle sheath cells, however, have also been 

shown to act as tissue domain-specific boundaries of antiviral silencing mechanisms (Deleris et 

al. 2006). This further supports the idea that mainly posttranscriptional gene silencing (PTGS), 

maybe in concerted action with transcriptional gene silencing (TGS) targeting the viral 

chromatin structures (Pilartz & Jeske 1992; Pilartz & Jeske 2003), impedes AbMV export from 

the phloem, unless a suitable helper suppressor protein is available. A combined 

complementation of silencing suppression and movement cannot be ruled out either, as it has 

been shown for a luteovirus which was mobilized into non-phloem cells only by co-delivered 

silencing suppression as well as movement functions (Ryabov et al. 2001). 

Recent experiments have compared the complex and in several aspects unexpected behaviour 

of the bipartite AbMV in mixed infections to that of a monopartite tomato-infecting 

begomovirus (TYLCSV); Sardo, Tavazza, Accotto, Noris & Wege; unpublished data). In 

symptom induction, cell-to-cell movement via putative ternary nucleoprotein complexes, and 

phloem-limited tissue tropism, TYLCSV strongly resembles the properties of AbMV (Wege 

2007, and references therein). Intriguingly, a parallel study on both TYLCSV and AbMV has 

236

Figure 1. AbMV/CMV doubly infected (A), singly AbMV- (B), or CMV-(C) infected 

Nicotiana benthamiana plants, respectively: begomoviral tissue tropism and 

symptoms at seven to eight weeks post infection (via agroinoculation). A1/2: 

In situ detection of AbMV DNA (dark stain) in consecutive cross sections of 

heavily affected, chlorotic young leaves double-invaded by AbMV and CMV. 

Signals on AbMV-infected nuclei in non-phloem tissues are indicated by 

arrows. In single infections, AbMV DNA was present in significantly lower 

numbers of nuclei, all of which were strictly confined to the phloem domain 

(not shown). Differential Contrast Microscopy (DIC); X: xylem, (e/i) ph: 

(external/internal) phloem, PalPar: palisade parenchyma, SpPar: spongy 

parenchyma (representative marking). Scale bars represent 50 µm. Right 

column: Symptoms of doubly (A3) and singly infected (B, C) plants: With 

respect to biomass (fresh as well as dry weight), plants co-infected with 

AbMV and CMV exhibited a synergistic increase in symptoms. Similar results 

were obtained for tomato. For those and further detailed data on mutual 

interactions between AbMV and CMV, refer to Wege & Siegmund (2007). 

revealed striking differences between the two begomoviruses' mutual interactions with coinfecting 

RNA viral pathogens. The results indicate that significant functional differences in 

the silencing suppression strategies pursued by distinct begomovirus species exist. This 

supports earlier molecular findings for different non-homologous geminiviral proteins, which 

obviously contribute to silencing suppression in a virus species-specific manner (Bisaro 2006). 

Irrespective of these probable differences in the PTGS/TGS suppression mechanism, distinct 

begomoviruses produce very similar diseases, with respect to phenotypic and molecular 

characteristics. The data illustrate that only a delicate interplay between a host plant's antiviral 

silencing machinery and interactive silencing suppression responses of co-invasive DNA and 

237

ssRNA viruses may uncover otherwise hidden differences in the tissue invasion tools utilized 

by distinct begomoviruses (Sardo, Tavazza, Accotto, Noris & Wege unpublished data). The 

mutual interdependencies are far from being understood, and are probably unique for every 

specific oligo- or multi-virus-host combination. After introduction of viruses or crop cultivars 

into a new environment, conditions of selection may therefore lead to unpredictable 

epidemiological risks. 

REFERENCES 

Aberle H J; Rütz M L; Karayavuz M; Frischmuth S; Wege C; Hülser D; Jeske H (2002). 

Localizing BC1 movement proteins of Abutilon mosaic geminivirus in yeasts by 

subcellular fractionation and freeze-fracture immunolabelling. Archives of Virology 

147, 103-107. 

Bisaro D M (2006). Silencing suppression by geminivirus proteins. Virology 344, 158-168. 

Briddon R W; Brown J K; Moriones E; Stanley J; Zerbini M; Zhou X; Fauquet C M (2008). 

Recommendations for the classification and nomenclature of the DNA-beta satellites of 

begomoviruses. Archives of Virology 153, 763-781. 

Briddon R W; Bull S E; Amin I; Idris A M; Mansoor S; Bedford I D; Dhawan P; Rishi N; 

Siwatch S S; Abdel-Salam A M; Brown J K; Zafar Y; Markham P G (2003). Diversity 

of DNA beta, a satellite molecule associated with some monopartite begomoviruses. 

Virology 312, 106-121. 

Briddon R W; Bull S E; Amin I; Mansoor S; Bedford I D; Rishi N; Siwatch S S; Zafar Y; 

Abdel-Salam A M; Markham P G (2004). Diversity of DNA 1: a satellite-like molecule 

associated with monopartite begomovirus-DNA beta complexes. Virology 324, 

462-474. 

Briddon R W; Stanley J (2006). Subviral agents associated with plant single-stranded DNA 

viruses. Virology 344, 198-210. 

Delatte H; Dalmon A; Rist D; Soustrade I; Wuster G; Lett J M; Goldbach R W; Peterschmitt 

M; Reynaud B (2003). Tomato yellow leaf curl virus can be acquired and transmitted 

by Bemisia tabaci (Gennadius) from tomato fruit. Plant Disease 87, 1297-1300. 

Delatte H; Holota H; Moury B; Reynaud B; Lett J M; Peterschmitt M (2007). Evidence for a 

founder effect after introduction of Tomato yellow leaf curl virus-mild in an insular 

environment. Journal of Molecular Evolution 65, 112-118. 

Deleris A; Gallego-Bartolome J; Bao J; Kasschau K D; Carrington J C; Voinnet O (2006). 

Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. 

Science 313, 68-71. 

Ding B; Itaya A; Qi Y (2003). Symplasmic protein and RNA traffic: regulatory points and 

regulatory factors. Current Opinion in Plant Biology 6, 596-602. 

Duffy S; Holmes E C (2007). Multiple introductions of the Old World begomovirus Tomato 

yellow leaf curl virus into the New World. Applied and Environmental Microbiology 

73, 7114-7117. 

Duffy S; Holmes E C (2008). Phylogenetic evidence for rapid rates of molecular evolution in 

the single-stranded DNA begomovirus tomato yellow leaf curl virus. Journal of 

Virology 82, 957-965. 

238

Fauquet C M; Briddon R W; Brown J K; Moriones E; Stanley J; Zerbini M; Zhou X (2008). 

Geminivirus strain demarcation and nomenclature. Archives of Virology 153, 783-821. 

Fernandes F R; de Albuquerque L C; de Britto Giordano L; Boiteux L S; de Avila A C; Inoue- 

Nagata A K (2008). Diversity and prevalence of Brazilian bipartite begomovirus 

species associated to tomatoes. Virus Genes 36, 251-258. 

Fondong V N; Pita J S; Rey M E C; de Kochko A; Beachy R N; Fauquet C M (2000). 

Evidence of synergism between African cassava mosaic virus and a new doublerecombinant 

geminivirus infecting cassava in Cameroon. Journal of General Virology 

81, 287-297. 

Frischmuth S; Kleinow T; Aberle H-J; Wege C; Hülser D; Jeske H (2004). Yeast two-hybrid 

systems confirm the membrane-association and oligomerization of BC1 but do not 

detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic 

geminivirus. Archives of Virology 149, 2349-2364. 

Frischmuth S; Wege C; Hülser D; Jeske H (2007). The movement protein BC1 promotes 

redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the 

plasma membrane in fission yeast. Protoplasma 230, 117-123. 

Garcia-Andres S; Accotto G P; Navas-Castillo J; Moriones E (2007a). Founder effect, plant 

host, and recombination shape the emergent population of begomoviruses that cause the 

tomato yellow leaf curl disease in the Mediterranean basin. Virology 359, 302-312. 

Garcia-Andres S; Monci F; Navas-Castillo J; Moriones E (2006). Begomovirus genetic 

diversity in the native plant reservoir Solanum nigrum: Evidence for the presence of a 

new virus species of recombinant nature. Virology 350, 433-442 

Garcia-Andres S; Tomas D M; Sanchez-Campos S; Navas-Castillo J; Moriones E (2007b). 

Frequent occurrence of recombinants in mixed infections of tomato yellow leaf curl 

disease-associated begomoviruses. Virology 365, 210-219. 

Hehnle S; Wege C; Jeske H (2004). The interaction of DNA with the movement proteins of 

geminiviruses revisited. Journal of Virology 78, 7698-7706. 

Horns T; Jeske H (1991). Localization of Abutilon mosaic virus DNA within leaf tissue by insitu 

hybridization. Virology 181, 580-588. 

Jeske H (2009). Geminiviruses. In: Torque Teno Virus: The Still Elusive Human Pathogens, 

eds H zur Hausen & E.-M.de Villiers, Vol 331, pp 185-226. Springer: Berlin. 

Jeske H; Lütgemeier M; Preiss W (2001). Distinct DNA forms indicate rolling circle and 

recombination-dependent replication of Abutilon mosaic geminivirus. EMBO Journal 

20, 6158-6167. 

Jiu M; Zhou X P; Tong L; Xu J; Yang X; Wan F H; Liu S S (2007). Vector-virus mutualism 

accelerates population increase of an invasive whitefly. PLoS ONE 2, e182. 

Kitamura K; Murayama A; Ikegami M (2004). Evidence for recombination among isolates of 

Tobacco leaf curl Japan virus and Honeysuckle yellow vein mosaic virus. Archives of 

Virology 149, 1221-1229. 

Kleinow T; Holeiter G; Nischang M; Stein M; Karayavuz M; Wege C; Jeske H (2008). Posttranslational 

modifications of Abutilon mosaic virus movement protein (BC1) in fission 

yeast. Virus Research 131, 86-94. 

Legg J P; Fauquet C M (2004). Cassava mosaic geminiviruses in Africa. Plant Molecular 

Biology 56, 585-599. 

Levy A; Dafny-Yelin M; Tzfira T (2008). Attacking the defenders: plant viruses fight back. 

Trends in Microbiology 16, 194-197. 

239

Liu S S; De Barro P J; Xu J; Luan J B; Zang L S; Ruan Y M; Wan F H (2007). Asymmetric 

mating interactions drive widespread invasion and displacement in a whitefly. Science 

318, 1769-1772. 

Lough T J; Lucas W J (2006). Integrative plant biology: Role of phloem long-distance 

macromolecular trafficking. Annual Review of Plant Biology 57, 203-232. 

Mansoor S; Zafar Y; Briddon R W (2006). Geminivirus disease complexes: the threat is 

spreading. Trends in Plant Science 11, 209-212. 

Monci F; Sanchez-Campos S; Navas-Castillo J; Moriones E (2002). A natural recombinant 

between the geminiviruses Tomato yellow leaf curl Sardinia virus and Tomato yellow 

leaf curl virus exhibits a novel pathogenic phenotype and is becoming prevalent in 

Spanish populations. Virology 303, 317-326. 

Morales F J (2006). History and current distribution of begomoviruses in Latin America. 

Advances in Virus Research 67, 127-162. 

Morales F J (2007). Tropical Whitefly IPM Project. Plant Virus Epidemiology 69, 249-311 

Morilla G; Krenz B; Jeske H; Bejarano E R; Wege C (2004). Tête à tête of Tomato yellow leaf 

curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in single 

nuclei. Journal of Virology 78, 10715–10723. 

Moriones E; Navas-Castillo J (2000). Tomato yellow leaf curl virus, an emerging virus 

complex causing epidemics worldwide. Virus Research 71, 123-134. 

Padidam M; Sawyer S; Fauquet C M (1999). Possible emergence of new geminiviruses by 

frequent recombination. Virology 285, 218-225. 

Palukaitis P; García-Arenal F (2003). Cucumber mosaic virus. AAB (Association of Applied 

Biologists) Descriptions of Plant Viruses 400, 1-20. www.dpvweb.net (date of access 

10.03.2009). 

Palukaitis P; MacFarlane S (2006). Viral Counter-Defense Molecules. In: Natural Resistance 

Mechanisms of Plants to Viruses, eds G Loebenstein & J P Carr, pp 165-185. Springer: 

The Netherlands. 

Pilartz M; Jeske H (1992). Abutilon mosaic geminivirus double-stranded DNA is packed into 

minichromosomes. Virology 189, 800-802 

Pilartz M; Jeske H (2003). Mapping of Abutilon mosaic geminivirus minichromosomes. 

Journal of Virology 77, 10808-10818. 

Pita J S; Fondong V N; Sangaré A; Otim-Nape G W; Ogwal S; Fauquet C M (2001). 

Recombination, pseudorecombination and synergism of geminiviruses are determinant 

keys to the epidemic of severe cassava mosaic disease in Uganda. Journal of General 

Virology 82, 655-665. 

Pohl D; Wege C (2007). Synergistic pathogenicity of a phloem-limited begomovirus and 

tobamoviruses, despite negative interference. Journal of General Virology 88, 1034- 

1040. 

Preiss W; Jeske H (2003). Multitasking in replication is common among geminiviruses. 

Journal of Virology 77, 2972-2980. 

Rochow W F (1972). Role of mixed infections in transmission of plant viruses by aphids. 

Annual Review of Phytopathology 10, 101-124. 

Ryabov E V; Fraser G; Mayo M A; Barker H; Taliansky M (2001). Umbravirus gene 

expression helps potato leafroll virus to invade mesophyll tissues and to be transmitted 

mechanically between plants. Virology 286, 363-372. 

240

Sanz A I; Fraile A; García-Arenal F; Zhou X; Robinson D J; Khalid S; Butt T; Harrison B D 

(2000). Multiple infection, recombination and genome relationships among 

begomovirus isolates found in cotton and other plants in Pakistan. Journal of General 

Virology 81, 1839-1849. 

Saunders K; Wege C; Karuppannan V; Jeske H; Stanley J (2001). The distinct disease 

phenotypes of the common and yellow vein strains of Tomato golden mosaic virus are 

determined by nucleotide differences in the 3'-terminal region of the gene encoding the 

movement protein. Journal of General Virology 82, 45-51. 

Ssekyewa C; Van Damme P L; Hofte M (2007). Relationship between Tomato yellow leaf curl 

viruses and the whitefly vector. Communications in Agricultural and Applied 

Biological Sciences 72, 1029-1048. 

Stanley J; Bisaro D M; Briddon R W; Brown J K; Fauquet C M; Harrison B D; Rybicki E P; 

Stenger D C (2005). Geminiviridae. In: Virus Taxonomy. VIIIth Report of the 

International Committee on Taxonomy of Viruses, eds C M Fauquet; M A Mayo; J 

Maniloff; U Desselberger & L A Ball, pp 301-326. Elsevier/Academic Press: London. 

Ueda S; Kimura T; Onuki M; Hanada K; Iwanami T (2004). Three distinct groups of isolates 

of Tomato yellow leaf curl virus in Japan and construction of an infectious clone. 

Journal of General Plant Pathology 70, 232-238. 

Waigmann E; Ueki S; Trutnyeva K; Citovsky V (2004). The ins and outs of nondestructive 

cell-to-cell and systemic movement of plant viruses. Critical Reviews in Plant Science 

23, 195-250. 

Walker K (2007). Tomato yellow leaf curl virus (TYLCV; Begomovirus). Pest and Diseases 

Image Library (PaDIL). www.padil.gov.au (date of access 10.03.2009). 

Wege C (2007). Movement and localization of tomato yellow leaf curl viruses in the infected 

plant. In: Tomato yellow leaf curl virus disease: management, molecular biology, 

breeding for resistance, ed H Czosnek, pp 185-206. Springer: Dordrecht/New York. 

Wege C; Gotthardt R D; Frischmuth T; Jeske H (2000). Fulfilling Koch's postulates for 

Abutilon mosaic virus. Archives of Virology 145, 2217-2225. 

Wege C; Jeske H (1998). Abutilon mosaic geminivirus proteins expressed and phosphorylated 

in Escherichia coli. Journal of Phytopathology 146, 613-621. 

Wege C; Pohl D (2007). Abutilon mosaic virus DNA B component supports mechanical virus 

transmission, but does not counteract begomoviral phloem limitation in transgenic 

plants. Virology 365, 173-186. 

Wege C; Saunders K; Stanley J; Jeske H (2001). Comparative analysis of tissue tropism of 

bipartite geminiviruses. Journal of Phytopathology 149, 359-368. 

Wege C; Siegmund D (2007). Synergism of a DNA and an RNA virus: Enhanced tissue 

infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber 

mosaic virus (CMV). Virology 357, 10-28. 

Yongping Z; Weimin Z; Huimei C; Yang Q; Kun S; Yanhui W; Longying Z; Li Y; Zhang H 

(2008). Molecular identification and the complete nucleotide sequence of TYLCV 

isolate from Shanghai of China. Virus Genes 36, 547-551. 

Zhang S C; Wege C; Jeske H (2001). Movement proteins (BC1 and BV1) of Abutilon mosaic 

geminivirus are cotransported in and between cells of sink but not of source leaves as 

detected by green fluorescent protein tagging. Virology 290, 249-260. 

241

Bargen S v, Arndt N, Grubits E, Büttner C, Jalkanen R: Cherry Leaf Roll Virus in birch – an old problem or an 

emerging virus in Finland? In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 


Germany 

4-2 Cherry Leaf Roll Virus in birch – an old problem or an emerging virus 

in Finland? 

Bargen S v, Arndt N, Grubits E, Büttner C, Jalkanen R 2 

Humboldt-Universität zu Berlin, Faculty of Agriculture and Horticulture, Section 

Phytomedicine, Lentzeallee 55/57, D-14195 Berlin, Germany 

2 

Finnish Forest Research Institute, Rovaniemi Research Unit, P.O. Box 16, FI-96301 

Rovaniemi, Finland 

Email: susanne.von.bargen@agrar.hu-berlin.de 

242 

ABSTRACT 

Cherry leaf roll virus (CLRV) was first mentioned to occur in Finland in 1980 by 

Cooper and Edwards who described an isolate from red elderberry (Sambucus 

racemosa). CLRV affecting birch species in the country have also been confirmed 

sporadically from single silver birch (Betula pendula) trees. Since 2002 the situation 

changed as virus-like symptoms in birch species native to Fennoscandia started to 

accumulate. Leaf roll, vein banding and chlorotic patterns with subsequent necrosis 

of birch leaves were increasingly observed. Disease symptoms affecting downy 

birch (B. pubescens ssp. pubescens), curly birch (B. pendula var. carelica), 

mountain birch (B. pubescens ssp. czerepanovii), dwarf birch (B. nana), the local 

variety Kiilopää birch (B. pendula var. appressa) and silver birch could be 

associated with an infection of CLRV. Since the incidence in 2002, virus-associated 

symptoms are spreading in Betula species and are distributed all over the country at 

present. As causes of the sudden appearance of the disease are still unknown, 

possible ways of transmission and viral dissemination as well as unique features of 

the CLRV population occurring in Finland are discussed. 

INTRODUCTION 

The birches (genus Betula) are common trees and shrubs of the boreal and north temperate 

zones of the Northern hemisphere; in Finland more than one fifth of the forest is birch forest 

and the genus represents the most abundant group of deciduous trees providing an important 

raw material in the mechanical and chemical forest industry (Peltola 2006). Downy birch

(Betula pubescens ssp. pubescens) and silver birch (B. pendula) are used industrially for 

plywood, veneer (Luostarinen & Verkasalo 2000) and paper production (Viherä-Aarnio & 

Velling 1999). Downy and silver birch are abundant throughout the country in towns, on 

roadsides and most common in mixed forests. North of the Arctic Circle dwarf birch (B. nana), 

Kiilopää birch (B. pubescens ssp. appressa) and mountain birch (B. pubescens ssp. 

czerepanovii) are dominant and important key components of the arctic ecosystem (Walker 

2000; van Wijk et al. 2005). 

Cherry leaf roll virus, CLRV, is a plant pathogen which belongs to the genus Nepovirus and 

the family Comoviridae. The virus is distributed worldwide and infects various deciduous trees 

and shrubs (Bandte & Büttner 2001). The seed and pollen-borne virus is also transmitted by 

mechanical means, grafting and root connation. 

Until recently CLRV has only been detected rarely in Finland and adjacent countries (Cooper 

& Edwards 1980; Bremer et al. 1991). However, since 2002 virus-related symptoms such as 

vein banding, leaf roll, chlorosis and subsequent necrosis on birch leaves were increasingly 

recorded throughout Fennoscandia. In a survey throughout Finland symptoms on birch were 

especially distinct during the dry summer of 2006 and it was found that several birch species 

were affected. Recently, CLRV was confirmed in Rovaniemi, northern Finland in several B. 

pubescens ssp. pubescens trees exhibiting symptoms of a viral disease (Jalkanen et al. 2007). 

Aims of the study were to determine CLRV distribution in Finland and occurrence of the virus 

in different Finnish birch species. Furthermore, genetic characteristics of individual CLRV 

isolates obtained from different locations in Finland and Betula species were assessed, in order 

to compare the Finnish CLRV population with other known CLRV isolates. 


More than seventy trees of the genus Betula exhibiting characteristic symptoms of a virus 

infection were sampled in 2007 and 2008 all over Finland. Furthermore, selected birch trees 

from stands in Kittilä and Läyliäinen used for seed production were included in the study as 

well as four water samples collected randomly in the vicinity of symptomatic trees. Singular 

rowan (Sorbus aucuparia) trees exhibiting ringspots and mottle were also sampled as well as 

red elderberry (Sambucus racemosa) exhibiting leaf deformations (Figure 1). 

Two twigs of individual trees were tested by a CLRV specific IC-RT-PCR (Jalkanen et al. 

2007) in duplicate by application of symptomatic leaves and buds, catkins or twig tips. Coat 

protein specific primers (CP188F and CP350R) were deduced and used alternatively in the IC- 

RT-PCR replacing the primer combination RW1 and RW2 established by Werner et al. (1997). 

Ten microliters per water sample were subjected to IC-RT-PCR and were also tested in 

duplicate. A tree/water sample was scored as CLRV positive, if a specific fragment of the 

expected size was amplified at least from one sample per tree/water sample. Partial fragments 

of the CLRV 3’ non-coding region (3´ NCR) fragments were digested with AluI, Bsp143I, or 

RsaI respectively to determine sequence variants of CLRV isolates detected in birch samples 

243

according to Buchhop et al. (in print). Selected PCR amplicons of the CLRV 3´ NCR as well 

as coat protein fragments obtained from CLRV contaminated samples in Finland were cloned 

and sequenced. PCR products were ligated into pBluescriptII SK(-)-vectors (Stratagene, USA) 

and transformed into chemocompetent E. coli using standard protocols (Sambrook et al. 1989). 

Constructs were purified from liquid bacterial cultures (InvisorbSpinPlasmid MiniII, Invitek, 

Germany) and inserts were sequenced from both directions by cycle sequencing and an ABI 

PRISM 310 Genetic Analyzer (Applied Biosystems, USA) by use of vector specific primers. 

Obtained sequences were analysed and compared to CLRV isolates characterised by 

Rebenstorf et al. (2006) applying ClustalX 1.83 (Thompson et al. 1997) using the incorporated 

neighbour-joining method for phylogenetic tree construction. 

244 

Figure 1. CLRV infected Betula sp. (A), Sorbus aucuparia (B), and Sambucus racemosa 

(C) exhibiting virus-like symptoms. 

RESULTS 

The birch trees sampled in 2007 from southern, central and northern Finland revealed 

numerous CLRV infections. Altogether, CLRV was proved to be in 55% of the symptomatic 

birch trees, thereof 18 downy birches (56%) and 12 silver birches (48%). Sampled dwarf 

birches (4), mountain birches (6) and Kiilopää birches (5) included in the study were limited; 

still, CLRV detection was successful at least in two trees per species. Additionally, the one 

curly birch sampled from a garden in Rovaniemi was CLRV positive (Table 1). Results 

confirmed that CLRV is widely distributed in different birch species throughout Finland, even 

north of Rovaniemi and the Arctic Circle up to northern and alpine tree line. Tree samples 

originated from rural areas, i.e. from alleys, parks (churchyards, schoolyard) and along 

roadsides in town centres but also from natural stands as for instance the samples collected in 

Inari. Furthermore, four symptomatic saplings — two B. pendula and two B. pubescens — 

originated from a 100-year-old seed-production stand in Kittilä (northern Finland) could be 

shown to be CLRV infected.

Table 1. Detection of CLRV by IC-RT-PCR in samples from Finland 

Sample Sampled, no. CLRV positive, no. 

symptomatic birch species 

B. pubescens subsp. Pubescens 32 18 

B. pendula 25 12 

B. pubescens subsp. Czerepanovii 6 2 

B. pubescens var. appressa 5 5 

B. nana 4 2 

B. pendula var. carelica 1 1 

birch seed-production stands 

Kittilä (B. pubescens subsp. pubescens, B. pendula) 4 4 

Läyliäinen (B. pendula) 5 2 

other species and environmental samples 

Sorbus aucuparia 6 2 

Sambucus racemosa 1 1 

water 4 1 

Total 93 50 

Figure 2. CLRV detection in six different birch trees by IC-RT-PCR amplification of the 

partial 3´ non-coding region (412 bp) with RW1 and RW2 primers developed 

by Werner et al. (1997) and subsequent RFLP analysis of CLRV specific 

fragments by use of the restriction endonucleases AluI, Bsp143I, and RsaI. 

Asterisks indicate shorter fragments of 404 bp. (Marker: 50 bp ladder, 

Fermentas) 

245

Similar results were obtained by testing asymptomatic silver birch trees from a seed-production 

stand, established in 1998 in southern Finland (Läyliäinen) comprising trees from a clone 

collection. In two out of five randomly sampled trees CLRV was detectable, which was 

confirmed by sequencing of the amplified fragments of the partial coat protein-coding region. 

In Finland the virus is not restricted to the genus Betula because it was also detected in two 

European mountain ash trees and a singular red elderberry exhibiting virus-like symptoms. 

Furthermore, a CLRV positive water sample (no. 152) taken from the lake Rautavesi in the 

Länsi-Suomi area in May 2008 revealed the contamination of surface water with the virus. 

CLRV infection of three downy, two silver birches and one mountain birch was also confirmed 

by restriction analysis and sequencing of the amplified 3´ NCR fragment. The samples 

originated from various locations in Finland, i.e. Rovaniemi and Inari (North), Lieksa (East) 

and Vaasa (West) and display size differences between 404 and 412 bp as well as sequence 

variability after restriction analysis (Figure 2). RFLP-types of CLRV strains from Finnish birch 

samples differed from virus variants characterised from other geographical origins. This was 

supported by sequence comparison of 3´ NCR fragments with CLRV strains characterised 

previously by Rebenstorf et al. (2006). Analysis revealed that fragments of CLRV strains 

obtained from Finnish birches shared highest sequence identities to CLRV isolates belonging 

to phylogenetic group B, D or E (data not shown). Sequencing of individual clones of the 

partial coat protein-coding region (161 bp) of selected B. pendula trees (no. 137, 140 and 256) 

and the positive water sample followed by phylogenetic analysis showed close relationships of 

samples from Finland (98.2–100% sequence identities); these samples are clearly distinguished 

from other CLRV strains characterised previously by Rebenstorf et al. (2006) (Figure 3). 

Partial coat protein sequences of CLRV strains obtained from birches in Germany and the 

United Kingdom enclosed in phylogenetic group A (I2, E441, E120) are most distantly related 

to virus strains found in Finnish birch species sharing only between 75.0–80.3% sequence 

identity at the nucleotide level of the analysed 112 bp. 

DISCUSSION 

CLRV has been confirmed in birch trees from several places in Finland by molecular means, 

revealing that the virus is widely distributed in the country and also affects at least six birch 

species or varieties native to Fennoscandia. The main route of CLRV dispersal in birch in 

natural habitats is assumed to be pollen and seed transmission, which has been studied in detail 

before (Cooper 1976, 1979; Cooper et al. 1984). Cross pollination resulting in Betula hybrids 

is commonly reported from the genus (Anamthawat-Jónsson & Thórsson 2003; Atkinson 1992) 

and may be a reason why the virus had been spread between different Betula species. In our 

investigations we found CLRV infected seedlings in a seed production stand in Kittilä in 

northern Finland as well as asymptomatic B. pendula trees harvested for seeds in the southern 

part of the country. Thus, contaminated seed could be a possible route of CLRV dispersal into 

planted birch populations. However, most trees with CLRV symptoms especially in the coun- 

246

Figure 3. Neighbour-joining phylogenetic tree calculated with ClustalX 1.83 from 112 

nucleotides of the partial CLRV coat protein-coding region amplified with 

primers CP188F/CP350R. Bootstrap analysis was performed with 1000 

replicates; values above 900 are indicated on branches. The bar length 

represents substitutions per nucleotide. Samples from Finland are boxed. 

Major groups (A–E) defined by Rebenstorf et al. (2006) are indicated by the 

respective character on the right side. 

247

tryside are naturally born. Further, most sampled birches from alleys and town birches are 

rather old, 40 to 80 years, suggesting that contaminated seed is not involved in the rapid spread 

of CLRV unless the trees were infected latently without symptom expression until recently. 

The role of insects as virus vectors has not been studied satisfactory, but the birch catkin bug 

Kleidocerys resedae as well as weevils (Polydrusus sp.) has been shown to carry the virus 

(Werner et al. 1997; Rebenstorf 2005). Potential insect vectors have never been under 

investigation in Finland; however, the contamination of surface water with CLRV may indicate 

towards an additional route of virus dissemination in the environment as was postulated 

already by Bandte et al. (2007). 

Sequence comparisons of the partial 3´ NCR revealed unusual phylogenetic relationships of 

Finnish CLRV isolates. Until now, phylogenetically characterised CLRV isolates of birch trees 

from the United Kingdom and Germany exclusively clustered within clade A (Rebenstorf et al. 

2006). They concluded that co-evolution of CLRV and host plant is a major factor that led to 

quick adaptation of virus populations within one host species, which could be genetically 

differentiated according to infected plant species. The majority of CLRV isolates from Finnish 

birch trees were found to relate to other phylogenetic clades which was supported by analysis 

of the partial coat-protein-coding region. CLRV isolates from Finland clustered with 

characterised strains originating from a wider range of host plant species including ash 

(Fraxinus excelsior), rowan and Sambucus sp. (Rebenstorf et al. 2006). These species are also 

native to southern Finnish ecosystems (Mikk & Mander 1995; Simola 2006) and may have 

been the source of CLRV strains now affecting birch species in Finland. This speculation is 

substantiated by our findings that mountain ash trees as well as a red elderberry sampled in 

2008 were found to be CLRV infected, and our analyses of sequence data which indicates 

towards a different virus population to be present in Finnish birches. Birches may have recently 

acquired CLRV from other host plants and the virus population is not adapted to specific host 

plant species yet. Lack of adaption of the virus to the host species may have induced severe 

symptom development in birches and the presence of a mixed virus population may be 

responsible for the aggressive spreading of the virus disease in Finland in a very short time. 

However, this may also be due to the new introduction of CLRV into birch species of this north 

European region and cannot be secured because of the few individuals tested and virus isolates 

characterised so far. 

It is of particular importance to monitor the dissemination of CLRV in the Finnish environment 

and the development of virus populations found in Finnish plant species, because they differ 

considerably from previous findings. The virus may even represent a threat to the Finnish 

forest industry relying on birch logs as source for pulpwood and therefore, the epidemiology of 

the virus has to be elucidated, and the impact of the pathogen for the Betula genus has to be 

investigated in future studies. 

248


We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support in the form of the 

projects BU 890/8-1 and BU890/8-2 and Mrs. R. Junge for skilled technical assistance. 

REFERENCES 

Anamthawat-Jónsson K; Thórsson A T (2003). Natural hybridisation in birch: triploid hybrids 

between Betula nana and B. pubescens. Plant Cell, Tissue and Organ Culture 75, 99– 

107. 

Atkinson M D (1992). Betula pendula Roth (B. verrucosa Ehrh.) and B. pubescens Ehrh. The 

Journal of Ecology 80, 837–870. 

Bandte M; Büttner C (2001). A review of an important virus of deciduous trees — cherry leaf 

roll virus: occurrence, transmission and diagnosis. Pflanzenschutzberichte 59, 1–19. 

Bandte M; Echevarria-Laza H J; Paschek U; Ulrichs C; Pestemer W; Schwarz D; Büttner C 

(2007). Transmission of plant pathogenic viruses by water. In:. Segundo Congreso 

Colombiano de Horticultura, eds G Fischer; S Magnitskiy; L E Flórez; D Miranda; A 

Medina, pp. 31–43. Sociedad Columbiana de Ciencias Horticolas: Bogota. 

Bremer K; Lehto K; Kurkela T (1991). Metsäpuiden virus- ja mykoplasmatauteja. Diseases 

caused by viruses and mycoplasmas in forest trees. Bulletins of the Finnish Forest 

Research Institute 382, 1–15. 

Buchhop J; von Bargen S; Büttner C. (2009). Differentiation of Cherry leaf roll virus isolates 

from various host plants by immunocapture-reverse transcription-polymerase chain 

reaction-restriction fragment length polymorphism according to phylogenetic relations. 

Journal of Virological Methods, in print, doi: 10.1016/j.jviromet.2008.12.010. 

Cooper J I (1976). The possible epidemiological significance of pollen and seed transmission 

in the cherry leaf roll virus/Betula spp. complex. Mitteilungen der Biologischen 

Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem 170, 17–27. 

Cooper J I (1979). Virus diseases of trees and shrubs. Institute of Terrestrial Ecology; 

Cambrian News Aberystwyth: Cambridge. 

Cooper J I; Edwards M L (1980). Cherry leaf roll virus in Juglans regia in the United 

Kingdom. Forestry 53, 41–50. 

Cooper J I; Massalski, P R; Edward M L (1984). Cherry leaf roll virus in the female 

gametophyte and its relevance to vertical virus transmission. Annals of Applied Biology 

105, 55–64. 

Jalkanen R; Büttner C; von Bargen S (2007). Cherry leaf roll virus abundant on Betula 

pubescens in Finland. Silva Fennica 41, 755–762. 

Luostarinen K; Verkasalo E (2000). Birch as sawn timber and in mechanical further processing 

in Finland. A literature study. Silva Fennica Monographs 1. 1–40. 

Mikk M; Mander Ü (1995). Species diversity of forest islands in agricultural landscapes of 

southern Finland, Estonia and Lithuania. Landscape and Urban Planning 31, 153–169. 

Peltola A (2007). Finnish Statistical Yearbook of Forestry. Metla: Helsinki. 

Rebenstorf K (2005). Untersuchungen zur Epidemiologie des Cherry leaf roll virus (CLRV): 

genetische und serologische Diversität in Abhängigkeit von der Wirtspflanzenart und 

der geographischen Herkunft. Dissertation Humboldt-Universität zu Berlin, 127 p. 

249

Rebenstorf K; Candresse T; Dulucq M J; Büttner C; Obermeier C (2006). Host speciesdependent 

population structure of a pollen-borne plant virus, Cherry leaf roll virus. 

Journal of Virology 80, 2453–2462. 

Sambrook J; Fritsch E F; Maniatis T (1989). Molecular cloning — a laboratory manual. Cold 

Spring Harbour: New York. 

Simola H (2006). Cultural land use history in Finland. The Finnish Environment 23, 163–172. 

Thompson J D; Gibson T J; Plewniak F; Jeanmougin F; Higgins D G (1997). The ClustalX 

windows interface: flexible strategies for multiple sequence alignment aided by quality 

analysis tools. Nucleic Acids Research 25, 4876–4882. 

Viherä-Aarnio A; Velling P (1999). Growth and stem quality of mature birches in a combined 

species and progeny trial. Silva Fennica 33, 225–234. 

Walker D A (2000). Hierarchical subdivision of Arctic tundra based on vegetation response to 

climate, parent material and topography. Global Change Biology 6, 19–34. 

Werner R; Mühlbach H-P; Büttner C (1997). Detection of cherry leaf roll nepovirus (CLRV) in 

birch, beech and petunia by immunocapture RT-PCR using a conserved primer pair. 

European Journal of Forest Pathology 27, 309–318. 

Wijk M T v; Williams M; Shaver G R (2005). Tight coupling between leaf area index and 

foliage N content in arctic plant communities. Oecologia 142, 421–427. 

250

Habekuß A, Riedel C, Schliephake E, Ordon F: Importance of Insect-Transmitted Viruses in Cereals and Breeding 

for Resistance. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 

251-261; ISBN 978-3-941261-05-1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

4-3 Importance of Insect-Transmitted Viruses in Cereals and Breeding for 

Resistance 

Habekuß A, Riedel C, Schliephake E, Ordon F 

Julius Kühn Institute, Erwin-Baur-Straße 27, D-6484 Quedlinburg, Germany 

Email: antje.habekuss@jki.bund.de 

Abstract 

Investigations on the incidence of barley yellow dwarf virus (BYDV) and wheat 

dwarf virus (WDV) carried out in Saxony-Anhalt from 1998 to 2008 revealed a 

periodic appearance of these viruses and a clear relation between the number of 

infection days in autumn and the BYDV-attack in winter barley fields in the 

following spring. In additional experiments carried out in growth chambers under 

controlled conditions it turned out that 10°C is a threshold level for an efficient 

transmission of BYDV by Rhopalosiphum padi. Due to global warming longer 

periods of higher temperature in autumn and winter are expected which may result 

in an increasing importance of insect-transmitted viruses. In order to enhance the 

level of resistance to BYDV, Ryd2, Ryd3 and a QTL derived from the cultivar 

‘Post’ located on chromosome 2HL were combined using DH-lines and molecular 

markers. Concerning symptom expression and virus extinction first results indicate 

a reduction in those lines combining especially Ryd2 and Ryd3. Concerning WDV 

extensive screening programmes were conducted, but tolerance was only detected 

in cv. ‘Post’. First results on the genetics give hint that this tolerance is inherited in 

a quantitative manner. 

INTRODUCTION 

Plant viruses transmitted by insects are important pathogens in cereals, i.e. the aphidtransmitted 

barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) 

belonging to the genera Luteovirus and Polerovirus, respectively as well as the leafhoppertransmitted 

wheat dwarf virus (WDV) being a member of the genus Mastrevirus. Barley yellow 

dwarf virus as the causal agent of dwarfing and leaf discoloration of barley was already 

251

detected in 1951 in California (Oswald & Houston 1951) and is known today worldwide as a 

serious disease on barley (Lister & Ranieri 1995) causing yield losses e.g. up to 25% (Pike 

1990). Main vectors are the bird cherry aphid (Rhopalosiphum padi) and the wheat aphid 

(Sitobion avenae). According to the transmission efficiency of aphid species, 5 different strains 

were distinguished (Rochow 1969, Rochow & Muller 1971), first. But today, these causal 

agents of barley yellow dwarf are classified as different viruses which are again sub-divided 

into different strains (Mayo & D’Arcy 1999). Wheat dwarf virus (WDV) is transmitted 

persistently by the leafhopper Psammotettix alienus. This virus was first detected in the 1960s 

in the former Czechoslovakia (Vacke 1961) and was later on detected in different parts of 

Europe (Lindsten et al. 1970; Lapierre et al. 1991; Huth 1994; Erlund 2007). For WDV also 

different strains are known, i.e. a barley strain and a wheat strain (Commandeur & Huth 1991), 

which according to Schubert et al. (2007) may be regarded as different viruses due to a 

different host range and sequence differences. In Germany the main infection of both viruses in 

the field takes place in autumn in case of winter wheat and winter barley. Long periods of mild 

temperatures in autumn increase the infection rate, in particular in case of an anholocyclic 

overwintering of the aphid-vectors. Such periods of mild temperatures enhance also the 

infection with WDV by the leafhopper P. alienus. Due to global warming it is expected that 

insect-transmitted viruses will become more important in the future, because longer and 

warmer periods in autumn will result in longer flight activities of the vectors leading to an 

increased risk of winter barley to get infected by these viruses. 

In this respect growing of tolerant/resistant cultivars has to be considered as the most 

environmental sound and effective method to control both viruses. In case of barley yellow 

dwarf three genes conferring tolerance to BYDV/CYDV are known, i.e. ryd1, which was 

detected in the spring barley cultivar 'Rojo' (Suneson 1955), Ryd2, which was detected in 

Ethiopian landraces and localized on the long arm of chromosome 3 near the centromere 

(Schaller et al. 1963, Collins et al. 1996) and Ryd3 which was detected in the Ethiopian barley 

landrace L94 and was mapped to chromosome 6H (Niks et al. 2004). Furthermore, quantitative 

trait loci (QTL) for tolerance to BYDV have been mapped on different barley chromosomes 

(Scheurer et al. 2001). Up to now, only Ryd2 has been successfully used in breeding tolerant 

spring and winter barley cultivars e.g. cv. ‘Vixen’ (Parry & Habgood 1986). 

In contrast to BYDV up to now only small quantitative differences in the degree of tolerance to 

WDV are reported (Vacke & Cibulka 2001; Širlová et al. 2005). 

Therefore, the aims of the present study are (i) to investigate the incidence of BYDV and WDV 

in the central part of Germany (Saxony-Anhalt), (ii) to get information on the effect of 

temperature on virus transmission, (iii) to get information whether pyramiding of different 

genes and QTL for tolerance to BYDV with help of molecular markers results in a higher level 

of tolerance and (iv) to identify sources of tolerance to WDV and get information on the mode 

of inheritance. 

252


Investigations on the incidence of BYDV and WDV and on the influence of temperature 

on virus transmission 

The incidence of BYDV and WDV was monitored in Saxony-Anhalt in about 10 to 15 winter 

barley fields in the period 1998 to 2008. In early spring 150 leaf samples per field (30 samples 

taken randomly at 5 points at each field) were analysed by double antibody sandwich - enzyme 

linked immunosorbent assay (DAS-ELISA) using polyclonal BYDV and WDV specific 

antibodies. 

Investigations on the influence of temperature on virus transmission were carried out using 

single aphids of Rhopalosiphum padi as vector to transmit BYDV-PAV in a growth chamber. 

An acquisition period of 4 days of the aphids from a virus-free permanent rearing was followed 

by an inoculation of 54 barley seedlings of cv. ‘Rubina’ for 1, 2 or 4 days, respectively at 10, 

15, 20 or 25°C. After inoculation the aphids were killed by insecticide spraying and the plants 

were cultivated in a greenhouse at 20°C. 6 weeks post inoculation the virus extinction of single 

plants was estimated by DAS-ELISA and the infection rate (%) was calculated. 

BYDV-resistance tests 

For pyramiding of genes and QTL encoding BYDV-tolerance doubled haploid lines (DHs) of 

the crosses ‘RIL K4-56’ (Ryd3) x ‘DH 21-136’ (Ryd2, QTL of cv. ‘Post’ located on 

chromosome 2H, winter barley) and DH-lines of ‘RIL K4-56’ (Ryd3) x ‘Coracle’ (Ryd2, spring 

barley) were produced by microspore or anthere culture technique, respectively by KWS- 

Lochow GmbH and the Saaten-Union Resistenzlabor. 

Phenotyping of DH-populations is carried out in four locations [Gudow (Nordsaat), Irlbach 

(Saatzucht Ackermann), Bernburg (Lochow-Petkus) and Quedlinburg (JKI)]. For this purpose 

24 plants of 281 winter barley DH-lines and 188 spring barley DH-lines were artificially 

infected in the greenhouse in the one leaf stage using BYDV-PAV bearing aphids (10 

aphids/plant) and simultaneously healthy control plants were grown. Plants of ‘RIL K4-56’ x 

‘DH 21-136’ were transferred to the field at the four locations in October 2007 in two 

replications (2x12 plants per infected and control variant) and the same was done for the spring 

barley cross ‘RIL K4-56’ x ‘Coracle’ in March 2008. The level of tolerance is estimated using 

the methods described by Scheurer et al. (2001). 

Genotyping 

DNA was extracted using a modified CTAB method according to Doyle & Doyle (1990). DNA 

concentration was adjusted to a final concentration of 30ng/µl for PCR. For the detection of 

Ryd2 the Capsmarker YlpPCRM was used according to Ford et al. (1998). Screening for the 

presence of Ryd3 was conducted using the microsatellite marker HVM74 (Niks et al. 2004). 

The QTL on chromosome 2H derived from cv. ‘Post’ was analysed by the SSR HVCSG 

253

(Scheurer et al. 2001). While YlpPCRM was detected on agarose gels, the SSRs were detected 

by means of a capillary electrophoresis (Beckman Coulter CEQ TM 8000). 

WDV-resistance tests 

In the period 2002 to 2006 248 winter barley accessions of the German genebank of the 

Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben as well as breeding 

lines and cultivars were evaluated for their reaction to WDV by artificial WDV-inoculation in 

the field using viruliferous leafhoppers of the species Psammotettix alienus. In the middle of 

September of each year, 12 seeds per accession were sown in two replications in an inoculated 

(I) and a non-inoculated control (C) variant in the field. To increase the infection pressure, 

single WDV-infected barley plants were planted between each row of the I-variant shortly 

before inoculation. At the 1- to 2-leaf stage the plots of the I-variant were covered with a tunnel 

made of cotton and viruliferous leafhoppers were distributed in the tunnel in a density of 

approximately 1 leafhopper per plant. To keep the C-variant virus free the plots were treated 

regularly with an insecticide. After 4 weeks the cover was removed and the whole test was 

sprayed with an insecticide. Scoring of symptom expression was conducted at heading stage 

using a scale from score 1 = without symptoms to 9 = plant died. On the basis of these data the 

degree of attack (DA) was calculated as follows: 

DAS-ELISA was used to determine the virus extinction of selected genotypes as described 

above. At harvest, plant height, number of ears per plant, kernel weight per plant and thousand 

kernel weight of both variants were determined. The level of tolerance was estimated as the 

results of the infected variant relative to the control of the same genotype. 

To get information on the genetics of the WDV-tolerance detected in cv. ‘Post’ in more detail, 

2 independent populations of doubled haploid lines of the cross ‘Post’ x ‘Vixen’ comprising 86 

(I) and 77 (II) lines were phenotypically analysed in gauze house tests in 2006 and 2007 as 

described above. 

Statistical analysis 

The statistical analysis was carried out using the package software SAS 9.1. ANOVA was 

conducted using the GLM procedure and Tukey-Test (α=0.05). The scores of symptom 

expression were compared by a bootstrap test using the MULTTEST procedure (Neuhäuser & 

Jöckel 2007). Frequency distributions concerning tolerance to WDV were analysed for the fit 

to a Gaussian distribution by the Kolmogorov-Smirnov-Test. QTLs for WDV-tolerance in the 

DH-population ‘Post’ x ‘Vixen’ (II) were mapped on the basis of the existing genetic map, 

developed by Scheurer et al. (2001) and the phenotypic data (relative values) scored for this 

254 

DA = 

9 

� � 

s = 2 

n * ( s − 1 ) 

N * 8 

n = number of plants per scoring class 

s = scoring class 

N = number of plants with symptoms

population. This first preliminary QTL-analyses concerning WDV-tolerance were carried out 

using the software MapQTL ® 5 (Plant Research International B.V. and Kyazma B.V., Benelux 

and USA). 


Incidence of insect-transmitted viruses and relation to temperature 

The cereal aphids dominate in the aphid population trapped by a suction trap at Aschersleben 

in Saxony-Anhalt (Schliephake & Karl 1995). Accordingly, the aphid-transmitted BYDV was 

detected in each year in Saxony-Anhalt but in different frequencies ranging from an average of 

0.1% to 45.0% infected plants/field in winter barley and 0.1% to 40.0% in winter wheat fields 

(Table 1). Besides this, the leafhopper-transmitted WDV has gained evident importance in 

winter barley and winter wheat. In the period 1998 to 2000, and in 2004, WDV was the 

predominant insect-transmitted virus in winter barley in Central Germany. 

Table 1. Average infection rate (%) of BYDV and WDV in winter barley and winter 

wheat fields in Saxony-Anhalt during 1998 to 2008 

Year 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 

Winter BYDV 2.0 3.0 3.0 30.0 45.0 6.0 0.1 13.0 12.0 24.0 20.0 

barley WDV 23.0 18.0 26.0 10.0 1.0 4.0 12.0 2.0 10.0 18.0 4.0 

Winter BYDV 40.0 10.0 2.0 0.1 10.0 4.0 17.0 6.0 

wheat WDV 10.0 0.1 1.0 6.0 0.4 3.0 16.0 15.0 

Table 2. Mean BYDV-PAV infection rate after transmission by single R. padi females 

to young plants of winter barley cv. ‘Rubina’ at different temperatures and 

number of days of inoculation 

Temperature 

Days of inoculation 

(°C) 1 2 4 

10 13.9 16.9 32.8 

15 86.6 93.0 92.0 

20 79.8 86.2 94.3 

25 72.4 92.9 95.6 

The temperature limit to which BYDV-PAV is transmitted by Rhopalosiphum padi is at about 

10°C as estimated in growth chamber experiments (Table 2). At a lower temperature only 

sporadic infections were detected on sensitive young plants. Higher temperatures and a longer 

255

inoculation period increase the transmission rate up to 95%. Therefore the time periods with 

daily average temperatures above this daily mean temperature of 10°C have a strong influence 

of the virus incidence in the fields. 

Comparing the number of days with >= 10°C mean temperature (infection days) in autumn 

(from the 1st of October to the first day with temperatures lower than -5°C) to the incidence of 

BYDV in the following spring an obvious relation was observed (Fig. 1). 

256 

Figure 1. Comparing the number of days with >= 10°C mean temperature (infection 

days) in autumn (from the 1st of October to the first day with temperatures 

lower than -5°C) with the average incidence of BYDV infected plants in 

winter barley fields in the following spring (infection rate) 

Pyramiding of BYDV-tolerance loci 

In the field tests 2007/2008 a clear phenotypic differentiation between DH-lines carrying no or 

two respectively three tolerance encoding alleles concerning symptom expression was detected 

in selected DH-lines of the different genotypes (3 winter barley lines and 6 spring barley lines 

of each genotypic class with 10 to 12 plants per replication from three different locations) 

(Table 3A and 3B). This holds true also for the virus concentration of the analysed lines. As 

can be seen, especially those DH-lines combining Ryd2 and Ryd3 showed a significantly lower 

virus titre in comparison to DH-lines carrying only Ryd2 or Ryd3. In contrast to this, the effect 

of the QTL on chromosome 2H on the virus concentration seems to be rather small. The same 

results concerning Ryd2 and Ryd3 were detected in the spring barley cross (Table 3B).

The genotypes of the DH-lines were analysed by using known molecular markers. Table 3 

shows the number of genotypes observed for each genotypic class by analysing 470 winter 

barley lines of the combination ‘RIL K4-56’ x ‘DH 21-136’ and of 295 spring barley lines of 

the combination ‘RIL K4-56’ x ‘Coracle’. In the spring barley combination a good fit to the 

expected segregation of 1:1:1:1 was observed (χ 2 =3,047), while in the winter barley population 

a significant deviation from the expected 1:1:1:1:1:1:1:1 was detected (χ 2 =74,612). 

Table 3. Symptom expression (mean score of different locations) and virus titre (DAS- 

ELISA, extinction at 405 nm) of selected DH-lines (A and B) representing 

different genotypic classes 

A) RILK4-56 x DH21-136 (means of the locations Gudow, Irlbach and Quedlinburg) 

Genotypic classes 1) 

Ryd2 Ryd2 Ryd2 Ryd2 ryd2 ryd2 ryd2 ryd2 

Ryd3 Ryd3 ryd3 ryd3 Ryd3 Ryd3 ryd3 ryd3 

QTL+ QTL- QTL+ QTL- QTL+ QTL- QTL+ QTL- 

Score 2.14 a 2) 2.07 a 2.56 a 3.05 b 2.52 a 2.15 a 4.10 c 5.79 d 

Extinction 

1.50 

0.30 e 0.38 e 1.37 bc ab 1.11 d 1.26 c 1.56 a 1.22 cd 

Number of lines *) 93 49 43 37 92 76 52 28 

B) RILK4-56 x ‘Coracle’ (means of the locations Bernburg, Irlbach and Quedlinburg) 

Genotypic classes 1) 

Ryd2 Ryd2 ryd2 ryd2 

Ryd3 ryd3 Ryd3 ryd3 

Score 2.35 a 2) 3.13 c 2.86 b 6.40 d 

Extinction 0.74 c 1.42 a 1.32 b 1.26 b 

Number of lines 68 66 76 85 

1) 

Capital letters and + represent alleles positively contributing to BYDV-tolerance 

2) 

means with the same letter are not significantly different 

Screening of WDV-resistance 

In the field evaluation of 248 barley accessions, only cv. ‘Post’ and 3 breeding lines, having 

this accession in their pedigree, revealed a higher level of tolerance to WDV. However, no 

reduction in virus concentration was detected in these genotypes (data not shown). 

Concerning all the characters investigated, cv. ‘Post’, which is also tolerant to BYDV, showed 

the highest level of tolerance, i.e. the smallest reduction in these traits after WDV-infection 

(Table 4). 

257

The results of a 2006 analysed DH-population of the cross ‘Post’ x ‘Vixen’ for the reaction to 

WDV gave good fit to a Gaussian distribution (P>0.15) for the frequency of the traits 

investigated, e.g. for the degree of attack (Table 5). These observations indicate a polygenic 

inheritance of the WDV tolerance detected in cv. ‘Post’. In the test of the second DHpopulation 

of this combination in 2007 the high level of tolerance of cv. ‘Post’ was confirmed 

(Table 6). Concerning the relative plant height after WDV infection also a good fit to a 

Gaussian distribution was observed (P>0.15). On the basis of these first phenotypic data of the 

77 DH-lines and the available genetic map for this population (Scheurer et al. 2001) up to now 

one QTL for the relative plant height after WDV infection could be detected on chromosome 

4H (LOD 4,76) explaining about 26% of the phenotypic variance. 

258 

Table 4. Performance of barley genotypes in the field concerning plant height, 

ears/plant and thousand kernel weight after WDV-infection in 2006 

Plant height Ears/plant Thousand kernel weight 

Cultivar Infected Control Infected Control Infected Control 

Post 81.6 ± 14.9 104.9 ± 7.4 8.4 ± 4.6 12.3 ± 4.7 29.1 ± 2.9 37.2 ± 1.6 

Erfa 29.1 ± 13.7 95.3 ± 8.6 1.7 ± 2.1 13.7 ± 5.8 16.0 ± 6.5 47.7 ± 1.0 

Lunet 37.8 ± 12.5 94.5 ± 5.5 4.3 ± 4.1 10.4 ± 3.2 15.1 ± 4.8 40.8 ± 2.2 

Luxor 27.6 ± 4.7 93.1 ± 8.5 2.2 ± 4.9 13.6 ± 3.9 8.3 ± 8.2 48.1 ± 0.8 

Okal 42.2 ± 18.0 90.1 ± 7.7 3.0 ± 2.7 11.5 ± 4.3 22.8 ± 15.5 44.8 ± 2.2 

Perry 47.8 ± 13.7 106.5 ± 10.2 3.3 ± 2.8 13.5 ± 7.1 23.5 ± 7.4 40.7 ± 1.5 

Rubina 54.8 ± 19.4 99.5 ± 10.9 3.7 ± 1.1 13.3 ± 5.2 21.6 ± 5.2 42.5 ± 1.4 

Sigra 51.3 ± 19.0 100.2 ± 7.5 5.3 ± 5.0 8.9 ± 3.3 18.5 ± 17.6 39.8 ± 0.4 

Vixen 35.4 ± 23.0 90.8 ± 7.9 2.4 ± 3.3 14.3 ± 5.8 21.5 ± 19.1 52.9 ± 0.7 

Table 5. Symptom expression (degree of attack) of DH-population (I) of the 

combination ‘Post’ x ‘Vixen’ to WDV-infection in the gauze house test 2006 

Degree of attack 

0-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 Total 

Number of lines 0 1 5 9 17 16 18 12 6 2 86 

‘Post’ = 32.0; ‘Vixen’ = 85.8 

Table 6. Relative plant height of DH-population (II) of the combination ‘Post’ x 

‘Vixen’ to WDV-infection in the gauze house test 2007 

Plant height of infected plant relative to the control (%) 

0-5 6-15 16-25 26-35 36-45 46-55 56-65 66-75 Total 

Number of lines 2 12 15 11 12 10 8 1 71 

‘Post’ = 64.5; ‘Vixen’ = 4.4

These first preliminary results give hint that due to global warming not only insects themselves 

will become more important with rising temperatures but also insect transmitted viruses. In this 

respect it also has to be taken into account, that aphids may survive the winter in an 

anholocyclic manner in the future causing permanent virus infections. 

With respect to BYDV, molecular markers are available facilitating efficient marker based 

selection and marker based backcrossing procedures (Ordon et al. 2003) as well as pyramiding 

strategies (Werner et al. 2005, 2007). These markers will be developed for WDV in the future. 

Applying such molecular breeding strategies in barley will considerably rise the level of 

tolerance to BYDV and WDV being a prerequisite for an environmental sound and consumer 

protecting barley production in case of rising average temperature in the future. 

ACKNOWLEDGMENT 

We thank the Federal Ministry of Education and Research, the Federal Ministry for Food, 

Agriculture and Consumer Protection and the Gemeinschaft zur Förderung der privaten 

deutschen Pflanzenzüchtung e.V. for financial support of parts of these studies (BMBF 

03i0607A, BLE-28-1-41.002-06). 

REFERENCES 

Collins N C; Paltridge N G; Ford C M; Symons H (1996). The Yd2 gene for barley yellow 

dwarf virus resistance maps close to the centromere on the long arm of barley 

chromosome 3. Theoretical and Applied Genetics 92, 858-864. 

Commandeur U; Huth W (1999). Differentiation of strains of wheat dwarf virus in infected 

wheat and barley plants by means of polymerase chain reaction. Zeitschrift für 

Pflanzenkrankheiten und Pflanzenschutz 106, 550-552. 

Doyle J F; Doyle J L (1990). Isolation of plant DNA from fresh tissue. Focus 12, 13-15. 

Erlund P (2007). Experiences of Wheat dwarf virus in Finland 2004-2007. Report of NJF 

Seminar 402, Virus vector management in a changing climate, Kristianstad, Sweden, 

09.-11.10.2007, p. 51. 

Ford C M; Paltridge N G; Rathjen J P; Moritz R L; Simpson R J; Symons R H (1998). Rapid 

and informative assays for Yd2, the barley yellow dwarf virus resistance gene, based on 

the nucleotide sequence of a closely linked gene. Molecular Breeding 4, 23-31. 

Friedt W; Scheurer K S; Huth W; Habekuss A; Ordon F (2003). Genetic analyses of BYDVtolerance 

in barley (Hordeum vulgare L.). Zeitschrift für Pflanzenkrankheiten und 

Pflanzenschutz 110, 278-286. 

Huth W (1994). Weizenverzwergung - bisher übersehen? Pflanzenschutz-Praxis 4, 37-39. 

IPCC (2007). Summary for Policymakers. In: Climate Change 2007: Impacts, Adaptation and 

Vulnerability. Contribution of Working Group II to the Fourth Assessment Report of the 

Intergovernmental Panel on Climate Change, eds M L Parry, O F Canziani, J P 

Palutikof, P J van der Linden & C.E. Hanson, Cambridge University Press: Cambridge, 

United Kingdom and New York, NY, USA. 

259

Lapierre H; Cousin M T H; Giustina W D; Moreau J P; Knogali M; Roux J; Gelie B; Ollier E 

(1991). Agent pathogéne et vecteur. Phytoma 432, 26-28. 

Lindsten K; Vacke J; Gerhardson B (1970). A preliminary report on three cereal virus diseases 

new to Sweden spread by Macrosteles- and Psammotettix leafhoppers. Swed. Stat. 

Vaxtskyddoanst. Med. 14, 281-297. 

Lister M R; Ranieri R (1995). Distribution and Economic Importance of Barley Yellow Dwarf. 

In: Barley Yellow Dwarf-40 Years of Progress, eds C J D'Arcy & P A Burnett, pp. 

29-53. APS Press: St. Paul. 

Mayo M A; D'Arcy C J (1999). Family Luteoviridae: A Reclassification of Luteoviruses. In: 

The Luteoviridae, eds H G Smith & H Barker, pp. 15-22. CABI Publishing. 

Neuhäuser M; Jöckel K H (2006). A bootstrap test for the analyses of microarray experiments 

with very small number of replications. Applied Bioinformatics 5, 173-179. 

Niks R E; Habekuss A; Bekele B; Ordon F (2004). A novel major gene on chromosome 6H for 

resistance of barley against the barley yellow dwarf virus. Theoretical and Applied 

Genetics 109, 1536-1543. 

Ordon F; Werner K; Pellio B; Schiemann A; Friedt W; Graner A (2003). Molecular breeding 

for resistance to soil-borne viruses (BaMMV, BaYMV, BaYMV-2) of barley (Hordeum 

vulgare L.). Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 110, 287-295. 

Oswald J W; Houston B R (1951). A new virus disease of cereals, transmissible by aphids. 

Plant Disease Reporter 11, 471-475. 

Parry A L; Habgood R M (1986). Field assessment of the effectiveness of a barley yellow 

dwarf virus resistance gene following its transference from spring to winter barley. 

Annals of Applied Biology 108, 395-401. 

Pike K S (1990). A review of barley yellow dwarf virus grain yield losses. In: World 

Perspectives on Barley Yellow Dwarf, ed P A Burnett, pp. 356-361, CIMMYT: Mexico. 

Rochow W F (1969). Biological properties of four isolates of barley yellow dwarf virus. 

Phytopathology 59, 1580-1589. 

Rochow W F; Muller I (1971). A fifth variant of barley yellow dwarf virus in New York. Plant 

Disease 55, 874-877. 

Schaller C W; Rasmusson D C; Qualset C O (1963). Sources of resistance to the yellow dwarf 

virus in barley. Crop Science 3, 342-344. 

Schaller C W; Qualset C O; Rutger J N (1964). Inheritance and linkage of the Yd2 gene 

conditioning resistance to barley yellow dwarf virus disease in barley. Crop Science 4, 

544-548. 

Scheurer K S; Friedt W; Huth W; Waugh R; Ordon F (2001) QTL analysis of tolerance to a 

German strain of BYDV-PAV in barley (Hordeum vulgare L.). Theoretical and Applied 

Genetics 103, 1074-1083. 

Schliephake E; Karl E (1995): Beobachtung des Blattlausfluges mittels Saugfalle. Mitteilungen 

derDeutschen Gesellschaft für Allgemeine und Angewandte Entomologie 10, 203-206 

Schubert J; Habekuss A; Rabenstein F (2002). Investigations of differences between wheat and 

barley forms of Wheat dwarf virus and their distribution in host plants. Plant Protection 

Science 38, 43-48. 

Schubert J; Habekuss A; Kazmaier K; Jeske H (2007). Surveying cereal-infecting 

geminiviruses in Germany. Diagnostics and direct sequencing using rolling circle 

amplification. Virus Research 127, 61-70. 

260

Širlová L; Vacke J; Chaloupková M (2005). Reaction of selected winter wheat varieties to 

autumnal infection with Wheat dwarf virus. Plant Protection Science 41, 1-7. 

Starling T M; Roane C W; Camper jr. H M (1987). Registration of Wysor barley. Crop Science 

27, 1306-1307. 

Suneson C A (1955). Breeding for resistance to yellow dwarf virus in barley. Agronomy 

Journal 47, 283. 

Vacke J (1961). Wheat dwarf virus. Biologia Plantarum (Praha) 3, 228-233. 

Vacke J; Cibulka R (2001). Reaction of registered winter wheat varieties to Wheat dwarf virus 

infection. Czech Journal of Genetics and Plant Breeding 37, 50-52. 

Werner K; Friedt W; Ordon F (2005). Strategies for pyramiding resistance genes against the 

barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2). Molecular 

Breeding 16, 45-55. 

Werner K; Friedt W; Ordon F (2007). Localisation and combination of resistance genes against 

soil-borne viruses of barley (BaMMV, BaYMV) using doubled haploids and molecular 

markers. Euphytica 158, 323-329. 

261

Vinogradova S, Rakitin A, Kamionskaya A, Agranovsky A, Ravin N: Strategy for pathogen-derived resistance in 

Nicotiana benthamiana to Beet yellows virus and Beet necrotic yellow vein virus. In: Feldmann F, Alford D V, 



4-4 Strategy for pathogen-derived resistance in Nicotiana benthamiana to 

Beet yellows virus and Beet necrotic yellow vein virus 

Vinogradova S, Rakitin A, Kamionskaya A, Agranovsky A, Ravin N 

Bioengineering Centre, Russian Academy of Sciences, 117312 Moscow, 60-letia Octyabrya 

prospect 7, Bldg 1 

Email: coatprotein@bk.ru 

262 

Abstract 

To obtain transgenic plants resistant to Beet yellows virus (Closterovirus, BYV) and 

Beet necrotic yellow vein (Benyvirus, BNYVV, cause of Rhizomania), we 

employed a strategy based on post-transcriptional gene silencing (PTGS). The 3’terminal 

untranslated regions (3’-UTR) of the BYV or BNYVV genomes were used 

as the PTGS targets. The cDNA inserts BYVsil and BNYVVsil contained the 

respective 3’-UTRs as sense and antisense, separated by maize intron ubi1. The 

efficiency of these inserts as potential PTGS inducers was confirmed by a newly 

developed method of 35S-promoter-driven transient co-expression of the inductor 

RNA (BYVsil or BNYVVsil) and a target (GFP mRNA with a viral 3’-UTR) in 

Nicotiana benthamiana. As detected by Western blotting, plants agroinoculated 

with the target and the inducer expressed 10 times less GFP compared to the 

controls inoculated with the target only. N. benthamiana was also used as model 

plant for agrobacterial transformation with vectors containing the inserts of bar 

marker (phosphinotricinacetyltransferase) and BYVsil or BNYVVsil cDNAs, each 

under the control of separate 35S promoters. PCR analysis of regenerated N. 

benthamiana confirmed the presence of both the virus-specific and bar inserts in 

some lines. Additionally, bar expression was detected serologically in these lines. 

Transgenic N. benthamiana plants were multiplied in vitro and adapted to soil 

growing for further testing of resistance to artificial inoculation with the viruses.

Varrelmann M, Thiel H: Current Status of Rhizomania Resistance in Sugar Beet - Still Holding or Breaking of 

Resistance? In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 


4-5 Current Status of Rhizomania Resistance in Sugar Beet - Still Holding 

or Breaking of Resistance? 

Varrelmann M, Thiel H 

Institute of Sugar Beet Research, Holtenser Landstr. 77, 37079 Göttingen 

Email: mvarrel@gwdg.de 

INTRODUCTION 

BNYVV the type member of the genus Benyvirus is a rod-shaped single-stranded (+) strand 

RNA virus with four to five genome components. The soil-borne virus is in vivo transmitted 

by the plasmodiophoromycete Polymyxa betae into root hairs of sugar beets, where it causes 

dwarfism of tap roots and induces rootlet proliferation. After its first description in Italy in 

1959 (Canova 1959) it was spread worldwide into numerous sugar beet producing countries 

(Japan, China, USA and Europe), where it severely threatened the sugar beet growing and 

processing industry due to generation of heavy yield losses (Asher 1993; Tamada 1999; 

Lennefors et al. 2000; Nielsen et al. 2001). To date within Europe 1.6 million hectares of sugar 

beet were examined for the occurrence of BNYVV. In 1990 15%, 2000 38% and for 2010 56% 

of the sugar beet production area were predicted to be BNYVV infected (Richard-Molard & 

Cariolle 2001). 

Extensive sugar beet breeding efforts finally saved the crop by selecting genetic sources of 

tolerance and resistance. Over a period of approximately 15 years, backcrossing, marker 

development and selection increased the yield of resistant cultivars to the level of susceptible 

cultivars under conditions of non-infestation, thereby minimizing the yield penalty. Only by 

use of BNYVV-resistant cultivars does sugar beet production under natural infection remains 

profitable. However, the more or less uniform growth of one resistance source in commercial 

cultivation is expected to exert a strong pressure on the pathogen for selection of 

resistance-breaking variants. Those isolates have been already identified at several independent 

locations and raise questions about their competitiveness, fitness and the selective agent. In 

case of possible widespread loss of resistance future strategies for durable virus control have to 

be developed. 

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THE HISTORY OF RHIZOMANIA RESISTANCE AND TOLERANCE SELECTION 

First breeding programmes started in the early 1970s in Italy, selecting genotypes on traits such 

as yield, white sugar yield and processing quality under disease pressure, but these did not 

consider the virus content or resistance to infection. At least differences in the occurrence of 

symptoms were considered (reviewed by Scholten et al. 2000). The resulting cultivars where 

more or less tolerating the virus replication by an unknown mechanism. In Germany as one of 

the first countries, the first BNYVV-tolerant cultivar was registered in 1983 (Bolz & Koch 

1983; Hecht 1989). As all other cultivars of the first generation, the cultivar displayed only 

slightly increased yields under diseased conditions compared with susceptible ones. The first 

cultivar carrying a BNYVV resistant phenotype and showing reduced virus content in the tap 

root was released in 1985 (Rizor) (Richard-Molard 1985, de Biaggi 1987). When a negative 

correlation of virus content and white sugar yield was detected (Giunchedi et al. 1985, 1987), 

the virus concentration in lateral roots was used as a selection criterion for the development of 

BNYVV-resistant sugar beet cultivars. Until today resistance tests which determine virus 

content in lateral roots of young sugar beet plants, as initially demonstrated by Bürcky & 

Büttner (1985, 1991), are widely used in BNYVV resistance breeding programs. Nevertheless, 

the BNYVV-resistance trait has not been introduced into cultivar registration. Until today only 

yields obtained under natural infection in field trials compared with reduced yields in 

susceptible cultivars as an indicator for the disease are criteria for registration of “rhizomania 

tolerant cultivars”. This has led to a spectrum of phenotypes from highly resistant cultivars 

with hardly detectable virus infection to cultivars displaying BNYVV concentrations 

comparable to susceptible genotypes (unpublished observations). 

BREEDING FOR BNYVV RESISTANCE 

The genotypes or cultivars used in the early stages of selection in several breeding efforts 

(Biancardi et. al. 2002) have not been analyzed or characterized for their inheritance, 

heritability or origin of the resistance source in detail. The monogenic dominant resistance 

gene Rz1 was used for the first time in a source of the Holly Sugar Company (Lewellen et al. 

1987). This Holly material proved to be highly heritable following several cycles of selection 

(Lewellen & Biancardi 1990) although showing incomplete dominance and various degrees of 

penetrance (Wisler et al. 1999; Meulemans et al. 2003). Rz1 however does not condition 

complete infection resistance to BNYVV but mediates a sort of partial or quantitative 

resistance, limiting virus replication in lateral roots and inhibiting systemic spread to the main 

tap root. To date this single resistance source is most widely used in all commercial cultivars 

from all breeders worldwide (reviewed in Biancardi et al. 2002). In addition to the resistance 

found in sugar beet, other sources have been identified in collections of wild beet germplasm 

(Beta vulgaris ssp. maritima) for example in WB41 and WB42 among others (Lewellen 1995) 

originating from Denmark (Lewellen et al. 1987; Whitney 1989). The WB42 resistance 

appeared to be more effective in limiting virus replication than Rz1 (Paul et al. 1993). 

However, in crosses between subsp. vulgaris and subsp. maritima, although being compatible 

264

and producing fertile progenies, problems in seed germination were observed (Lewellen & 

Whitney 1993). This might be a reason that breeding efforts concentrated mainly on Rz1 for 

introgression into commercial cultivars. The resistance in WB42 was named Rz2 after showing 

that the resistance is based on a different mechanism and a major gene (Scholten et al. 1999). 

Recently, molecular mapping studies performed by Gidner et al. (2005) provided evidence that 

the WB41 resistance, named Rz3, like Rz2 is different from Rz1 and possibly represents a third 

major resistance gene. 

However, although being mainly determined by monogenes, the strength of the resistance, 

estimated by the virus content in lateral roots in resistance tests seems to be strongly influenced 

by additional minor genes (Gidner et al. 2005; Lennefors 2006). In addition to this observation 

different independent breeding approaches have led to a combination of two or three Rz-genes 

which lead to additive affects, providing a higher level of resistance indicated by lower virus 

contents (Lennefors et al. 2006; Liu et al. 2005; Pferdmenges et al. 2008; Pferdmenges & 

Varrelmann 2008). 

Molecular linkage maps of sugar beet have been published by several authors (reviewed in 

Scholten et al. 2000). Based on this information, molecular mapping studies (Scholten et al. 

1994, 1999; Amiri et al. 2003; Gidner et al. 2005) have shown that all three major resistance 

genes are located in small distances on chromosome III (Butterfass 1964). In the breeding and 

selection process, Rz1 is followed using molecular markers with tight linkage (Barzen et al. 

1997). 

ALTERNATIVE STRATEGIES TO GENERATE RHIZOMANIA RESISTANCE IN 

SUGAR BEET 

The only way to inhibit virus spread in the host plant, symptom expression and yield reduction 

is the use of BNYVV-resistant cultivars. Because of the soil-borne nature of the disease 

consisting of virus and a plasmodiophoromycete vector, no agronomical or chemical measures 

are effective to prevent BNYVV infections. One possible alternative in addition to virus 

resistance might be resistance to the vector P. betae as a means of preventing or at least 

reducing entry of the virus. Asher et al. (2008) identified such a resistance to the 

plasmodiophoromycete in B. vulgaris ssp. maritima and introduced the resistance into sugar 

beet already carrying Rz1 resistance gene. The authors showed that the trait is heritable and 

amenable and quantitatively reduces BNYVV concentrations in addition to the Rz1 effect. 

Another alternative represents the generation of virus resistance in transgenic plants. Although 

several approaches to generate pathogen derived resistance by for example using translatable 

coat protein genes were successfully carried out as well in sugar beets (Mechelke & Kraus 

1998; Büttner & Mangold 1998), the method to transform plants with virus-derived sequences 

in inverted-repeat orientation to produce non-translatable dsRNA, has been proven the most 

successful and is assumed to burrow the least biological risks. 

All plants possess the adaptive virus resistance system, named RNA silencing, directed against 

invasive nucleic acids such as transposons and viruses. This mechanism is as well known as 

265

post-transcriptional gene silencing (PTGS) in plants (Baulcombe 2004) and is triggered in a 

sequence-specific manner by dsRNA, which is formed in the replication cycle in most viruses 

(Voinnet 2005). dsRNA can also be expressed in transgenic plants transformed with a construct 

encoding self-complementary RNA of the sequence aimed to be targeted (Smith et al. 2000). 

When viral sequences are constitutively expressed (for example in inverted repeat orientation 

in transgenic plants), RNA silencing of this sequence is triggered with high efficiency already 

before virus infection and the plant is protected when the virus containing this sequence is first 

entering a host cell. The RNA degradation mechanism is sequence specific. According to Prins 

(2003) the resistance is effective against isolates with up to 10% sequence divergence. Multiple 

studies have shown that inverted repeat constructs of a length of approximately 400 bp can be 

efficiently used for the generation of resistance and as well can be stacked for the generation of 

multivirus-resistance (Jan et al. 2000). A recently published methodological variant of this 

approach uses artificial microRNAs to confer virus resistance in transgenic plants (Niu et al. 

2006). MicroRNAs, known to be important regulators of plant development (Jones-Rhoades et 

al. 2006) express short (20-24 bp) single stranded RNA of plant genes which extensively form 

base-pairing and target the plants own mRNAs for degradation by RNA silencing to downregulate 

gene expression (Brodersen & Voinnet 2009). If these sequences are replaced with 

short virus derived sequences, the coding sequences can be used for generation of virus 

resistance in transgenic plants too. Concerning the width of efficiency against sequence 

variable isolates and durability of this virus resistance the sequence divergence is an important 

factor that needs to be evaluated. 

The strategy of using inverted repeat constructs of a virus gene fragment was used for 

production of BNYVV resistance in sugar beet by Lennefors et al. (2006). The authors 

generated highly BNYVV-resistant sugar beets by expression of a 0.4 kb inverted repeat 

construct based on a partial replicase gene derived sequence. The transgenic resistance 

provided high protection levels and significant lower virus contents than observed in plants 

carrying conventional resistance sources Rz1, Rz2 or a combination of both (Lennefors et al. 

2006a). Most interesting was the finding that a phenomenon representing a main concern of 

transgenic virus-resistant plants based on RNA silencing, the suppression of RNA silencing by 

superinfecting viruses of different species, was not observed in these plants (Lennefors et al. 

2007). Many viruses encode quite diverse proteins which are able to suppress a component of 

the plants RNA silencing machinery (Voinnet 2005) and a compromise of transgenic RNA 

silencing-based resistance by co-infecting viruses has been evidenced (Mitter et al. 2003; 

Savenkov & Valkonen 2001). As another resistance strategy Lauber et al. (2001) showed that 

transgenic sugar beets expressing mutated forms of one of the three movement proteins of the 

BNYVV generate higher protection levels than Rz1. 

Taken together transgenic BNYVV resistant sugar beets represent a very attractive alternative 

compared to conventional resistance because of the lower virus content which can be achieved 

and therefore can be additionally supposed to reduce the size of the BNYVV population in soil 

on a long-term perspective. Predictions of durability of transgenic virus resistance based on 

RNA silencing and comparisons with conventional resistance are difficult to draw. It seems 

266

quite improbable that the virus can change about 10% of the nucleotide sequence to be able to 

overcome the targeting, if a conserved sequence like replicase gene is chosen, however other 

mechanisms to adopt and overcome the resistance like modifications of silencing suppressor 

proteins or other yet unknown mechanisms are conceivable. 

PLANT RESISTANCE VERSUS VIRUS PATHOGENICITY 

Although being indispensable for sugar beet cultivation and profitable sugar production, 

several efforts to isolate the major resistance gene Rz1 by map-based cloning failed. Therefore, 

the resistance mechanism which limits virus replication and spread is still completely 

unknown. Recently Tian and co-workers (2004) isolated analogues of classic plant resistance 

genes named resistance gene analogues (RGAs) from the sugar beet genome which contain Rgene 

product components such as nucleotide binding sites (NBS) and Leucine-rich repeat 

(LRR) motifs clustering over the sugar beet genome. Lein et al. (2007) supplied evidence that 

several of them cosegregate with quantitative trait loci of rhizomania resistance. As an 

involvement of these RGAs in BNYVV resistance has not been demonstrated, this attractive 

approach still awaitings further functional characterisation and proof of concept. 

The re-modelling of the host metabolism to favour virus replication or a host resistance 

reaction which is unable to inhibit virus spread in susceptible genotypes has been analyzed in 

part using different approaches. The systemic necrosis in tap roots and rootlets is indicative for 

a failed hypersensitive resistance response. Histological observations on infected taproots 

revealed reprogramming of pericycle cells to meristematic cells caused by the infection and/or 

the necrosis (Pollini & Giunchedi 1989). The change in root hair morphogenesis suggests a 

change in phytohormone balance and indeed Pollini et al. (1990) detected increased auxin 

levels in infected plants and tolerant plants displayed lower auxin contents than susceptible 

ones. When the root transcriptome of infected plants was compared with healthy plants, a 

change in expression of auxin, cell-cycle, defence signalling and ubiquitin-related regulated 

genes was observed (Schmidlin et al. 2008). 

RNA3 encoded P25 and P26 in RNA5-containing isolates play a central role in virus 

pathogenicity and interaction with the resistance in sugar beet. Presence of RNA3-encoded P25 

is not only indispensable for symptom induction, virus replication and spread in susceptible 

genotypes as demonstrated by artificial infection experiments (Koenig et al. 1991; Tamada et 

al. 1989 and 1999) but as well involved in a necrotic host reaction resembling a hypersensitive 

resistance response in resistant Beta vulgaris and maritima plants being leaf inoculated 

(Tamada 2007). Therefore, Chiba et al. (2008) suggested P25 to act as pathogenicity factor in 

susceptible and as an avirulence gene product in resistant plants. Thiel & Varrelmann used P25 

to screen the Rz2 sugar beet proteome for proteins physically interacting with the viral 

pathogenicity factor, searching for proteins involved in and necessary for the virus life cycle as 

well as putative resistance components. Interestingly, P25 seems to interfere with the plants 

ubiquitin-proteasome, cell-cycle, defence signalling and phytohormone metabolism 

(unpublished). 

267

OCCURRENCE OF RESISTANCE BREAKING ISOLATES 

Following the observation that P25 represents the pathogenicity factor/avirulence gene product, 

it is no surprise that P25 displays amino acids with high variability (tetrad on position 67-70) 

and different geographic distribution. Schirmer et al. (2005) found evidence for strong positive 

selection on this P25 tetrad. There are numerous examples in the history of virus control in 

crop plants using monogenic dominant genes and their affected durability over time (Harrison 

2002) and, therefore, it was expected that the uniform cultivation of a single resistance gene or 

source would exert a strong selection pressure on the virus for the generation of 

resistance-breaking isolates. 

In addition to this P25 tetrad hypervariability several groups independently observed the 

occurrence of resistance-breaking isolates in US (California, Minnesota) and Spain (Liu et al, 

2005; Liu & Lewellen 2006; Pferdmenges et al. 2008) and detected specific P25 tetrad 

mutations. In addition to these findings, very recently it was shown that a single alanin to valin 

exchange at tetrad position 67 was responsible for increased virus contents in mechanically 

root-inoculated Rz1 plants (Koenig et al. 2009). It remains quite speculative, if 

resistance-breaking virus variants are already present in the soil population and enriched by the 

cultivation or occur first when several rotations of Rz1 plants have been cultivated, because 

until today, sequence variation of the virus soil population has not been investigated in detail. 

A first attempt to characterize BNYVV populations in soil in dependence of the plant´s 

resistance trait suggested that incompatible virus host interactions exert a strong selection 

pressure on the virus population, leading to changes in the intragenic isolate structure 

exemplified by P25 tetrad composition (Acosta-Leal et al. 2008). However, it is well-known 

that pathogen mutation, leading to increased pathogenicity or aggressiveness in many cases, 

lead to a significant loss of fitness (Roossinck 1997). Because the viral population in soil can 

be very large (long resting in P. betae spores and compared with air-borne diseases spreading 

relatively slowly), it is quite speculative if resistance-breaking isolates will outcompete the 

isolates presently dominating the populations. The spread of more aggressive BNYVV isolates 

with a fifth RNA component which occurred more than 20 years ago (Koenig et al. 1995 and 

1997) was observed in a limited region in France (Pithiviers) surrounded by soils containing 

only B-type BNYVV with four RNA components. Observations over the years have shown 

that the isolate has not spread very far and has not replaced the prevalent isolates in that region 

(B. Holtschulte, personal communication). Isolates carrying an additional fifth RNA 

component encoding the pathogenicity protein P26 do not seem to be selected by the 

cultivation of Rz1 containing cultivars. An argument for this assumption is the fact that RNA5 

containing BNYVV isolates have been widely spread already for a long time in Asian countries 

like China and Japan (Tamada et al. 1989). These so-called J-type isolates (Schirmer et al. 

2005) are known to spread faster from infected lateral roots to the main tap root (Tamada et al. 

1997). Pferdmenges et al. (2008) supplied evidence for incomplete Rz1 cultivar resistance 

breaking abilities of European BNYVV P-type isolates carrying RNA5 as well. 

268

In addition to these observations another phenomenon is reported regularly from many 

different countries: the occurrence of plants in rhizomania infested field where resistant 

cultivars are grown which display strong systemic BNYVV symptoms and high virus contents. 

These plants are called “blinkers”. Although those plants might be infected with 

resistance-breaking isolates (which has never been proven experimentally until today), most 

probably these plants are susceptible and do not carry the resistance gene (Lennefors 2006). 

According to this work, sugar beet hybrids are produced by crossing a pollinator line to a male 

sterile female line. As long as the Rz-gene is carried by the pollinator, in case of producing 

rhizomania and non rhizomania-resistant hybrids in the same seed production area, cross 

fertilization with non-resistant pollen is likely to occur up to a certain quantity, leading to a 

proportion of susceptible seeds in the seeds of the resistant hybrid. This could be circumvented 

if the female parent would carry the Rz-gene. However, the transfer of the resistance to the 

female parent is much more complicated and time consuming. 

STRATEGIES TO CIRCUMVENT SELECTION AND ACCUMULATION OF 

RESISTANCE BREAKING BNYVV STRAINS 

Although experimental information is lacking about the causal connection of growth of 

resistant cultivars and selection of resistance-breaking BNYVV isolates in sugar beet, this has 

been experienced in many host virus interactions (Harrison 2002; García-Arenal & MacDonald 

2003). Anticipating this relationship, strategies for durable use of the limited number of 

resistance sources available need to be rethought and evaluated. The use of multiple different 

Rz-genes for sure will strongly reduce the selection pressure as they target different sites of the 

pathogenicity factor what strongly decreases the probability for the occurrence of one virus 

mutant which carries both mutations necessary to overcome the resistance. Still a matter of 

debate is the fact that cultivar registration is based on yield tolerance rather than resistance 

based on reduction of virus content in roots. It appears logical that less virus replication means 

lower probability for the selection of resistant breaking mutations and reduction of the 

population size in soil. The latter was shown by Lennefors (2006) who demonstrated in field 

experiments that susceptible hybrids compared to highly resistant cultivars strongly increased 

the virus inoculum concentration in the soil which in contrast was reduced when resistant 

genotypes were cultivated. However as long as no general association between the type of 

virus resistance category and the durability can be demonstrated (García-Arenal & MacDonald 

2003) it has to be anticipated that none of the resistance phenotypes in use is superior. This still 

needs an experimental proof of principle which then must be extrapolated to the field situation. 

SUMMARY & CONCLUSION 

The rhizomania infested area is worldwide, increasing in all sugar beet growing countries 

despite the cultivation of cultivars carrying quantitative resistance traits (Richard-Molard & 

Cariolle 2001). Natural resistance sources which supply resistance to virus infection in the 

initially infected lateral root cell (based on extreme virus resistance or complete resistance to 

269

infection with P. betae) have not been identified yet. The transgenic virus-resistant sugar beets 

based on RNA silencing which show phenotypes of infection resistance are on a research stage 

and up to date their broad use is mainly restricted due to reasons of public acceptance. The 

extensive cultivation of a single monogenic resistance source probably exerts a strong selection 

pressure on the virus pathogenicity factor(s) although it has not been experimentally proven. 

To unknown reasons, Rz1 resistance breaking isolates have been detected in few fields 

independently in different countries (Liu et al. 2005; Liu & Lewellen 2006; Pferdmenges et al. 

2008) and outbreaks of RNA5 containing isolates in different European countries in restricted 

areas have been reported several times (Koenig et al. 1995; Koenig & Lennefors 2000; Harju et 

al. 2002). The combination of different Rz-genes will render the rhizomania resistance more 

durable and transgenic virus resistance targeted against strongly conserved virus sequence 

promise to be durable as well, although experience under practical conditions is lacking. 

More research is needed to understand the virus life cycle and the way the sugar beet host cells 

are modified to favour replication, spread and vector transmission as well as the mode of action 

of the different resistances (if based on different mechanisms). To achieve this, the isolation of 

resistance genes and the identification of novel additional resistance sources are requested. 

The extremely high replication rates and error-prone replication enzymes of viruses in general 

allow them to quickly adapt to changing environments and select new variants which enable 

the virus population to survive. Therefore, multiple and flexible resistance strategies are a 

prerequisite for effective virus control. 

REFERENCES 

Acosta-Leal R; Fawley M W; Rush C M (2008). Changes in the Intraisolate Genetic Structure 

of Beet Necrotic Yellow Vein Virus Populations Associated with Plant Resistance 

Breakdown. Virology 20, 60-68. 

Amiri R; Moghaddam M; Mesbah M; Sadeghian S Y; Ghannadha M R; Izadpanah K (2003). 

The inheritance of resistance to Beet necrotic yellow vein virus (BNYVV) in B. 

vulgaris subsp. maritima, accession WB42: Statistical comparisons with Holly-1-4. 

Euphytica 132, 363-373. 

Asher M J C (1993). The sugar beet crop. In: Cooke, D.A. & Scott, R.K. (eds), Science into 

practice. Chapman & Hall, London, 311-346. 

Asher M J C; Grimmer M K; Mutasa-Goettgens E S (2008). Selection and characterization of 

resistance to Polymyxa betae, vector of Beet necrotic yellow vein virus, derived from 

wild sea beet. Plant Pathology DOI:10.1111/j.1365-3059.01978.x. 

Baulcombe D (2004). RNA silencing in plants. Nature 431, 356-363. 

Barzen E; Stahl R; Fuchs E; Borchardt D C; Salamini F (1997). Development of couplingrepulsion-phase 

SCAR markers diagnostic for the sugar beet Rr1 allele conferring 

resistance to rhizomania. Molecular Breeding 3,231–238. 

Biancardi E; Lewellen R T; De Biaggi M; Erichsen A W; Stevanato P (2002). The origin of 

rhizomania resistance in sugar beet. Euphytica 127, 383-397. 

Bolz G; Koch G (1983). Aussichten der Resistenz-(Toleranz) Züchtung im Rahmen der 

Bekämpfung der Rizomania. Gesunde Pflanze 35, 275–278. 

270

Bürcky K; Büttner G (1985). Ansätze zur Selektion rizomaniatoleranter Zuckerrüben während 

der Jugendentwicklung – I. Virustiter. Zuckerindustrie 110, 997–1000. 

Bürcky K; Büttner G (1991). Gehalt an beet necrotic yellow vein virus (BNYVV) in der 

Hauptwurzel von Zuckerrübenpflanzen verschiedener Sorten und deren Leistung unter 

Rizomaniabefall im Feld. Journal of Phytopathology 131, 1–10. 

Büttner G; Mangold B (1998). Tolerance, resistance, immunity – terms and their significance 

with reference to rhizomania. Zuckerindustrie 123, 694–701 (in German). 

Butterfass T (1964). Die Chloroplastenzahlen in verschiedenartigen Zellen trisomer 

Zuckerrüben (Beta vulgaris L.). Zeitschrift für Botanik 52, 46–77. 

Brodersen P, Voinnet O (2009). Revisiting the principles of microRNA target recognition and 

mode of action. Nature Reviews Molecular Cell Biology 10, 141-148. 

Canova A (1959). Appunti di patologia della barbabietola. Informatore Fitopatologico 20, 390- 

396. 

Chiba S; Miyanishi M; Andika I B; Kondo H; Tamada T (2008). Identification of amino acids 

of the beet necrotic yellow vein virus p25 protein required for induction of the 

resistance response in leaves of Beta vulgaris plants. Journal of General Virology 89, 

1314-1323. 

de Biaggi M (1987). Methodes de selection – un cas concret. In: Proceedings of the 50th IIRB 

Winter Congress. Vol II, Brussels, 157–163. 

García-Arenal F; McDonald B A (2003). An analysis of the durability of resistance to plant 

viruses. Phytopathology 93, 941–952. 

Gidner S; Lennefors B L; Nilsson N O; Bensefelt J; Johansson E; Gyllenspetz U; Kraft T 

(2005). QTL mapping of BNYVV resistance from the WB41 source in sugar beet. 

Genome 48, 279-285. 

Giunchedi L; De Biaggi M; Polini C (1987). Correlation between tolerance and Beet necrotic 

yellow vein virus in sugar beet genotypes. Phytopathologia Mediterreanea 26, 23–28. 

Giunchedi L; De Biaggi M; Polini C (1985). Evaluation of ELISA technique for the screening 

of Rhizomania-tolerant sugar beet genotypes. Proc. IIRB 48th Winter Cong., Brussels: 

385–390. 

Harju V A; Mumford R A; Bockley A; Boonham N; Clover G R G (2002). Occurrence in the 

United Kingdom of beet necrotic yellow vein virus isolates which contain RNA 5. 

Plant Pathology 51, 811. 

Harrison D B (2002). Virus variation in relation to resistance-breaking in plants. Euphytica 

124, 181-192. 

Hecht H (1989). Rhizomania – ‚Bekämpfung‘ durch den züchterischen Fortschritt im Rahmen 

des Spektrums toleranter Zuckerrüben-Sorten: Wirksamkeit bei fehlendem, schwachem 

und starkem Beet necrotic yellow vein virus Befall. Bayerisches Landwirtschaftliches 

Jahrbuch 66, 515–527. 

Jan F J; Fagoaga C; Pang S Z; Gonsalves D (2000). A single chimeric transgene derived from 

two distinct viruses confers multi-virus resistance in transgenic plants through 

homology dependent gene silencing. Journal of General Virology. 81, 2103-2109. 

Jones-Rhoades M W; Bartel D P; Bartel B (2006). MicroRNAs and their regulatory roles in 

plants. Annual Reviews of Plant Biology 57, 19–53. 

271

Koenig R; Haeberle A M; Commandeur U (1997). Detection and Characterization of a Distinct 

Type of Beet Necrotic Yellow Vein Virus RNA 5 in a Sugarbeet growing Area in 

Europe. Archives of Virology 142, 1499-1504. 

Koenig R; Jarausch W; Li Y; Commandeur U; Burgermeister W; Gehrke M; Luddecke P 

(1991). Effect of Recombinant Beet Necrotic Yellow Vein Virus with Different RNA 

Compositions on Mechanically Inoculated Sugarbeets. Journal of General Virology 72, 

2243-2246. 

Koenig R; Lennefors B L (2000). Molecular analyses of European A, B and P type sources of 

Beet necrotic yellow vein virus and detection of the rare P type in Kazakhstan. Archives 

of Virology 145, 1561-1570. 

Koenig R; Loss S; Specht J; Varrelmann M; Lueddecke P; Deml G (2009). A single U/C 

nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus 

P25 protein promotes increased virus accumulation in roots of mechanically inoculated, 

partially resistant sugar beet seedlings. Journal of General Virology DOI 

10.1099/vir.0.007112-0. 

Koenig R; Luddecke P; Haeberle A M (1995). Detection of Beet Necrotic Yellow Vein Virus 

Strains, Variants and Mixed Infections by Examining Single-Strand Conformation 

Polymorphisms of Immunocapture Rt-PCR Products. Journal of General of Virology 

76, 2051-2055. 

Lauber E; Janssens L; Weyens G; Jonard G; Richards K E; Lefebvre M; Guilley H (2001). 

Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus 

triple gene block proteins p13 and p15 using a viral replicon. Transgenic Research 10, 

293-302. 

Lein J C; Asbach K; Tian Y; Schulte D; Li C; Koch G; Jung C; Cai D (2007). Resistance gene 

analogues are clustered on chromosome 3 of sugar beet and cosegregate with QTL for 

rhizomania resistance. Genome 50, 61-71. 

Lennefors, B (2006). Molecular breeding for resistance to Rhizomania in sugar beets. Acta 

Universitatis Agriculturae Sueciae, SLU, Doctoral thesis No. 2006:106. 

Lennefors B L; Savenkov E L; Bensefelt J; Wremerth-Weich E; van Roggen P; Tuvesson S; 

Valkonen J P T; Gielen J (2006). dsRNA-mediated resistance to Beet necrotic yellow 

vein virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris). Molecular Breeding 

18, 313–325. 

Lennefors B L; Lindsten K; Koenig R (2000). First record of A and B type Beet necrotic 

yellow vein virus in sugar beets in Sweden. European Journal of Plant Pathology 106, 

199-201. 

Lennefors B L; van Roggen P M; Yndgaard F, Savenkov E L, Valkonen J P (2007). Efficient 

dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets 

is not affected by other soilborne and aphid-transmitted viruses. Transgenic Research 

17, 219-28. 

Lewellen R T (1995). Performance of near-isolines of sugarbeet with resistance to rhizomania 

from different sources. In: Proc 58th IIRB cong, pp 83-92. 

Lewellen R T; E Biancardi (1990). Breeding and performance of rhizomania resistant sugar 

beet. Proc IIRB 53, 79–87. 

Lewellen R T; Skoyen I O; Erichsen A W (1987). Breeding sugar beet for resistance to 

Rhizomania: Evaluation of host–plant reactions and selection for and inheritance of 

resistance. Proc. IIRB 50th Winter Cong., Brussels, Belgium. Vol. II. 139-156. 

272

Lewellen R T; Whitney E D (1993) Registration of germplasm lines derived from composite 

crosses of sugarbeet x Beta maritima. Crop Science 33, 882–883. 

Liu H Y; Lewellen R T (2006). Distribution and molecular characterization of resistancebreaking 

isolates of Beet necrotic yellow vein virus in the United States. Plant Disease 

19, 847-851. 

Liu H Y; Sears J L; Lewellen R T (2005). Occurrence of resistance-breaking Beet necrotic 

yellow vein virus of sugar beet. Plant Disease 89, 464–468. 

Mechelke W; J Kraus (1998). Ergebnisse von Freilandversuchen mit Zuckerrüben mit 

transgener Rizomaniaresistenz. Proceedings of the 61th Congress of the IIRB 

(International Institute for Beet Research), Brussels: 351–355. 

Meulemans M; Janssens L; Horemans S (2003). Interactions between major genes and 

influence of the genetic background in the expression of rhizomania resistance. 1st 

IIRB-ASSBT Congress, 26 February to 1 March 2003, San Antonio, Tex. 161-174. 

Mitter N; Sulistyowati E; Dietzgen R G (2003). Cucumber mosaic virus infection transiently 

breaks dsRNA-induced transgenic immunity to Potato virus Y in tobacco. Molecular 

Plant Microbe Interactions 16, 936–944. 

Nielsen S L; Nicolaisen M; Scheel C; Dinesen I G (2001). First record of beet necrotic yellow 

vein virus in Denmark. Plant Disease 85, 559. 

Niu Q W; Lin S S; Reyes J L, Chen K. C; Wu H W; Yeh S D; Chua N H (2006). Expression of 

artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. 

Nature Biotechnology 24, 1420-1428. 

Paul H; Henken B; Scholten O E; Lange W (1993). Use of zoospores of Polymyxa betae in 

screening beet seedlings for resistance to beet necrotic yellow vein virus. Netherland 

Journal Plant Pathology 99, 3, 151–160. 

Pferdmenges F; Korf H; Varrelmann M (2008). Identification of rhizomania infected soil in 

Europe able to overcome Rz1 resistance in sugar beet and comparison with other 

resistance-breaking soils from different geographic. European Journal of Plant 

Pathology 10.1007/s10658-008-9388-9. 

Pferdmenges F; Varrelmann M, (2008). Breaking of Beet necrotic yellow vein virus resistance 

in sugar beet is independent of virus and vector inoculums densities. European Journal 

of Plant Pathology 10.1007/s10658-008-9408-9. 

Pollini C P; Giunchedi L (1989). Comparative Histopathology of Sugar Beets That Are 

Susceptible and Partially Resistant to Rhizomania. Phytopathologia Mediteranea 28, 

16-21. 

Pollini C P; Masia A; Giunchedi L (1990). Free Indole-3-Acetic Acid in Sugar-Beet Root of 

Rhizomania-Susceptible and Moderately Resistant Cultivars. Phytopathologia 

Mediteranea 29, 191-195. 

Prins M (2003) Broad virus resistance in transgenic plants Trends in Biotechnology 21, 373- 

375. 

Richard-Molard M (1985) Beet rhizomania disease-the problem in Europe. Report of the 1984 

British Crop Protection Conference-Pests and Diseases, 837-845. 

Richard-Molard M S; Cariolle M (2001). Stress hydrique et abiotique et amélioration 

genetique. Proc. IIRB 64th Cong. Bruges, Belgium, 153-158. 

Roossinck M J (1997) Mechanisms of plant virus evolution. Annual Review of Phytopathology 

35, 191-209. 

273

Savenkov E L; Valkonen J P T (2001). Coat protein gene-mediated resistance to Potato virus A 

in transgenic plants is suppressed following infection with another potyvirus. Journal of 

General Virology 82, 2275–2278. 

Schmidlin L; de Bruyne E; Weyens G; Lefebvre M; Gilmer D (2008) Identification of 

differentially expressed root genes upon rhizomania disease. Molecular Plant 

Pathology 9, 741-751. 

Schirmer A; Link D; Cognat V; Moury B; Beuve M; Meunier A; Bragard C; Gilmer D; 

Lemaire O (2005). Phylogenetic Analysis of Isolates of Beet Necrotic Yellow Vein 

Virus Collected Worldwide. Journal of General Virology 86, 2897-2911. 

Smith N A; Singh S P; Wang M B; Stoutjesdijk P A; Green A G; Waterhouse P M (2000). 

Total silencing by intron-spliced hairpin RNAs. Nature 407, 319-320. 

Scholten O E; de Bock T S M; Klein-Lankhorst R M; Lange W (1999). Inheritance of 

resistance to Beet necrotic yellow vein virus in Beta vulgaris, conferred by a second 

gene for resistance. Theoretical and Applied Genetics 99, 740-746. 

Scholten O E; Lange W (2000). Breeding for resistance to Rhizomania in sugar beet: a review. 

Euphytica 112, 219–231. 

Scholten O E; Paul H; Peters D; Van Lent J W M; Goldbach; R W (1994). In situ localisation 

of beet necrotic yellow vein virus (BNYVV) in rootlets of susceptible and resistant beet 

plants. Archives of Virology 136, 349–361. 

Tamada T; Uchino H; Kusume T; Saito M (1999). RNA 3 Deletion Mutants of Beet Necrotic 

Yellow Vein Virus Do Not Cause Rhizomania Disease in Sugar Beets. Phytopathology 

89, 1000-1006. 

Tamada T; Abe, H (1989). Evidence That Beet Necrotic Yellow Vein Virus RNA-4 Is 

Essential for Transmission by the Fungus Polymyxa betae. Journal of General Virology 

70, 3391-3398. 

Tamada T (2007). Susceptibility and resistance of Beta vulgaris subsp. maritima to foliar rubinoculation 

with Beet necrotic yellow vein virus. Journal of General Plant Pathology 

73, 76-80. 

Tian Y; Fan L J; Thurau T; Jung C; Cai D (2004). The absence of TIR type resistance gene 

analouges in the sugar beet (Beta vulgaris L.) genome. Journal of Molecular Evolution 

58, 40-53. 

Voinnet O (2005). Induction and suppression of RNA silencing: insights from viral infections. 

Nature 6, 206-221. 

Wisler G C; Lewellen R T; Sears J L; Liu H Y; Duffus J E (1999). Specificity of DAS-ELISA 

for beet necrotic yellow vein virus and its application for determining rhizomania 

resistance in field-grown sugar beets. Plant Disease 83, 864-870. 

Whitney E D (1989). Identification, distribution, and testing for resistance to Rhizomania in 

Beta maritima. Plant Disease 73, 287-290. 

274

Jeske H, Krenz B, Paprotka T, Wyant P: Geminivirology in the Age of Rolling Circle Amplification. In: 



4-6 Geminivirology in the Age of Rolling Circle Amplification 

Jeske H, Krenz B, Paprotka T, Wyant P 

Universität Stuttgart, Biologisches Institut, Abt. für Molekularbiologie und Virologie der 

Pflanzen, Pfaffenwaldring 57, D-70550 Stuttgart 

Email: holger.jeske@bio.uni-stuttgart.de 

Abstract 

Geminiviruses have caused devastating consequences on many important crop 

plants of the tropics and subtropics and they are migrating to temperate zones with 

climate change. Global transportation of plant material has boosted their epidemics 

and it is therefore highly desirable to developed strict quarantine measures. Easy 

and low cost diagnostic tools to detect geminiviruses in tranported plant material are 

necessary. Although efficient methods are available using antibodies or polymerase 

chain reaction, these techniques rely on prior knowledge on the virus. Rolling circle 

amplification (RCA) combined with Restriction fragment length polymorphism 

(RFLP) and direct sequencing has now been developed in our laboratory as an 

efficient alternative for future geminivirus diagnostics allowing the identification of 

circular DNA viruses without any a priori knowledge (Haible et al., 2006; Homs et 

al., 2008; Schubert et al., 2007). A trilateral European Project including partners of 

Spain (E. Bejarano) and France (B. Gronenborn) has been established under the title 

“International Reference Centre for the Genomics and Diagnosis of Viruses with 

Small Circular DNA” on the basis of these techniques in order to collect and 

identify geminiviruses from all over the world (see http://www.unistuttgart.de/bio/bioinst/molbio/). 

The current progress of this project will be 

explained and international collaborations in this context will be offered. 

References: Haible, D., Kober, S., and Jeske, H. (2006). Rolling circle amplification 

revolutionizes diagnosis and genomics of geminiviruses. J. Virol. Methods 135, 9- 

16. Homs, M., Kober, S., Kepp, G., and Jeske, H. (2008). Mitochondrial plasmids 

of sugar beet amplified via rolling circle method detected during curtovirus 

screening. Virus Res. 136, 124-129. Schubert, J., Habekuß, A., Kazmaier, K., and 

Jeske, H. (2007). Surveying cereal-infecting geminiviruses in Germany - 

diagnostics and direct sequencing using rolling circle amplification. Virus Res. 127, 

61–70. 

275

Haggag W M, Mahmoud Y S, Farag W M: Elicitor-Induced Sugar Beet Defence Pathways Against Beet Mosaic 

Virus (BtMV) infection. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

4-7 Elicitor-Induced Sugar Beet Defence Pathways Against Beet Mosaic 

Virus (BtMV) infection 

Haggag W M 1 , Mahmoud Y S 2 , Farag W M 3 

1 

Department of Plant Pathology, National Research Center, Dokki, Cairo, Egypt, 

2 Botany Department, Faculty of Science, Sohag University, Sohag 82524, Egypt. 

3 Botany Department, (Plant Pathology) South Valley University, Egypt. 

276 

Abstract 

Induced systemic resistance (ISR) in plants against pathogens is a widespread 

phenomenon that has been intensively investigated with respect to the underlying 

signaling pathways as well as to its potential use in plant protection. In the present 

study, sugar beet plants treated with methyl jasmonate (MJ) exhibited enhancement 

resistance to Beet Mosaic Virus (BtMV), also increased polyamines (PAs) 

accumulation and salicylic acid (SA). BtMV-inoculated plants showed symptoms 

including severe mosaic, mottling and deformations. Spraying sugar beet with MJ 

on leaves helped to prevent the harmful effects produced. Double antibody 

sandwich-enzyme linked immunosorbent assay (DAS-ELISA) values were 

decreased in extracts of MJ-treated plants. In addition, the lesion numbers and 

concentration of BtMV- were reduced by polyamines i.e. L-Ornithine; L- 

Ornithine.Hydro; L-Ornithine.monohyd; Pentamidine and Diminidine treatment. 

SDS–PAGE analysis of proteins accumulation in leaf tissue revealed that MJ at 3.0 

µg/ml concentration completely inhibited BtMV protein accumulation. The changes 

of some biochemical and molecular parameters in sugar beet leaves associated with 

BtMV infection and the effect of exogenous application of MJ were showed, 15 

days following treatment. Significant increases in levels of free and conjugated 

putrescine, spermidin and spermine was noticed after treatment of the leaves with 

MJ. These changes were accompanied by increasing in activity of soluble ornithine 

decarboxylase (ODC) and polyamine oxidase (PAO), in leaves following 

treatment with MJ. Analysis of soluble protein, salicylic acid (SA), peroxidase, 

chitinase, and phenols in protected plants revealed enhancement accumulation of 

these substances. In addition, protein patterns represent some newly synthesized

polypeptides which reflect formation of pathogenesis related proteins in MJ 

treatment. All results show significant changes in metabolism affected by either 

viral infection or MJ treatments and also indicate that exogenous MJ plays an 

important role in induction of defense mechanism against BYMV infection. 

INTRODUCTION 

Sugar beet (Beta vulgaris) ranks the second important sugar crop after sugar cane, producing 

annually about 40 % of sugar production all over the world (Mirvat Gobarah & Mekki 2005). 

Viruses are one of the most destructive plant pathogens, whereas a considerable numbers of 

viruses have been isolated from sugar beet plants around the world (Sutic et al. 1999). In 

Egypt, cucumber mosaic virus (CMV) (Omar et al. 1995); BtMV (Abdel-Ghaffar et al. 2003 

and Beet curly-top virus (BCTV) (Mahmoud et al. 2004) were isolated. One of the viruses 

occurring in this crop all over the world is BtMV. This virus belongs to potyviridae, easly 

aphid transmitted, filament-shaped and single-stranded RNA viruses (Russell 1971). BtMV 

causes a mosaic in sugar beet, red beet and spinach (Juretic 1999). Usually, this potyvirus 

occurs in the form of mild strains that don't cause significant economic damage in sugar beet or 

spinach. However, sever strains of BtMV that cause significant yield losses of sugar beet have 

been found as (Abde-Ghaffar, et al. 2003). Plants defend themselves against pathogen invasion 

through the action of specific resistance (R) genes and various nonspecific host responses (Li, 

et al. 1999). Most of the plants possess defense mechanisms against pathogen attack, which 

triggered by a stimulus prior to the pathogen attack, reduces the disease. The stimulus can 

increase the concentration of existing defense compounds that induce production a new 

defensive structures and chemicals (Baileya et al. 2005). Methyl jasmonate (MJ) is widespread 

natural regulators involved in many processes during plant development and defense (Cheong 

& Yang 2003; Thaler et al. 2004). Jasmonates have been reported to induce systemic 

protection against plant fungal diseases such as rust and powdery mildew in wheat and barley, 

respectivilly (Haggag & Abd-Kreem 2009; Walters et al. 2002). The major forms of 

polyamines are putrescine, spermidine and spermine. The three polyamines are normal 

constituents of eukaryotic and prokanytic cells and are important regulators of growth and 

differentiation as well as in plant responses to stress (Walters 2000). Polyamines occur in 

plants in free form, bound electrostatically to negatively charged molecular, and conjugated to 

small molecules and proteins (Walters et al. (2002). Consequently, they modulate DNA-protein 

(Shah et al. 1999), and protein-protein interactions (Thomas et al. 1999). In general, polyamine 

metabolism has long been known to be altered in plants responding to profound changes in 

plants interacting with fungal and viral pathogens (Walters 2003). Accumulation of polyamines 

has been observed in tobacco cultivars resistant to TMV, but not in TMV-susceptible 

counterparts (Marini et al. 2001). Polyamines conjugated to phenolic compounds, 

hydroxycinnamic acid amides (HCAs), have been shown to accumulate in incompatible 

interactions between plants and a variety of pathogens, while changes in the diamine catabolic 

277

enzyme diamine oxidase suggest a role for this enzyme in the production of hydrogen 

peroxidase during plant defense responses (Walters 2003). 

Another line of evidence for the role of jasmonates in disease resistance comes from their 

stimulatory effect on secondary metabolite production including ribosome-inactivating protein, 

serine proteinase inhibitors, phenylalanine ammonia lyase, alkaloids, thionin, terpenes, 

phenolics and including hydroxycinnamic acid amides (HCAs) (Biondi et al. 2000; Martin et 

al. 2002). HCAs are formed from the covalent binding of polyamines (putrescine, spermidine 

and spermine) to hydroxycinnamic acids like caffeic acid and coumaric acid (Martin et al. 

2002). This form of induced resistance is generally referred to as systemic acquired resistance 

(SAR). SAR is an inducible plant defence response involving a cascade of transcriptional 

events induced by salicylic acid (SA) (Naylor et al. 1998; Madhusudhan et al. 2008). This 

paper reports, induce systemic protection against sever isolate of BtMV in sugar beet. Also 

show that components of the MJ/ PAs–mediated resistance pathway are required for plant 

resistance. 


Virus strains and plant material 

Isolate of BtMV which was isolated in previous work by Abdel-Ghaffar et al. (2003) was used 

in this study. Symptoms caused by this isolate was small chlorotic lesions, mosaic, apical 

necrosis and mottle mosaic. For inoculum preparation, young BtMV-infected leaves of 

greenhouse- grown plants of the cultivar Kawemira were harvested about 25 days after 

inoculation. Leaves were ground in 0.1 M phosphate buffer pH 7.4 (1:10, w/ v) and the sap was 

filtered through two layers of cheesecloth and mixed with Carborundum (600-mesh) at 2% 

(w/v). Healthy sugar beet (Beta vulgaris L. cv Kawemira) and Chenopodium amaranticolor 

plants were maintained under greenhouse conditions at 23 ±2°C were sprayed with solutions of 

Methyl jasmonate and polyamines i.e. L-Ornithine.monohyd; L-Ornithine.Hydro; L- 

Ornithine.monohyd; Pentamidine and Diminidine (1.5 µg/ml ), (Sigma chemicals) as described 

by Wafaa and Abd-El-Kareem 2009. The sprayed plants were viral inoculated 2 dayes after 

treatment. Up to 25 days post inoculation (DPI), disease incidences were observed every day in 

sugar beet as the number of plants showing symptoms. Also, samples of leaves were taken for 

DAS-ELISA test. On the other hand, local lesion numbers were counted in C. amaranticolor 

leaf plants. 

Enzyme-linked Immunosorbent Assay (ELISA) 

Double-antibody sandwich (DAS)-ELISA test according to Clark et al. 1977 with polyclonal 

antisera was used to determine BtMV presence in plants treated with MJ (from 0.08 to 3.0 

µg/ml) or PAs (1.5 µg/ml). The uppermost expanded leaves of sugar beet plants were collected 

at 15 DPI, and sap was expressed using phosphate buffer saline (PBS) containing 0.05% 

Tween-20 at a ratio of 1:10 (w/v). Plates were coated with anti-BtMV obtained from previous 

278

study (Abde-Ghaffar, et al. 2003) then diluted at 1:200 in phosphate buffer. Plates were 

incubated for 4 h at room temperature (RT).Plant samples were incubated in the coated plate at 

RT for 2 h before adding alkaline phosphatase-conjugated anti-BtMV diluted at 1:200 in PBS- 

T. After a 2-h incubation at RT, substrate (p-nitrophenylphosphate at 1 mg/ml in 

diethanolamine, pH 9.8) was added and incubated at room temperature for 1 h. Absorbance 

values were determined at 405 nm. 

Chemical analysis 

After three days of inoculation, three leaves/ plant treated with MJ were separately collected, 

frozen for 36 hrs, dried and powdered. Generally, 100 mg of dried samples was employed for 

analysis. 

Quantification of polyamines (PAs) 

Determination of free polyamines and polyamine conjugates 

Free and conjugated PAs in sugar beet leaves were quantified. Free polyamines were extracted 

and hydrolysed using the method described by Slocum and Galston (1985). This yielded a non 

–hydrolysed perchloric acid (used at 10%) supernatant, containing the free polyamines, and the 

hydrolysed supernatant and pellet fractions, containing polyamines liberated from various 

types of conjugates. Polyamines were extracted with 2ml of 0.5M HClO4 overnight at room 

temperature, derivatized with benzoyl chloride and quantitated with high performance liquid 

chromatography (HPLC) using standard chemicals (Sigma chemicals). Separation and 

quantification of derivatized polyamines were performed with a Shimadzn Lc-6A HPLC 

equipped with a UV detector. The analytical condition was as follows: 6×150 mm in column 

size; 45 0 C column temperature; 64% methanol mobile phase and detection on 254 nm. 

Activities of polyamine biosynthetic enzymes 

Ornithine decarboxylase (ODC) and polyamine oxidase (PAO) activityies were determined 

according to as described previously ( Zarb and Walters 1993). 

Evaluation of MJ-Induced Resistance against BtMV Infection 

Measurement of Protein 

Protein in leaves treated with MJ was extracted by the method of Bollag and Eldelstein (1992). 

Fifty μg protein of each treatment was analyzed by 12% sodium dodecyl sulfate (SDS-PAGE) 

according to the method described by Laemmli, (1970) using 10% acrylamide in the separating 

gel and 3% in the stacking gel. Molecular weights of polypeptide bands (KDa) were calculated 

from a calibration curve of low molecular weight marker kit of Phramacia (Uppsala, Sweden). 

279

Measurement of Salicylic Acid (SA) 

Changes in the level of free SA were determined in the leaves by using a modified 

spectrophotometric method (Li et al. 1999). Leaves were ground in liquid nitrogen with a 

mortar and pestle then extracted with 2 ml of 50% ethanol. The supernatant was centrifuged 

(3,000 rpm for 15 min), filtered through four layers of cheesecloth, and then 0.5 ml of 6MHCl 

was added for SA hydrolysis. To extract SA, 10 ml of tetrachloride was added to each sample, 

and the extract was mixed with 5 ml of ferric nitrate solution for 2 min. After centrifugation, 

the aqueous phase was analyzed by spectrophotometry (530 nm). For quantitative analysis, a 

standard curve was established with commercial SA (Sigma, St. Louis, MO) suspended in 50% 

ethanol. 

Measurement of Chitinase, Peroxidase and Polyphenoloxidase 

Chitinase activity was evaluated according to the methods described by Boller and Mauch 

(1988). Colloidal chitin was used as substrate and dinitrosalicylic acid as reagent to measure 

reducing sugars. Chitinase activity was expressed as mM N-acetylglucose amine equivalent 

released / gram fresh weight tissue / 60 minutes. Peroxidase activity was evaluated according 

to the methods described by Allam & Hollis (1972) as one unit of perioxidase activity was 

expressed for the change in absorbance at 425 nm/minute /g fresh weight. Polyphenoloxidase 

activity was quantitatively determined according to the method described by Matta & Dimond 

(1963). One unit of polyphenoloxidase was expressed as the change in absorbance at 420 nm 

for 30 min at 25 0 C/ g fresh weight. 

Determination of free and conjugated phenol contents 

Free and conjugated phenols were determined in treated leaves after 15 days of plant spraying, 

according to the A.O.A.C . (1975). In this study the Folin–Danis reagent phenols identified by 

High Performance Liquid Chromatography (HPLC) was used. Also, a reverse phase C8 

column was used then compared with a standard (Sigma chemicals). 

Western blotting for BtMV-coat protein analysis 

Leaf samples of sugar beet were collected 15 days after viral inoculation and then extracted 

according to the method of Donald et al. 1993. Samples were centrifuged at 15.000 rpm for 3 

min and the supernatant were separated using polyacrylamide gel electrophoresis (SDS- 

PAGE). Proteins were transferred onto nitrocellulose membrane using transfer buffer. The 

membranes were blocked overnight in 3% (w/v) milk powder in TBS buffer at 4 °C, followed 

by washing in TBS three times, 5 min each. The membranes were probed with polyclonal 

antibody diluted (1:1000) raised in rabbits against BtMV coat protein then washed 3 times, 5 

min each. The membranes were probed using a secondary antibody, goat antrabbits conjugate 

with horseradish peroxidase (1:5000), and then developed using West Dura extracted (Pierce) 

and photographed by x0Ray film. 

280


For data analysis the statistical computer application package SPSS 10.0 was employed. The 

data generated were average of three independent experiments. Data were subjected to analysis 

of variance (ANOVA) and the means were compared for significance using Duncan's Multiple 

Range Test (DMRT; P = 0.05). 

RESULTS 

The effects of MJ and PAs on BtMV infection in inoculated sugar beet. 

The experiments were performed to evaluate the effect of MJ and PAs on infection and 

presence of BtMV in sugar beet leaves through symptoms (Fig. 1); infectivity assay (Fig. 2A) 

and DAS-ELISA confirmation (Fig. 2B). Twenty five days after inoculation by BtMV, treated 

plants showed symptoms ranged from severe mosaic (Fig. 1C and D), (wheares L- 

Ornithine.Hydro and Pentamidine were sprayied) to healthy one (Fig, 1E), (wheares MJ was 

used). Treated sugar beet leaves with 1.5 µg/ml of MJ as a foliar spray, prevented the 

appearance of disease symptoms caused by BtMV. Furthermore, the number of local lesions 

which appeared on C. amaranticolor leaves after inoculation with sap extracted from treatedviral 

inoculated plants, not showed when MJ was used. But greatly decreased with PAs, when 

L-Ornithine, L-Ornithine.monohyd and Diminidine. On the other hand, there is no variation on 

local lesion numbers when sap extracted from L-Ornithine.Hydro and Pentamidine treated and 

non-treated plants. L-Ornithine. at 1.5 µg/ml is the most effective polyamine tested compared 

with control. BtMV accumulation in MJ-treated and nontreated plants was evaluated using 

DAS-ELISA analyses. The results of ELISA represent the mean value for 5 samples in each 

treatment was showed in (Fig. 2B). When the mean ELISA absorbance values for those plants 

infected with BtMV was compared, MJ treatment showed lower values in treated plants. MJ 

treatment significantly reduced virus accumulation compared with the no treated plants. In 

addition, PAs treatment had less effect on virus multiplication with that of the control one that 

showing mosaic symptom. 

A B C D E 

Figure 1. Effect of MJ and PAs treatments on symptoms induced by BtMV on sugar 

beet plants A: Diminidine; B: L-Ornithine; C: L-Ornithine Hydro; D: 

Pentamidine and E: MJ. 

281

To examine whether MJ-mediated activation of the defense responses, different concentrations 

from 3 to 0.08 µg/ml concentrations were used and tested by DAS-ELISA (Fig. 3) . The mean 

values of DAS-ELISA were decreased gradually with increasing concentration of MJ. Greatest 

reduction and non significant results were achieved by using MJ as foliar spray at 3.0; 1.5; 0.75 

and 0.35 µg/ml (Fig. 3). 

282 

DAS- ELISA Value 

number of lesion /leaf 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 

132 

38 

145 

A 

128 

112 

39 

117 118 

139 

Methyl Jasmonate 

L-Ornithine 

L-Ornithine. hydro 

L-Ornithine.monohyd 

Pentamidine 

Diminidine 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 


0.071 

1.352 

L-Ornithine 

0.983 

1.246 

Antivirus compound 

B 

L-Ornithine. hydro 

1.052 

1.305 

L-Ornithine.monohyd 

0.591 

Antivirus compound 

1.144 1.055 

Pentamidine 

1.28 

Diminidine 

37 

0.343 

126 

1.193 

Tested 

Control 

Tested 

Control 

Figure 2. Survey of antiviral activity of MJ and PAs measured biologically in C. 

amaranticolor plant as local lesion numbers (A) or serologically by DAS- 

ELISA test(B)

DAS-ELISA value 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

0.066 

1.27 

0.075 

1.35 

0.089 

1.28 

0.094 

1.21 

0.122 

1.34 

0.151 

3 1.5 0.75 0.35 0.17 0.08 

MJ concentrations 

1.22 

Test 

Control 

Figure 3. ELISA-values in inducer treated host plants challenge inoculated with BtMV 

and sprayed with methyl jasmonate concentrations 

Free and conjugated polyamines in plants treated with MJ 

The relationship between MJ and the polyamines biosynthesis was then examined (Table1). 

Data estimated of polyamine levels showed significant change in polyamine biosynthesis either 

MJ-treated and/or BtMV-inoculated leaves. Putrescine, spermidine and spermine were greatly 

decreased in leaves following inoculation with BtMV. On contrast, treated sugar beet leaves 

with MJ produced moderate significant effect on levels of free polyamines in compared with 

untreated control. Moreover, levels of conjugates of spermidine and spermine were 

significantly increased in leaves after exposure to MJ. 

Table 1. Concentrations of free and conjugated forms of polyamines in leaves of 

sugarbeet plants treated with different concentrations of methyl jasmonate and 

inoculated with BYMV under greenhouse conditions. 

Methyl 

Jasmonate 

concentration 

Putrescine 

(µg/ml) Free Conjugated 

Polyamine concentration y 

(nmol g -1 FW) 

Spermidin Spermine 

Free Conjugated 

Free Conjugated 

3.0 97.9a 351.2a 84.5a 253.3a 98.9a 298.5a 

0.17 82.9b 319.6b 71.9b 229.4b 71.8b 251.7b 

0.0 71.3c 276.5c 60.5c 141.2c 66.5c 201.2c 

Inoculated 42.8d 121.7d 32.8d 89.4d 44.7d 121.9d 

Every value represents the mean of three replicates with standard error and values with the different letters are 

significantly different according to Duncan's Multiple Range Test (P = 0.05). 

Activities of polyamine biosynthetic enzymes 

ODC and PAO activities were determined in leaves of sugar beet following exposure of the 

leaves to MJ and BtMV-inoculated leaves (Table 2). Activity of both enzymes was decreased 

in leaves following treatment to BtMV. Very large and significant increases in activities of both 

283

ODC and PAL were found in leaves after treated with MJ and inoculated with BtMV in 

compared with untreated control. 

Induction of resistance to BtMV in sugar beet by MJ 

Protein patterns represent some newly synthesized polypeptides which reflect formation of 

pathogenesis related proteins in MJ treatment (Fig. 4). Induction of PR proteins by MJ was also 

confirmed at the protein level. Two bands were observed by MJ treatment. Large and 

significant increases in soluble protein activity were found in leaves of sugar beet following 

treatment of the leaves with MJ, with a 5-fold increase. A biochemical assay for SA revealed 

accumulation of SA in leaves treated with MJ and inoculated with BtMV (Fig. 5). To reveal the 

possible involvement of plant defense enzymes in MJ-induced protection against BtMV in 

284 

Table 2 Concentrations of ornithine decarboxylase (ODC) and polyamine oxidase 

(PAO) in leaves of sugarbeet plants treated with different concentrations of 

methyl jasmonate and inoculated BYMV under greenhouse conditions. 


concentration (µg/ml) 

3.0 

0.17 

0.00 

Inoculated 

ODC activity y 

(nmol CO2 [ mg protein] -1 h -1 ) 

39.2a 

21.5b 

12.5c 

5.87d 

Enzymes activities 

PAO activity 

(pmol product [ mg protein] -1 h -1 ) 

178.2a 

93.5b 

18.65c 

7.65d 



SA 

ng/mg 

F.W. 

80 

70 

60 

50 

40 

30 

20 

10 

0 

SA Soluble protein 

70.8 

39.1 

48.829.4 

35.7 

11.4 

23.7 

9.65 

3 0.17 0 Inoculated 

Methyl jasmonate conc. ug/ml 

Fig.4 Concentrations of plant defense related protein, SA in leaves of sugarbeet 

plants treated with different concentrations of methyl jasmonate and inoculated 

BYMV under greenhouse conditions 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0

Figure 5. Methyl jasmonate induced virus resistance in sugar beet. Line 1: untreated 

control plants; Line 2: infected plants with BtMV; Line 3: Leaves treated with 

MJ at 3.0 µg/ml and inoculated with BtMV; Line 4: Leaves treated with MJ at 

3.0 µg/ml only and M: molecular weight marker 

sugar beet, the activities of chitinase, peroxidase and polyphenoloxidase were monitored 

(Table 3). Inoculation of the leaves with BtMV led to a decrease in enzymes activities. 

Activities of the plant defence-related enzymes chitinase, peroxidase and polyphenoloxidase 

were increased significantly in leaves following treatment leaves with MJ. Chitinase activity 

was significantly increased in leaves following treatment with MJ. Activity of peroxidase was 

greatly increased in treated leaves with MJ. Data also show that the polyphenoloxidase activity 

was similar to that of peroxidase activity. Polyphenoloxidase activity was greatly decreased in 

plants inoculated with BYMV in comparison with untreated control. The BtMV inoculated 

plants showed slightly significant difference in phenol contents compared with the healthy 

plants (Table 4). MJ at 3.0 µg/ml sprayed on sugar beet plants, resulted in an increase in 

conjugated phenols content in compared with free phenols. Since, the greatest increase in 

conjugated phenol contents were 39.1 Catechol /g/ /F.W. compared with free phenol 27.4 

Catechol /g/ /F.W. inoculated control 8.9 and 11.4 Catechol /g/ /F.W and untreated control 

10.3 and 11.4 Catechol /g/ /F.W., respectively. 

Table 3. Concentrations of chitinase and peroxidase in leaves of sugarbeet plants 

treated with different concentrations of methyl jasmonate and inoculated 

BYMV under greenhouse conditions. 


concentration (µg/ml) 

Chitinase 

(unit) 

Peroxidase 

(unit) 

Polyphenoloxidase 

(unit) 

3.0 7.0a 31.8a 0.6a 

0.17 6.0b 21.1b 0.5b 

0.0 4.87c 9.40c 0.3c 

Inoculated 3.21d 5.87d 0.2d 



285

286 

Table 4. Concentrations of Phenols and SA in leaves of sugarbeet plants treated with 

different concentrations of methyl jasmonate and inoculated BYMV under 

greenhouse conditions. 

Methyl Jasmonate concentration 

(µg/ml) 

Free Phenols 

(Catechol /g/ /F.W.) 

Conjugated phenols 

(Catechol /g/ /F.W.) 

3.0 27.4a 33.1a 

0.17 25.2b 17.2b 

0.00 14.2c 10.3c 

Inoculated 11.4cd 8.9cd 



Western blotting for viral coat protein 

The coat protein of the BtMV purified by SDS-PAGE was cross reacted specifically to the 

antiserum raised against the coat protein of the Egyptian isolate of BtMV, using western 

blotting analysis. One out of three MJ-treated plants negatively reacted (as negative control) in 

the presence of the coat protein of BtMV isolate (Figure 6; lanes 6). On the other hand, tow 

plants give faint reaction (Figure 6; lanes 7 and 8) In this experiment BtMV-infected sugar beet 

and not treated with MJ was used as a positive control (Figure 6) 

1 2 3 4 5 6 7 8 9 

Figure 6. Effect of methyl jasmonate (MJ) treatment on accumulation of BYMV 

coat proteins in inoculated sugar beet tissue. Lanes 1-5: samples from 

MJ-untreted and BtMV-infected plants (as a positive control); Lanes 6-8: 

samples from BtMV-infected and then MJ-treated plants and Lane 9: 

sample from BtMV-uninfected and MJ-untreated plant (as a negative 

control) 

DISCUSSION 

We have demonstrated that treatment with the MJ induces the resistance in the sugar beet 

plants against BtMV. These results were confirmed by reduction in the lesions numbers and 

DAS-ELISA test. Sprayed leaves of sugar beet with 1.5 µg/ml of MJ prevented the appearance 

of disease symptoms caused by BtMV and reduced its concentration compared with nontreated 

control as evident by the results of indirect ELISA and indicator plant tests. The typical

dominant resistance response is associated with several defense-related events, including rapid 

activation of polyamines, PR protein, biosynthesis of SA, oxidative enzymes, and phenols 

contents. There is limited evidence that PAs play a role in plant self-defense. When leaves 

were sprayed with MJ or PAs, and then inoculated with BtMV, lesions became much fewer in 

comparison with those of the untreated controls. This result, may be indicated that treatment of 

host plants with MJ and PAs led to reduce on viral concentration in comparison with controls. 

Our results from independent pharmacological experiments strongly indicated that polyamines 

contribute to plant resistance. Treated leaves of sugar beet with MJ great increases in free and 

conjugated putrescine spermidine and spermine, were observed. The increase in soluble 

polyamine free and conjugates found here in treated leaves agrees with our previous reports in 

wheat plants treated with MJ (Haggag, Wafaa & Abd-El-Kareem 2009). Also, in the present 

work, MJ treatment of leaves led to increased activities of the polyamine biosynthetic enzymes 

ODC and an increase of PAL activity, it would appear that both substrates for the formation of 

soluble polyamine free and conjugates were likely to have been increased in amount in these 

tissues. The increase in activities of the three polyamine biosynthetic enzymes noted here 

agrees with other work (Biondi et al. 2000) which also found increased activities of ODC in 

MJ-treated tobacco thin layers. Also, all concentrations of MJ treatments increased PR protein 

and SA. Accumulations of PR-proteins and SA have been correlated with systemic resistance 

in plants. These results obtained by various groups shows that SA treatment inhibit the 

replication, cell-to-cell movement, and long distance movement of plant viruses (Shekara et al. 

2004; Madhusudhan et al. 2008). Moreover, results in this study indicate that all treatments 

stimulated the enzymes activities. Several studies have demonstrated that over-expression of 

chitinases , ß-1,3- glucanase , peroxidase and polyphenoloxidase in transgenic plants is 

associated with enhanced resistance to various viral pathogens (Thomas et al. 1999). The 

results of our experiments showed a higher level of free and conjugated phenols was induced 

by the treatment with MJ, indicating the possible role in viral resistance. Interestingly, 

jasmonates are known to increase the formation of phenolic compounds by stimulating the 

phenylpropanoid pathway. In many cases, resistance is associated with increased expression of 

defense genes, including the pathogenesis related (PR) genes and the accumulation of SA in 

the inoculated leaf; localized host cell death at the site of pathogen entry, a phenomenon known 

as the hypersensitive response (HR), also occurs (Shekara et al. 2004). Salicylic acid is an 

important component in the signal transduction pathway leading to systemic acquired 

resistance (SAR) to the entire spectrum of plant pathogens: bacteria, fungi, and viruses (Naylor 

et al. 1998). Our results show that MJ can inhibit the development of virus disease in plants in 

two ways: either by inhibiting replication of the virus at the initial point of infection, or by 

stimulating PR protein, PAs and SA. 

REFERENCES 

Abdel-Ghaffar M H; Salama M I; Mahmoud S Y M (2003). Electron microscopy, serological 

and molecular studies on an Egyptian isolate of beet mosaic potyvirus. Arab University 

J. of Agric. Sci. 11(2), 469-484. 

287

Allam A; Hollis S (1972). Sulfide inhibition of oxidases in rice root. Phytopathology 62, 

634-639. 

A.O.A.C. (1975). Official Methods of Analysis of Association Official Agricultural Chemists 

(12 th Ed.). Washington, DC. 

Baileya A; Strema D; Baea H; Mayolob G; Guiltinan J (2005). Gene expression in leaves of 

Theobroma cacao in response to mechanical wounding, ethylene, and/or methyl 

jasmonate. Plant Science 168, 1247–1258. 

Biondi S; Fornalé S; Oksman K; Eeva M; Agostani S; Bagni N (2000). Jasmonates induce 

over-accumulation of methyl putrescine and conjugated polyamines in Hyoscyamus 

muticus L. root cultures. Plant Cell Reports 19, 691–697. 

Bollag D M; Eldelstein S J (1992). Protein extraction. In: Protein methods, eds D M Bollag & 

S J Eldelstein, pp 27 – 42. Wiley-Liss: New York. 

Boller T; Mauch F (1988). Colourimetric assay for chitinase. Methods in Enzymology 161, 

430-435. 

Boye K; Jensen P; Stummann B; Henningsen K (1990). Nucleotide sequence of cDNA 

encoding the BYMV coat protein gene. Nucleic Acids Res. 25(16), 4926. 

Cheong J J; Yang D C (2003). Methyl jasmonate as a vital sub-stance in plants. Trends in 

Genetics 19, 409 – 413. 

Clark M F; Adams A N (1977). Characteristics of the microplate method of enzyme-linked 

immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34, 475-483. 

Donald R G K; Zhou H; Jackson A O (1993). Serological analysis of Barly stripe mosaic virusencoded 

proteins in infected barly. Virology 195, 659-668. 

Gallagher S R (1996). One-dimensional SDS gel electrophoresis of proteins. In: Current 

Protocols in Molecular Biology, eds F M Ausubel, R Brent, R E Kingston, D D Moore, 

J G Seidman, J A Smith, K Struhl, pp. 10.2.1–10.2.35. New York: Greene Publishing 

and Wiley-Interscience. 

Gillaspie AG; Hopkins M S; Pinnow D L; Jordan R L (1998). Characteristics of a potyvirus of 

the bean yellow mosaic virus subgroup in Sesbania speciosa germ plasm. Plant Dis. 82, 

807-810. 

Haggag W M (2005). Polyamines: induction and effect on rust disease control of bean. Plant 

Pathol. Bull. 14, 89-102. 

Haggag, W M; Abd-El-Kareem F. (2009). Application of methyl jasmonate increases 

polyamines, chemical defense and resistance against leaf rust infection in wheat plants. 

Achieves Journal of Phytopathology and Plant Protection 42(1), 16–31. 

Juretic N (1999). First report of beet mosaic potyvirus on sugar beet in Croatia. Acta Bot. 

Croat. 58, 141-145. 

Laemmli U K (1970). Cleavage of structural proteins during the assembly of the head 

bacteriophage T4. Nature 224, 680 – 685. 

Li X; Zhang Y; Clarke J D; Li Y; Dong X (1999). Identification and cloning of a negative 

regulator of systemic acquired resistance, SNI1, through a screen for suppressors of 

npr1-1. Cell 98, 329–339. 

Madhusudhan1 K; Deepak A; Prakash K; Agrawal K; Jwa N; Rakwal, R (2008). Acibenzolar- 

S-Methyl (ASM)-Induced Resistance against Tobamo viruses Involves Induction of 

RNA Dependent RNA Polymerase (RdRp) and Alternative Oxidase (AOX) Genes. 

J. Crop Sci. Biotech. 11 (2), 127- 134. 

288

Mahmoud S; Galal A M; Abdel-Ghaffar M H (2005). Biological and molecular studies on an 

Egyptian isolate of beet curly top geminivirus. Egypt. J. Biotechnol. (20), 236-253. 

Marini F; Betti L; Scaramagli S; Biondi S; Torrigiani P (2001). Polyamine metabolism is 

upregulated in response to tobacco mosaic virus in hypersensitive, but not in 

susceptible, tobacco. New Phytol 149, 301–309. 

Martin D; Tholl D; Gershenzon J; Jörg J (2002). Methyl jasmonate induces traumatic resin 

ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of 

norway spruce stems. Plant Physiol 129, 1003-1018. 

Matta A; Dimond A (1963). Symptoms of Fusarium wilt in relation to quantity of fungus and 

enzyme activity in tomato stems. Phytopathology 53, 574-587. 

Gobarah M E; Mekki B B (2005). Influence of Boron Application on Yield and Juice Quality 

of Some Sugar Beet Cultivars Grown under Saline Soil Conditions. Journal of Applied 

Sciences Research 1(5), 373-379. 

Naylor M; Murphy M;O. Berry J; Carr P (1998). Salicylic acid can induce resistance to plant 

virus movement. Mol. Plant-Microbe Interactions 11(9), 860–868. 

Radwan D; Lu G; Fayez K; Younis S M (2008). Protective action of salicylic acid against bean 

yellow mosaic virus infection in Vicia faba leaves. J. Plant Physiol. 165, 845-857. 

Ross W; Walters D; Robins D (2004). Synthesis and antifungal activity of five classes of 

diamines. Pest Management Sci. 60 (2), 143-148. 

Russell G (1971). Beet mosaic virus. C.M.I/A.A.B. Descriptions of plant viruses, No. 53. 

Shah N; Thomas T; Shirahata A; Sigal L; Thomas T (1999). Activation of nuclear factor B by 

polyamines in breast cancer cells. Biochemistry 38, 14763–14774. 

Shekara S; Navarre D; Kachroo1 A; Kang H; Klessig D; Kachroo R. (2004). Signaling 

requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip 

crinkle virus in Arabidopsis. The Plant Journal 40, 647–659. 

Shukla D; Ward C; Brunt A (1994). The potyviridae. CAB International: Wallingford. 

Slocum R; Galston A (1985. Change in polyamine biosynthesis associated with postfertilization 

growth and development in tobacco ovary tissue. Plant Physiol. 79, 

336-343. 

Sutic D D; Ford R E; Tosic M T (1999). Handbook of plant virus diseases. CRC Press: Boca 

Raton. 

Thaler J; Owen B; Higgins V (2004). The role of the jasmonate response in plant susceptibility 

to diverse pathogens with a range of lifestyles. Plant Physiol. 135, 530-538. 

Thomas T; Shah N; Klinge C; Faaland C; Adihkarakunnathu S; Gallo M A; Thomas T J 

(1999). Polyamine biosynthesis inhibitors alter protein-protein interactions involving 

estrogen receptor in MCF-7 breast cancer cells. J. Mol Endocrinol 22, 131–139. 

Walters D R (2000). Polyamines in plant microbe interaction. Physiol. Mol. Plant Pathol. 57, 

137-146. 

Walters D R (2003). Polyamines and Plant Disease. Photochemistry 64, 97-107. 

Walters D; Cowley T; Mitchell A (2002). Methyl jasmonate alters polyamine metabolism and 

induces systemic protection against powdery mildew infection in barley seedlings. 

J. Experimental Botany 53 (369), 747-756. 

Zarb J; Walters D (1993). The effect of ornithine decarboxylase inhibition on growth, enzyme 

activities and polyamine concentrations in Crinipellis perniciosa. Pestic. Biochem. 

Physiol. 47(1), 44-50. 

289

Deising H B, Horbach R, Ludwig N, Münch S, Schweizer P : Mechanisms of Fungal Infection. In: Feldmann F, 



5-1 Mechanisms of Fungal Infection 

Deising H B 1, 2 , Horbach R 1 , Ludwig N 1 , Münch S 1 , Schweizer P 3 

1 

Martin-Luther-Universität Halle-Wittenberg, Naturwissenschaftliche Fakultät III, Institut für 

Agrar- und Ernährungswissenschaften, Phytopathologie und Pflanzenschutz, Ludwig- 

Wucherer-Str. 2, D-06108 Halle (Saale), Germany 

2 

Martin-Luther-Universität Halle-Wittenberg, Interdisziplinäres Zentrum für Nutzpflanzenforschung 

(IZN), Ludwig-Wucherer-Str. 2, D-06108 Halle (Saale), Germany 

3 

Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben, Corrensstraße 

3, D-06466 Gatersleben, Germany 

INTRODUCTION 

The kingdom of fungi is comprised of a highly diverse array of species. Estimates suggest that 

more than 1.5 million fungal species may exist (Carlile & Watkinson 1994; Hawksworth 

2001). Thus, the number of fungal species exceeds that of flowering plants (approximately 

270,000 species) approximately six-fold. Fungi exhibit a remarkable ability to adapt to new 

environmental conditions and to develop either mutualistic or pathogenic relationships with 

plants or other hosts such as man, animals and microbes (Deising 2009; Dix & Webster 1995). 

In crop plants, fungi cause more damage than any other group of microorganisms, with annual 

losses estimated at more than 200 billion $US (Birren et al. 2002; Oerke et al. 1994). 

Reduction of yield, however, represents only part of the problems associated with fungal plant 

pathogens. Several pathogenic species produce highly toxic secondary metabolites called 

mycotoxins, and uptake of contaminated food may result in increased incidence of cancer 

(Desjardins et al. 2000; Richard 2007). In spite of the availability of efficient fungicides 

against many but not all fungal diseases, both quantitative and qualitative yield losses 

continued to occur in the last decades, emphasizing the need to develop novel plant protection 

strategies. One of the key elements helping to achieve this goal is to understand infection 

processes of different pathogenic fungi at the molecular level and to identify genes essential for 

fungal pathogenicity. Products of pathogenicity genes may represent novel specific or broad 

spectrum targets, depending on the degree of conservation of the gene(s) in phylogenetically 

remote species. Recently, an RNA interference- (RNAi)-based technology called host-induced 

gene silencing (HIGS) has been developed, which relies on the transfer of silencing-inducing 

RNA molecules, probably short interfering RNA (siRNA), from host plants into the pathogens 

290

(Gay et al. 2008; Schweizer et al. 2006). RNAi constructs carry DNA strands of fungal genes 

to be silenced in sense and antisense orientation, linked by a loop region. Consequently, 

transcripts of RNAi constructs form double-stranded RNA, which are efficiently degraded by a 

double-strand (ds) RNA-specific RNAse called DICER, resulting in the formation of siRNAs 

of 21 or 22 bp. The siRNAs are directed to the RNA-induced silencing complex (RISC), and 

fungal transcripts corresponding to these siRNAs are recognized and degraded (Fig. 1) 

(Cottrell & Doering 2003). 

Figure 1. Mechanisms of host-induced gene silencing (HIGS). An RNAi construct 

targeting an essential fungal gene (e.g. a pathogenicity gene) is expressed in 

the plant cell. The dsRNA formed is degraded into 21-22 bp short interfering 

RNA fragments (siRNAs) by a dsRNA-specific RNase called DICER. Either 

the fragments or the entire dsRNA is transferred into the pathogen and directed 

to the RNA-induced silencing complex (RISC), mediating degradation of the 

transcripts encoding an essential protein. 

Although it is presently unclear whether siRNAs or the undigested dsRNAi are taken up by the 

pathogen, expression of RNAi constructs in plants that target fungal genes essential for 

pathogenic development led to significantly reduced disease incidence, suggesting that 

degradation of transcripts required for pathogenicity had occurred (Gay et al. 2008; Schweizer, 

Novara, Douchkov, patent WO2006/097465 A2 ; BayerCropScience patent 

WO2005/071091A1). An important aspect of HIGS is its unique mechanism conferring 

resistance, independent of functional proteins. HIGS represents a methodological breakthrough 

in plant protection as, depending on the RNAi constructs used, individual or groups of 

291

pathogen species may be efficiently controlled. In case this technique should prove applicable 

in several crops to provide protection against biotrophic, hemibiotrophic and necrotrophic 

fungi, significant effort should be made to identify large numbers of pathogenicity genes in 

various pathogens belonging to different taxa. In the following paragraphs we will focus on the 

identification of pathogenicity genes of the maize pathogen Colletotrichum graminicola. 

THE INFECTION PROCESS AND KNOWN PATHOGENICITY GENES OF THE 

MAIZE PATHOGEN COLLETOTRICHUM GRAMINICOLA 

Colletotrichum graminicola (Cesati) Wilson [teleomorph Glomerella graminicola (Politis)] is 

the causal agent of maize (Zea mays L.) anthracnose and stalk rot disease (Bergstrom & 

Nicholson 1999). C. graminicola exhibits a hemibiotrophic lifestyle, combining an initial 

biotrophic with a subsequent highly destructive necrotrophic phase of development. While host 

cells remain alive during the biotrophic phase, necrotrophy leads to extended areas of killed 

host tissue. In contrast to obligate biotrophs like powdery mildews and rust fungi, for which 

robust transformation protocols are not yet available, the hemibiotroph C. graminicola allows 

to study the biotrophic lifestyle on the molecular level, side by side with the mechanisms of 

necrotrophy. 

In order to infect and colonize maize leaves, C. graminicola sequentially differentiates specific 

infection structures. The pre-invasive phase of pathogenesis is characterized by attachment and 

germination of conidia and, upon recognition of the host surface, formation of a highly 

specialized infection cell called an appressorium (Deising et al. 2000). As the appressorium 

matures, a melanin layer is incorporated into the appressorial cell wall and osmotically active 

compounds are concomitantly synthesized to high concentrations (Deising 1993). As nonmelanized 

mutants generated by UV mutagenesis are non-pathogenic, melanin can be regarded 

as a pathogenicity factor in C. graminicola (Rasmussen & Hanau 1989). The requirement for 

melanin for appressorium function has also been reported for other plant pathogenic fungi such 

as Magnaporthe grisea (Chumley & Valent 1990) and C. lagenarium (Tsuji et al. 2000). 

Melanized appressoria generate enormous turgor pressure, which is translated into force 

driving penetration of the host cell wall. Indeed, measurements with optical wave guides have 

demonstrated that single appressoria of C. graminicola exert force of approximately 17 µN, 

corresponding to an appressorial turgor pressure of more than 53 bar (5.3 MPa) (Bechinger et 

al. 1999; Latunde-Dada 2001). It is as yet unclear to which extent cell wall-degrading enzymes 

assist the penetration process of C. graminicola. However, pectin-degrading-enzymes have 

been found in infected maize tissue (Nicholson et al. 1976), and a yeast secretion signal trap 

(YSST) screen used to identify genes encoding secreted proteins, in combination with macroarray 

and quantitative RT-PCR studies, showed that a large number of genes encoding host cell 

wall-degrading enzymes is expressed during infection and colonization of maize tissue (Krijger 

et al. 2008). 

After penetration into the host cell, most Colletotrichum species establish a biotrophic 

interaction. It is assumed that different strategies to avoid defense responses, i.e. masking of 

292

invading hyphae or active suppression of defense, are essential for establishment of a 

biotrophic lifestyle. Interestingly, fluorescence microscopy studies involving chitin-specific 

wheat germ agglutinin and antibodies specific for chitosan indicated that C. graminicola, like 

the biotrophic rust fungi Uromyces fabae and Puccinia graminis, mask their infection 

structures by converting the surface-exposed chitin by deacetylation (El Gueddari et al. 2002). 

The resulting chitosan is significantly less accessible to plant chitinases, and chitosan 

fragments, if they occur, are less elicitor active (Barber et al. 1989; Vander et al. 1998). Fungal 

chitin deacetylases may thus help avoiding degradation of chitin by plant chitinases, 

recognition of chitin fragments and elicitation of defense responses (El Gueddari et al. 2002). 

In addition to masking hyphal surfaces, proteins capable of suppressing defense responses may 

be secreted into the host tissue. A nitrogen starvation-induced gene of C. gloeosporioides, 

CgDN3, is expressed at the early stages of infection of the host plant Stylosanthes guianensis. 

CgDN3-deficient mutants of C. gloeosporioides formed normal appressoria on the leaf surface, 

but these mutants were unable to suppress a localized hypersensitive-like response in the host 

(Stephenson et al. 2000). A REMI mutant of C. graminicola mutant containing a plasmid 

integration in the 3' untranslated region of the CPR1 gene, encoding a eukaryotic microsomal 

signal peptidase, had significantly reduced transcript levels and failed to form secondary 

hyphae and to enter the necrotrophic phase (Thon et al. 2002). Low CPR1 levels may be 

sufficient for secretion of effectors suppressing or delaying host defense responses, but the 

mutant may be unable to secrete sufficient amounts of proteins indispensable for necrotrophic 

development (Thon et al. 2002). The YSST screen mentioned above identified several genes 

encoding secreted peptidases and small cystein-rich proteins, some of which might function as 

suppressors of host defense (Krijger et al. 2008), and it would be interesting to analyze the 

function of these genes by targeted mutagenesis (see below). 

Between 48 and 72 hours post inoculation, depending on environmental conditions, the 

infection hyphae of Colletotrichum species enter the destructive, necrotrophic phase. 

Secondary hyphae representing this phase are smaller in diameter, breach the plasma 

membrane, kill the host cells and ramify within the tissue. During necrotrophic development 

the pathogen actively kills the host tissue, e.g. by secretion of toxins (Thines et al. 2006). 

Several secondary metabolites of Colletotrichum species have been identified, but only a few 

of them possess phytotoxic activity (García-Pajón & Collado 2003). For example, terpenoid 

compounds called colletotrichins function as non-host specific toxins of the tobacco pathogen 

C. nicotina. When colletotrichins were applied to tobacco leaves, they induced symptoms 

similar to those of tobacco anthracnose caused by C. nicotina (Thines et al. 2006, and 

references therein). However, it should be mentioned that toxins have only rarely been reported 

in Colletotrichum species so far. Alternatively, reactive oxygen species (ROS) might be 

generated by the fungus to induce host cell death. While ROS production has been reported for 

Botrytis cinerea and other necrotrophic fungi (Govrin & Levine 2000; Tudzynski & Kokkelink 

2009, and references therein) no experimental proof for this strategy to induce death of host 

cells has been reported for Colletotrichum species so far. 

293

Taken together, the features of infection biology of C. graminicola indicate that this pathogen 

is an excellent model organism for studying mechanisms of hemibiotrophy. Importantly, early 

DNA reassociation studies have indicated a relatively small size of the nuclear genome of C. 

graminicola of 48 Mbp (Randhir & Hanau 1997), and the annotated genome sequence will be 

available soon (http://www.colletotrichum.org/?p=54). As methods allowing identification and 

functional characterization of genes, including Agrobacterium tumefaciens-mediated 

transformation (ATMT), protoplast transformation, and restriction enzyme-mediated 

integration of DNA (REMI) have been established (Epstein et al. 1998; Flowers & 

Vaillancourt 2005; Münch et al. 2008; Thon et al. 2000; Werner et al. 2007), several additional 

novel genes involved in pathogenicity or virulence are likely to be discovered in the near 

future. 

IDENTIFICATION OF NOVEL PATHOGENICITY GENES BY TARGETED AND 

RANDOM MUTAGENESIS IN THE MAIZE PATHOGEN COLLETOTRICHUM 

GRAMINICOLA 

Genes involved in the infection process of a fungus can be identified by directed or nondirected 

approaches. New pathogenicity genes can be identified by random mutagenesis 

without a priory knowledge of the function(s) of a gene. Random mutagenesis has been 

performed with UV light or chemicals, but in these mutants, as affected genes are not tagged, it 

is difficult to identify genes of interest. In comparison, genes inactivated by disruption of the 

coding region or the promoter by integration of a marker gene, can easily be identified by 

sequencing into the disrupted gene, e.g. by amplification of genomic DNA ends after 

endonuclease digestion and polynucleotide tailing (Liu & Baird 2001). In contrast to random 

mutagenesis approaches, targeted mutagenesis of a gene that is assumed to be required for 

pathogenicity may verify a function in the infection process. The hypothesis that a gene may be 

involved in pathogenicity may come from reported function of heterologous genes in other 

pathogens. Alternatively in non-pathogens, genes may serve functions that could also be 

required in pathogens during certain steps in pathogenesis. Examples for identification of 

candidate genes by random and targeted mutagenesis are given below. 

Random mutagenesis in C. graminicola by ATMT 

Depending on the lifestyle and the genome size of the fungus, plant pathogenic fungi likely 

harbor between 60 and 360 virulence or pathogenicity genes (Idnurm & Howlett 2001). Only 

few of these, however, have been identified yet. 

Initially REMI mutagenesis has been used for random mutagenesis in order to tag fungal genes 

required for the establishment of a pathogenic interaction with the host plant (Kahmann & 

Basse 1999; Maier & Schäfer 1999; Sweigard et al. 1998; Thon et al. 2000). In REMI 

mutagenesis experiments performed with the plant pathogens Ustilago maydis, Magnaporthe 

grisea, and Cochliobolus heterostrophus the percentage of transformants with virulence defects 

ranged from 0.5 to 2% (Bölker et al. 1995; Lu et al. 1994;Sweigard et al. 1998). For 

294

comparison, in similar experiments with C. graminicola only 0.3% of the transformants were 

affected in virulence (Epstein et al. 1998; Thon et al. 2000). It is important to note that 

analyses of REMI mutants of different fungi have shown that up to 50% of the mutations were 

not tagged (Kahmann & Basse 1999; Maier & Schäfer 1999), raising doubts whether REMI 

mutagenesis is suited well enough for efficient identification of virulence genes. 

Agrobacterium tumefaciens-mediated transformation (ATMT) is thought to avoid most of the 

problems associated with REMI mutagenesis and related protoplast transformation techniques. 

After initial application to plants (Escobar & Dandekar 2003) ATMT has been used to 

transform yeast (de Groot et al. 1998) and filamentous fungi (de Groot et al. 1998), including a 

number of plant pathogens, e.g. M. grisea, M. fructicola, and different Fusarium and 

Colletotrichum species (Covert et al. 2001; Flowers & Vaillancourt 2005; Huser et al. 2009; 

Lee & Bostock 2006; Maruthachalam et al. 2008; Mullins et al. 2001; Münch et al. 2008; Rho 

et al. 2001; Tsuji et al. 2003). 

Applying an ATMT protocol to C. graminicola allowed generation of a collection of 

transformants, approx. 70% of which showed single T-DNA integrations. Of 500 independent 

transformants tested in virulence assays on whole plants, 19 showed virulence defects. Seven 

transformants have been studied in detail, including identification of T-DNA integration sites. 

In six transformants T-DNA integration had occurred into 5’-flanks or coding regions of 

putative genes with unknown functions. In one transformant T-DNA had integrated into the 5’flank 

of a gene with similarity to the allantoicase genes of other Ascomycota such as M. grisea 

or Neurospora crassa (Münch S, Sode B & Deising H.B., unpublished data). The genes of C. 

graminicola tagged by ATMT will be analyzed by targeted gene deletion. In case the 

pathogenicity function of the genes tested is confirmed, these genes represent excellent 

candidate genes to be used for HIGS approaches. 

Targeted mutagenesis in C. graminicola 

Numerous genes are essential for fungal pathogenicity (Idnurm & Howlett 2001). Examples 

are genes encoding enzymes involved in fungal cell wall biogenesis, signal transduction, or 

toxin biosynthesis (Deising 2009; Werner et al. 2007). If a pathogenicity gene has been 

identified in a fungus, a comparable gene function can be confirmed in other pathogens, 

provided that targeted mutagenesis experiments can be performed. Interestingly, in nonpathogenic 

model fungi genes have been identified that might have homologs in pathogens, 

playing an essential role in pathogenesis. An excellent example is the identification of the 4'phosphopantetheinyl 

transferase (PPTase) gene of the filamentous Ascomycete Aspergillus 

nidulans. This fungus is closely related to the human pathogen A. fumigates and several plant 

pathogenic Ascomycota, including C. graminicola. Studies with A. nidulans have shown that 

the PPTase gene is a central regulator of secondary metabolism (Marquez-Fernandez et al. 

2007) (Fig. 2). 

Polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) are central 

components of secondary metabolism not only in fungi, but also in bacteria and plants, and in 

295

several fungi PKSs and/or NRPSs contribute to virulence on plants. PKSs and NRPSs are 

involved in the biosynthesis of pigments (melanin), siderophores and toxins. Siderophores are 

produced by NRPSs and detailed studies in various plant pathogenic fungi such as M. grisea, 

Cochliobolus heterostrophus, Gibberella zeae and Ustilago maydis have shown that these 

compounds are required for recruitment of iron and pathogenicity (Eichhorn et al. 2006; 

Greenshields et al. 2007; Hof et al. 2007; Oide et al. 2006). 

296 

Figure 2. 4'-Phosphopantetheinyl transferase (PPTase) covalently attaches a 4'phosphopantetheinyl 

(CoA-) residue to peptidyl carrier proteins of nonribosomal 

peptide synthetases (NRPSs) and to acyl carrier proteins of 

polyketide synthases (PKSs) and α-amino adipate reductase (AAR). The 

attachment of a CoA-residue is required for activation of the enzymes. 

Modules of PKSs: acyltransferase (AT); acyl carrier protein (ACP) with an SH 

group on the cofactor, a serine-attached CoA; keto-synthase (KS); 

ketoreductase (KR); thioesterase dehydratase (T). Modules of NRPSs: 

adenylation domain (AD); thiolation and peptide carrier protein (PCP) with 

attached 4'-phospho-pantetheine; condensation domain (CO); thio-esterase (T). 

Modules of AARs: adenylation domain (AD); peptide carrier protein (PCP); 

reductase (R). Transmission electron micrograph insert shows median section 

appressorium of M. grisea (from: (Howard et al. 1991). 

Accordingly, several host-specific and non host-specific toxins formed either by NRPSs or 

PKSs contribute to fungal pathogenicity (Thines et al. 2006), as these secondary metabolites 

kill the host cells before resistance responses can be activated. In spite of the enormous

diversity of the metabolites produced, all PKSs and NRPSs share a common regulatory step, as 

they require activation by the enzyme PPTase (Fig. 2). PPTases covalently attach a 4'phosphopantetheinyl 

(CoA-) residue to the peptidyl carrier proteins of NRPSs and to the acyl 

carrier proteins of PKSs. Interestingly, α-amino adipate reductase (AAR) also requires 

activation by 4'-phosphopantetheinylation. Thus, PPTase activity is not only required for 

secondary metabolism, but –specifically in fungi – also for lysine biosynthesis. Based on these 

considerations, targeted deletions of the PPTase genes of different plant pathogens should be 

performed to verify the role of these genes as central regulators of secondary metabolism and 

pathogenicity. These studies are in progress with C. graminicola. 

CONCLUSIONS 

This chapter summarized mechanisms of fungal pathogenicity, with special emphasis on the 

maize pathogen C. graminicola. Unique aspects of pathogens exhibiting a hemibiotrophic life 

style have been discussed, and targeted and random mutagenesis have been introduced as tools 

allowing identifying novel pathogenicity determinants. As chemical control of plant pathogenic 

fungi becomes increasingly difficult due to occurrence and spread of fungicide resistant strains 

(Deising et al. 2002) but also because of increasing concerns about pesticide-contaminated 

food, novel strategies are urgently needed in plant protection. As outlined above, HIGS may 

allow highly efficient and specific control of pathogenic microorganisms. As this strategy 

requires expression of RNAi constructs that target genes essential for the development and/or 

pathogenicity of pathogens, such genes need to be identified in many fungi with economical 

impact in different crop plants. 

REFERENCES 

Barber M S; Bertram R E; Ride J P (1989). Chitin oligosaccharides elicit lignification in 

wounded wheat leaves. Physiological and Molecular Plant Pathology 34, 3-12. 

Bechinger C; Giebel K-F; Schnell M; Leiderer P; Deising H B; Bastmeyer M (1999). Optical 

measurements of invasive forces exerted by appressoria of a plant pathogenic fungus. 

Science 285, 1896-1899. 

Bergstrom G C; Nicholson R L (1999). The biology of corn anthracnose. Knowledge to exploit 

for improved management. Plant Disease 83, 596-608. 

Birren B; Fink G; Lander E (2002). Fungal genome initiative: white paper developed by the 

fugal research community. In: Whitehead Institue Center for Genome Research, 

Cambridge, MA. 

Bölker M; Böhnert H U; Braun K H; Görl J; Kahmann R (1995). Tagging pathogenicity genes 

in Ustilago maydis by restriction enzyme-mediated integration (REMI). Molecular and 

General Genetics 248, 547-552. 

Carlile M J; Watkinson S C (1994). The Fungi. Academic Press: London, Boston, San Diego. 

Chumley F G; Valent B (1990). Genetic analysis of melanin-deficient, non-pathogenic mutants 

of Magnaporthe grisea. Molecular Plant-Microbe Interactions 3, 135-143. 

297

Cottrell T R; Doering T L (2003). Silence of the strands: RNA interference in eukaryotic 

pathogens. Trends in Microbiology 11, 37-43. 

Covert S F; Kapoor P; Lee M; Briley A; Nairn C J (2001). Agrobacterium tumefaciensmediated 

transformation of Fusarium circinatum. Mycological Research 105, 259-264. 

de Groot M J; Bundock P; Hooykaas P J; Beijersbergen A G (1998). Agrobacterium 

tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16, 

839-842. 

Deising H B (2009). The Mycota V. Plant Relationships. Springer: Berlin, Heidelberg. 

Deising H B; Reimann S; Peil A; Weber W E (2002). Disease management of rusts and 

powdery mildews. In: The Mycota XI. Application in Agriculture, ed F Kempken, pp 

243-269. Springer: Berlin. 

Deising H B; Werner S; Wernitz M (2000). The role of fungal appressoria in plant infection. 

Microbes and Infection 2, 1631-1641. 

Desjardins A E; Manandhar G; Plattner R D; Maragos C M; Shrestha K; McCormick S P 

(2000). Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat 

and the effect of traditional processing methods on mycotoxin levels. Journal of 

Agricultural and Food Chemistry 48, 1377-1383. 

Dix N J; Webster J (1995). Fungal Ecology. Chapman & Hall: London. 

Eichhorn H; Lessing F; Winterberg B; Schirawski J; Kämper J; Müller P; Kahmann R (2006). 

A ferroxidation/permeation iron uptake system is required for virulence in Ustilago 

maydis. Plant Cell 18, 3332-3345. 

El Gueddari N E; Rauchhaus U; Moerschbacher B M; Deising H B (2002). Developmentally 

regulated conversion of surface-exposed chitin to chitosan in cell walls of plant 

pathogenic fungi. New Phytologist 156, 103–112. 

Epstein L; Lusnak K; Kaur S (1998). Transformation-mediated developmental mutants of 

Glomerella graminicola. Fungal Genetics and Biology 23, 189-203. 

Escobar M A; Dandekar A M (2003). Agrobacterium tumefaciens as an agent of disease. 

Trends in Plant Science 8, 380-386. 

Flowers J L, Vaillancourt L J (2005). Parameters affecting the efficiency of Agrobacterium 

tumefaciens-mediated transformation of Colletotrichum graminicola. Current Genetics 

48, 380-388. 

García-Pajón C M; Collado I G (2003). Secondary metabolites isolated from Colletotrichum 

species. Natural Product Reports 20, 426–431. 

Gay A; Zimmermann G; Nowara D; Kumlehn J; Schweizer P; Hensel G (2008). Triple RNAiconstructs 

for broard-range resistance against fungal leaf diseases. 9th International 

Congress of Plant Pathology, August 24-29. Torino, Italy. 

Govrin E M; Levine A (2000). The hypersensitive response facilitates plant infection by the 

necrotrophic pathogen Botrytis cinerea. Current Biology 10, 751-757. 

Greenshields D L; Liu G; Feng J; Selvaraj G; Wei Y (2007). The siderophore biosynthetic gene 

SID1, but not the ferroxidase gene FET3, is required for full Fusarium graminearum 

virulence. Molecular Plant Pathology 8, 411-421. 

Hawksworth D L (2001). The magnitude of fungal diversity: The 1.5 million species estimate 

revisited. Mycological Research 105, 1422-1432. 

298

Hof C; Eisfeld K; Welzel K; Antelo L; Foster A J; Anke H (2007). Ferricrocin synthesis in 

Magnaporthe grisea and its role in pathogenicity in rice. Molecular Plant Pathology 8, 

163-172. 

Howard R J; Bourette T M; Ferrari M A (1991). Infection by Magnaporthe: an in vitro 

analysis. In: Electron Microscopy of Plant Pathogens, eds K Mendgen; D-E Lesemann, 

pp 251-264. Springer: Berlin. 

Huser A; Takahara H; Schmalenbach W; O'Connell R (2009). Discovery of pathogenicity 

genes in the crucifer anthracnose fungus Colletotrichum higginsianum, using random 

insertional mutagenesis. Molecular Plant-Microbe Interactions 22, 143-156. 

Idnurm A; Howlett B J (2001). Pathogenicity genes of phytopathogenic fungi. Molecular Plant 

Pathology 2, 241-255. 

Kahmann R; Basse C (1999). REMI (restriction enzyme mediated integration) and its impact 

on the isolation of pathogenicity genes in fungi attacking plants. European Journal of 

Plant Pathology 105, 221-229. 

Krijger J-J; Horbach R; Behr M; Schweizer P; Deising H B; Wirsel S G R (2008). The yeast 

signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen 

Colletotrichum graminicola. Molecular Plant-Microbe Interactions 21, 1325-1336. 

Latunde-Dada A O (2001). Pathogen profile Colletotrichum: Tales of forcible entry, stealth, 

transient confinement and breakout. Molecular Plant Pathology 2, 187-198. 

Lee M-H; Bostock R M (2006). Agrobacterium T-DNA-mediated integration and gene 

replacement in the brown rot pathogen Monilinia fructicola. Current Genetics 49, 

309-322. 

Liu X; Baird V (2001). Rapid amplification of genomic DNA ends by NlaIII partial digestion 

and polynucleotide tailing. Plant Molecular Biology Reporter 19, 261-267. 

Lu S; Lyngholm L; Yang G; Bronson C; Yoder O C; Turgeon B G (1994). Tagged mutations 

at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated 

integration. Proceedings of the National Academy of Sciences of the USA 91, 12649- 

12653. 

Maier F J; Schäfer W (1999). Mutagenesis via insertional - or restriction enzyme-mediated - 

integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic 

fungi. Biological Chemistry 380, 855-864. 

Marquez-Fernandez O; Trigos A; Ramos-Balderas J L; Viniegra-Gonzalez G; Deising H B; 

Aguirre J (2007). Phosphopantetheinyl transferase CfwA/NpgA is required for 

Aspergillus nidulans secondary metabolism and asexual development. Eukaryotic Cell 

6, 710-720. 

Maruthachalam K; Nair V; Rho H S; Choi J; Kim S; Lee Y H (2008). Agrobacterium 

tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum. 

Journal of Microbiology and Biotechnology 18, 234-241. 

Mendgen K; Deising H (1993). Infection structures of fungal plant pathogens - a cytological 

and physiological evaluation. The New Phytologist 124, 193-213. 

Mullins E D; Chen X; Romaine P; Raina R; Geiser D M; Kang S (2001). Agrobacteriummediated 

transformation of Fusarium oxysporum: an efficient tool for insertional 

mutagenesis and gene transfer. Phytopathology 91, 173-180. 

Münch S; Lingner U; Floss D S; Ludwig N; Sauer N; Deising H B (2008). The hemibiotrophic 

lifestyle of Colletotrichum species. Journal of Plant Physiology 165, 41-51. 

299

Nicholson RL; Turpin CA; Warren HL (1976). Role of pectic enzymes in susceptibility of 

living maize pith to Colletotrichum graminicola. Phytopathologische Zeitschrift 87, 

324-336. 

Oerke E C; Dehne H W; Schönbeck F; Weber A (1994). Crop Production and Crop 

Protection. Elsevier: Amsterdam. 

Oide S; Moeder W; Krasnoff S; Gibson D; Haas H; Yoshioka K; Turgeon B G (2006). NPS6, 

encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron 

metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. 

Plant Cell 18, 2836-2853. 

Randhir R J; Hanau R M (1997). Size and complexity of the nuclear genome of Colletotrichum 

graminicola. Applied and Environmental Microbiology 63, 4001-4004. 

Rasmussen J B; Hanau R M (1989). Exogenous scytalone restores appressorial melanization 

and pathogenicity in albino mutants of Colletotrichum graminicola. Canadian Journal 

of Plant Pathology 11, 349-352. 

Rho H S; Kang S; Lee Y H (2001). Agrobacterium tumefaciens-mediated transformation of the 

plant pathogenic fungus, Magnaporthe grisea. Molecules and Cells 12, 407-411. 

Richard J L (2007). Some major mycotoxins and their mycotoxicoses - An overview. 

International Journal of Food Microbiology 119, 3-10. 

Stephenson S A; Hatfield J; Rusu A G; Maclean D J; Manners J M (2000). CgDN3: an 

essential pathogenicity gene of Colletotrichum gloeosporioides necessary to avert a 

hypersensitive-like response in the host Stylosanthes guianensis. Molecular Plant- 

Microbe Interactions 13, 929-941. 

Sweigard J A; Carroll A M; Farrall L; Chumley F G; Valent B (1998). Magnaporthe grisea 

pathogenicity genes obtained through insertional mutagenesis. Molecular Plant- 

Microbe Interactions 11, 404-412. 

Thines E; Aguirre J; Foster A J; Deising H B (2006). Secondary metabolites as virulence 

determinants of fungal plant pathogens. In: Progress in Botany, eds K Esser, U E 

Lüttge, W Beyschlag, J Murata, pp 132-159. Springer: Berlin, Heidelberg. 

Thon M R; Nuckles E M; Takach J E; Vaillancourt L J (2002). CPR1: a gene encoding a 

putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola 

to maize. Molecular Plant-Microbe Interactions 15, 120-128. 

Thon M R; Nuckles E M; Vaillancourt L J (2000). Restriction enzyme-mediated integration 

used to produce pathogenicity mutants of Colletotrichum graminicola. Molecular 

Plant-Microbe Interactions 13, 1356-1365. 

Tsuji G; Fujii S; Fujihara N; Hirose C; Tsuge S; Shiraishi T; Kubo Y (2003). Agrobacterium 

tumefaciens-mediated transformation for random insertional mutagenesis in 

Colletotrichum lagenarium. Journal of General Plant Pathology 69, 230-239. 

Tsuji G; Kenmochi Y; Takano Y; Sweigard J; Farrall L, Furusawa I, Horino O, Kubo Y 

(2000). Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium 

and pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 

binuclear cluster DNA-binding motifs and regulate transcription of melanin 

biosynthesis genes in a developmentally specific manner. Molecular Microbiology 38, 

940-954. 

Tudzynski P; Kokkelink L (2009). Botrytis cinerea: Molecular aspects of a necrotrophic life 

style. In: Plant Relationships V, ed H B Deising, pp 29-50. Springer: Berlin, 

Heidelberg. 

300

Vander P; Vårum K M; Domard A; El Gueddari N E, Moerschbacher B M (1998). Comparison 

of the ability of partially N-acetylated chitosans and chitoologosaccharides to elicit 

resistance reactions in wheat leaves. Plant Physiology 118, 1353-1359. 

Werner S; Sugui J A; Steinberg G; Deising H B (2007). A chitin synthase with a myosin-like 

motor domain is essential for hyphal growth, appressorium differentiation and 

pathogenicity of the maize anthracnose fungus Colletotrichum graminicola. Molecular 

Plant-Microbe Interactions 20, 1555-1567. 

301

Evans N, Gladders P, Fitt B D L, & Tiedemann A v: Altered Distribution and Life Cycles of Major Pathogens in 

Europe. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 302- 


5-2 Altered Distribution and Life Cycles of Major Pathogens in Europe 

Evans N 1 , Gladders P 2 , Fitt B D L 1 , & Tiedemann A v 3 

1 

Rothamsted Research, Harpenden, Herts. AL5 2JQ, UK; 

2 

ADAS, Boxworth, Cambridge, CB23 8NN, UK; 

3 

Department of Crop Sciences, University of Goettingen, Germany 

Email: atiedem@gwdg.de 

302 

Abstract 

Climate is a major determinant of crop productivity. Besides its direct impact on 

plant growth it has a major effect on the prevalence and incidence of plant diseases. 

The sensitivity of pathogens to climate is pronounced. While the local climate 

determines the general pattern of prevailing pathogen populations, specific weather 

conditions are important drivers in distinct phases of the life cycles of pathogens 

such as dormancy and survival, asexual or sexual propagation, and infection and 

colonization of host plants. Knowledge about the impact of weather variables on 

pathogens and diseases is an important part in predictive crop protection strategies. 

There is considerable data available from the past 30 to 40 years on climate-disease 

relationships, which have been used to develop weather-based forecasting models 

with the aim of predicting epidemic severity to maximise economic use of 

pesticides. A large number of such models have been constructed for specific 

diseases to help farmers and advisers in making their decisions about crop 

protection to save unnecessary sprays. Such weather-based disease forecast models 

can also be used to simulate epidemic severity and/or the geographic spread of a 

particular pathogen under future climatic change scenarios. However, there are 

some important constraints in making climate change-disease projections, the first 

resulting from the large variability and uncertainty of current climate prediction 

models themselves. Further complicating factors arise from the fact that climate not 

only affects pathogen or pest populations directly but also induces changes in the 

crop production systems and cropping techniques (soil tillage, irrigation, sowing 

dates, cultivars, crop species) that indirectly alter the prevalence of pathogens or 

pests. It is difficult to separate direct from indirect climate effects. Climate change

effects can be exemplified with major pathogens of oilseed rape. Greater mean 

temperatures may be associated with spread of phoma stem canker further north and 

altered temporal pattern of the fungal life cycle and disease stages in the UK. There 

is also some indication that sclerotinia stem rot, after mild winter conditions, may 

perform a pre-seasonal sclerotial stage. In addition, root infection has occurred 

more frequently with yet unknown relation to recent climate shifts. Yield losses 

from ascospore infections at early flowering stages may increase compared to late 

infections. Soil-borne diseases will specifically be affected by altered temperature 

profiles in the soils. After mild winters, V. longisporum caused greater yield losses 

in Germany. Clubroot has recently become a serious threat in Germany, but its 

relationship to recent climatic changes is still unknown. Research is needed to 

improve and combine climate and disease prediction models to provide a realistic 

forecast of disease risks associated with climate change. As climate models are 

likely to continue to lack sufficient accuracy, thorough surveillance of disease 

epidemics will be more crucial than ever before, in order to detect changes and 

adaptation in pathogen (and pest) populations and to establish the appropriate crop 

protection systems early enough. 

INTRODUCTION 

Climate change has been a consistent phenomenon throughout the earth’s history over the past 

millions of years. Living organisms are sensitive to climatic factors and thus all species always 

have had to cope with or adapt to climate changes. This is specifically true for agriculture, 

which is a particularly climate-dependent industry. Crop production systems have to adapt to 

local conditions which has always been a key prerequisite for successful crop production. Pests 

and diseases may significantly limit crop productivity. They are highly sensitive to climate on a 

long-term scale and to weather conditions within short-term interactions (Chakraborty et al. 

2000; Boland et al. 2004; Garrett et al. 2006). Consequently, the impact of climate on pests and 

diseases is a key factor for long-term strategies in crop protection, while weather-disease 

relationships are important for immediate agricultural measures in disease and pest control 

under local conditions. 

There is a considerable amount of data available from crop pathologists having studied 

weather-disease relationships in the past 30 - 40 years in order to understand climate-disease 

relationships. More recently, much of this data has been utilized to construct weather-based 

forecasting models with the aim of epidemic prediction and decision support under practical 

conditions in crop protection (Kluge et al. 1996; Kleinhenz & Jörg 1998; Kleinhenz & 

Rossberg 2000; Delinxhe et al. 2003; Koch et al. 2007; Rossi & Giosue 2005; Racca & Jörg 

2007). Such disease forecast models can now be adapted to simulate the epidemic behaviour or 

geographic spread of a particular pathogen under hypothetical future climatic conditions. 

However, this approach is hampered by some complicating factors. Firstly, results from 

climate models still vary considerably and have a low spatial and temporal resolution (IPPC 

303

2007). Thus the most uncertain factor in global change research with regard to crop diseases is 

global change prediction itself. Secondly, there may be short-term quantitative effects on 

pathogen populations such as the amount of pathogen inoculum shifting the relative importance 

of specific diseases in a certain area. This relates to pre-existing pathogens of crops in a certain 

location and is to be separated from long-term effects of a changed climate, which may induce 

the invasion and establishment of novel pathogen or pest species, causing significant changes 

in the species composition. These direct effects on diseases may all be overlapped by indirect 

effects, which derive from a parallel adaptation of crop production techniques to climate 

change. In the short-term, this may result in altered sowing dates, reduced soil tillage or 

enhanced irrigation. All such changes will of course have an impact on diseases by themselves. 

In the long term, local crop production may adapt by changing to alternative crops or 

increasing the proportion of individual crops in the rotation scheme. Such changes will have a 

considerable impact on pests and diseases and are a response to climate change. In conclusion, 

direct and indirect effects of climate change are difficult to separate and will be affected by the 

technological changes in agricultural practices. 

ALTERED LIFE CYCLES OF PATHOGENS CAUSING ABOVE-GROUND 

DISEASES ON OILSEED RAPE 

This paper focusses on two important above-ground diseases of oilseed rape, Sclerotinia stem 

rot and Leptosphaeria phoma stem canker, and two soil-borne diseases, Verticillium 

longisporum and clubroot (Plasmodiophora brassicae). Sclerotinia stem rot is typically a 

monocyclic disease with sclerotia of Sclerotinia sclerotiorum ripening in early spring to 

produce apothecia which release sexual ascospores at the time of flowering; these ascospores 

initiate the process that leads to infection of stems of oilseed rape plants. Recently, there have 

been observations indicating that the sclerotinia life cycle may change in various ways. Firstly, 

due to mild winter conditions (e.g. in 2006/2007) a pre-seasonal epidemic in late winter/early 

spring (February/March) has been observed in France and Germany. This caused significant 

disease on the winter oilseed rape crops before flowering. As a result, early production of 

sclerotia occurred. This may significantly increase the local sclerotial inoculum and increase 

ascospore inoculum concentration during flowering. Therefore, milder winter weather under 

future climates may significantly extend the period of time for infection and thus increase the 

disease incidence and damage potential of this pathogen. 

Secondly, there may be changes in the timing of infection during flowering. The environmental 

requirements for infection to occur have recently been studied in order to develop sclerotinia 

stem rot forecasting systems such as SkleroPro (Koch et al. 2007). If conditions for infection 

are fulfilled earlier during flowering, infection may happen earlier and cause greater damage. 

In a recent study, early infections were found to cause twice as much yield loss per unit disease 

incidence (% plants affected, DI) as late infections (0.45% vs. 0.23 % loss per percent DI, 

respectively). Consequently the threshold DI for economic damage changed from 12 to 27 % 

(Dunker et al. 2005). Thirdly, we recently observed a new disease caused by sclerotinia, 

304

namely root infection deriving from myceliogenic germination of sclerotia in the soil and direct 

hyphal infection of the roots. Its potential relationship to altered soil temperatures awaits 

further investigation. Phoma stem canker is the most severe disease of oilseed rape in many 

parts of the world, causing significant economic losses (Fitt et al. 2008). A survey of the UK 

by the Department for the Environment Food & Rural Affairs showed that canker incidence 

was greatest in the south-east region of the main oilseed rape growing area of England 

(www.cropmonitor.co.uk/). Disease prediction models have been updated with meteorological 

data from many sites across the UK, in order to make predictions in autumn of the date of 

increase in incidence of phoma leaf spotting (www.rothamsted.bbsrc.ac.uk/ppi/phoma/). These 

predictions of the date of increase in phoma leaf spotting can then be used to predict the date of 

canker onset in spring, canker severity at harvest (Evans et al. 2008) and potential yield loss. 

Growers can use this information to make spray application decisions in the autumn at the best 

time to control the initial leaf spotting and prevent stem canker development. 

A weather-based prediction model for phoma stem canker was run with data sets from climate 

change models for different years and CO2 emission scenarios in the UK. For example, a 

climate model based simulation was done for the high CO2 emission scenario and the 2020s 

compared to present times. If 1200 degree-days after sowing is used as the threshold for canker 

onset in spring, the prediction is that start of disease will be a significantly earlier (by about 40 

days). Thresholds from disease forecasting models can also be connected with geographic 

climate scenarios, under the predicted weather for future periods (2020s, 2050s) under low or 

high CO2 emission scenarios. These predictions suggest that the range of the disease will 

extend northwards, so that farmers in Scotland, who currently have phoma leaf spotting may 

start to have problems with stem canker. 

ALTERATIONS IN OCCURRENCE OF SOIL-BORNE DISEASES OF OILSEED 

RAPE 

Altered mean temperatures in the soil are very likely to affect soil-borne diseases. Root 

infection and survival of resting spores or microsclerotia are crucial stages in the life cycle of 

pathogens such as Plasmodiophora brassicae and Verticillium longisporum. Currently, it is not 

known what impact temperature increases will have on this type of pathogen but recent 

observations indicate that there may be serious effects. Verticillium is a disease which has 

recently increased in importance, particularly in the cooler oilseed rape growing regions in 

Europe. V. longisporum accumulates microslerotia in the soil; mycelium originating from these 

microsclerotia then infects the crop roots. Although V. longisporum is a xylem-invading 

fungus, there is some evidence that it is very weather-dependent. In Northern Germany, yield 

losses on single plants were up to 70%. Although these losses were partly compensated by 

increases in plant biomass per m 2 , yield losses of 10 to 30% can be expected where there is a 

high incidence of the disease on susceptible cultivars (Dunker et al. 2008). Given a temperature 

threshold for infection of 15°C and a 2°C increase in mean soil temperature, the critical period 

when the crop is susceptible to infection would extend by about 4 weeks in autumn and 2 

305

weeks in spring, based on the 2006/2007 weather data from Rosemaund, UK. This may 

significantly aggravate the impact of this disease in the future. 

Another emerging threat is clubroot, which has recently increased in importance as a disease of 

oilseed rape in Germany, particularly but not only in the traditional oilseed rape growing 

regions. Taking the Rosemaund data for 2006/07 and a threshold temperature for root infection 

of 16°C, this implies an increase in the length of the infection period in autumn by about 6 

weeks and may have a considerable impact on the incidence of disease and the resulting 

economic losses. Overall, the impact of changed soil conditions on soil-borne diseases is not 

yet understood and requires particular research efforts in the future. 

CONCLUSIONS 

In conclusion, the long-term impact of climate and the short-term effects of weather conditions 

on diseases are among the major factors determining crop productivity in a changed climate. 

Climate prediction models are likely to remain imprecise and thus make it difficult to predict 

long-term changes in severity of diseases or to make risk assessments for crop protection. The 

same is true for short-term based weather predictions. Therefore, the best course of action is 

surveillance and adaptation. There is a great need for detailed disease surveys which will allow 

us to detect changes and adaptation in pathogen life cycles sufficiently early to develop 

appropriate crop protection measures. This will in part be the task of farmers and advisers in 

their continuing efforts to improve crop protection systems but needs substantial support from 

crop protection research to identify the underlying biological principles. 


We thank the UK Biotechnology and Biological Sciences Research Council (BBSRC) and 

Department for Environment, Food and Rural Affairs (Defra, OREGIN) and Sustainable 

Arable LINK programme (PASSWORD, CLIMDIS) for funding this research. 

REFERENCES 

Boland G J, Melzer M S, Hopkin A, Higgins V, Nassuth A (2004). Climate change and plant 

diseases in Ontario. Can. J. Plant Pathol. 26, 335-350. 

Chakraborty S, Tiedemann A v, Teng P S (2000). Climate change: potential impact on plant 

diseases. Environmental Pollution 108, 317-326. 

Detrixhe P, Chandelier A, Cavalier M, Buffet D, Oger R (2003). Development of an 

agrometeorological model integrating leaf wetness duration estimation to asses the risk 

of head blight infection in wheat. Aspects of Applied Biology 68, 1-6. 

Dunker S, Tiedemann A. v (2004). Disease/yield loss analysis for Sclerotinia stem rot in winter 

oilseed rape. IOBC wprs Bulletin (2004). ‘Integrated Control in Oilseed Crops’ Vol. 27 

(10), 59-66. 

306

Dunker S, Keunecke H, Steinbach P, Tiedemann A v (2008). Impact of Verticillium 

longisporum on yield and morphology of winter oilseed rape (Brassica napus) in 

relation to systemic spread in the plant. Journal of Phytopathology 156, 698–707. 

Evans N, Baierl A, Semenov M A, Gladders P, Fitt B D L (2008). Range and severity of a 

plant disease increased by global warming. J. R. Soc. Interface 5, 525–531. 

Fitt BDL, Hu B C, Li Z Q, Liu S Y, Lange R M, Kharbanda P D, Butterworth M H, White R P 

(2008). Strategies to prevent spread of Leptosphaeria maculans (phoma stem canker) 

onto oilseed rape crops in China; costs and benefits. Plant Pathol. 57, 652-664. 

Garrett K A, Dendy S P, Frank E E, Rouse M N, Travers S E (2006). Climate change effects on 

plant disease: genomes to ecosystems. Annu. Rev. Phytopathol. 44, 489-509. 

IPCC (Intergovernmental Panel on Climate Change). 2007. Climate change 2007: Impacts, 

adaptation and vulnerability. Contribution of working group II to the fourth assessment 

report of the Intergovernmental Panel on Climate Change, ed. M.L. Parry, O.F. 

Canziani, J.P. Palutikof, P.J. van der Linden, and C.E. Hanson. Cambridge: Cambridge 

University Press. 

Kleinhenz B, Jörg E (1998). Integrierter Pflanzenschutz - Rechnergestützte Entscheidungshilfen. 

Schriftenreihe des Bundesministerium für Ernährung Landwirtschaft und 

Forsten: Angewandte Wissenschaft Vol. 473. Bonn: Bundesministerium für Ernährung 

Landwirtschaft und Forsten. 

Kleinhenz B, Roßberg D (2000). Structure and development of decision-support systems and 

their use by the State Plant Protection Services in Germany. Bulletin OEPP/EPPO 

Bulletin 30, 93-97. 

Koch S, Dunker S, Kleinhenz B, Röhrig M, Tiedemann A v (2007). A crop loss-related 

forecasting model for Sclerotinia stem rot in winter oilseed rape. Phytopathology 97, 

1186-1194. 

Racca P, Jörg E (2007). CERCBET 3 – a forecaster for epidemic development of Cercospora 

beticola. Bulletin OEPP/EPPO Bulletin 37, 344–349. 

Rossi V, Giosue S (2005). A dynamic simulation model for powdery mildew epidemics on 

winter wheat. EPPO Bulletin 2003 33, 389-396. 

307

Diederichsen E, Werner S, Frauen M: Genetics of the Plasmodiophora brassicae - Brassica napus interaction. In: 

Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), …-…… ISBN 978- 


5-3 Genetics of the Plasmodiophora brassicae - Brassica napus interaction 

Diederichsen E 1 , Werner S 2 , Frauen M 3 

1 

Institut für Biologie - Angewandte Genetik, Freie Universität Berlin, Albrecht-Thaer-Weg 6, 

14195 Berlin 

2 

Plant Breeding Institute, Christian-Albrechts-University of Kiel, Olshausenstr. 40, D-24098 

Kiel, Germany 

3 

Norddeutsche Pflanzenzucht H.G. Lembke KG, Hohenlieth, 24363 Holtsee 

Email: elked@zedat.fu-berlin.de 

308 

Abstract 

Clubroot caused by Plasmodiophora brassicae has gained increasing importance in 

major Brassica crops. Interspecific hybridizations and breeding efforts did lead to 

the release of the clubroot resistant B. napus cultivar ‘Mendel’ in many European 

countries. The resistance in ‘Mendel’ is race-specific and monogenic, and therefore, 

demands resistance management. To check for the occurence of compatible 

pathotypes in infected oilseed rape crops, greenhouse tests were made including a 

new set of differential hosts. Results from differential testing will be presented 

giving a more detailed picture of ‘Mendel’s resistance. To evaluate additional 

resistance sources, a set of different B. napus lines representing major race-specific 

resistance QTL from a DH mapping population has been tested with different P. 

brassicae collections. A summary of map positions and race-specificity will be 

given. In contrast to previous reports, a clear differentiation into major QTL from B. 

rapa and minor race-independent QTL from B. oleracea could not be found. One 

QTL originating from the susceptible parent “Express” was identified conferring 

resistance to one P. brassicae isolate, although clubroot resistance had never been 

observed in this cultivar. Some QTL co-localize with QTL that were found by other 

groups. In general, genetics of clubroot resistance in Brassica is complex, with 

race-specificity being the rule and only a few QTL having a broader effect.

Müllenborn C, Krause J-H, Muktiono B, Cerboncin C: Differential Gene Expression in Wild Sunflower with 

Resistance to the Necrotrophic Pathogen Sclerotinia Sclerotiorum. In: Feldmann F, Alford D V, Furk C: Crop 

Plant Resistance to Biotic and Abiotic Factors (2009), 309-318; ISBN 978-3-941261-05-1; © Deutsche 


5-4 Differential Gene Expression in Wild Sunflower with Resistance to the 

Necrotrophic Pathogen Sclerotinia Sclerotiorum 

Müllenborn C, Krause J-H, Muktiono B, Cerboncin C 

Forschungszentrum Jülich GmbH, Institut für Chemie und Dynamik der Geosphäre, 

Phytosphäre ICG 3, D-52425 Jülich, Germany 

Email: c.muellenborn@fz-juelich.de 

ABSTRACT 

Sclerotinia sclerotiorum is a pathogen causing devastating yield losses in cultivated 

sunflower. Durable resistance to this necrotrophic fungus does not exist in 

cultivated sunflower. However, resistance to S. sclerotiorum has been demonstrated 

in some wild sunflower species. Up to now, there is still marginal information on 

the molecular background of this resistance complex. In order to go into detail 

according to the traits of the resistance, two Sclerotinia-resistant wild sunflower 

accessions, AC7 and ACM, and one Sclerotinia-susceptible sunflower cultivar were 

examined in greenhouse trials with a major consideration on transcriptomic changes 

after inoculation with the pathogen. The transcriptomic approach was accomplished 

by means of Differential Display RT-PCR to amplify cDNA fragments coupled to 

fragmentation of cDNA fragments by capillary gel electrophoresis. In consideration 

of the transcriptomic approach, the evidence was given that an initial pathogen 

induced gene expression is possibly critical for pathogen defense in wild sunflower. 

Furthermore, it was shown that genotypic differences between the two wild 

sunflower species AC 7 and AC M exist regarding number of differential expressed 

transcripts and total phenol content although phenotypic reaction was similar in 

both accessions. Overall, three up- and one down-regulated transcripts originating 

from different pathways in plants were isolated and characterized. In addition, 

further evidence was given that the phenylpropanoid pathway most likely plays a 

crucial role in mechanisms of Sclerotinia-resistance in wild sunflower. 

309

INTRODUCTION 

Sclerotinia sclerotiorum is a necrotrophic pathogen causing devastating yield losses in a wide 

range of dicotyledonous crops, e.g. rapeseed, bean, soybean (Purdy 1979; Boland & Hall 

1994). In cultivated sunflower (Helianthus annuus), Sclerotinia-infection can lead to rot 

symptoms in every plant organ (Masirevic & Gulya 1992). S. sclerotiorum utilizes specific 

mechanisms, e.g. secretion of oxalic acid, cellulolytic and pectinolytic enzymes, that permit 

maceration of the plant tissue and active penetration into the host (Riou et al. 1991; Godoy et 

al. 1990). Natural durable resistance to this pathogen does not exist in cultivated sunflower. 

Solely, genotypes with differing susceptibility have been detected. It has been shown in 

previous studies that the level of susceptibility in sunflower is possibly associated with 

differing levels of phenolic compounds (Bazzalo et al. 1985). Nevertheless, a high genetic 

diversity revealing a potential source for valuable agronomic traits is present in the genus 

Helianthus (Seiler 1992). In particular, the perennial wild sunflower species dispose of a 

comprehensive gene reservoir with diverse resistances towards biotic factors (Seiler 1992; 

Skoric 1993). Resistance to S. sclerotiorum is also specified in a number of wild sunflower 

species, e.g. H. mollis, H. nuttallii, H. giganteus and H. maximiliani (Skoric 1987). Despite this 

high genetic variability of the wild sunflower gene pool, sources of resistance to Sclerotinia 

have been rarely utilized in breeding programs in cultivated sunflower so far. This is mainly 

attributed to somatic and physiological incompatibilities existing between the perennial and 

annual sunflower species (Schuster 1993). In addition, there is still marginal information on the 

molecular background of Sclerotinia-resistance in wild sunflower up to now. However, the 

resistance to Sclerotinia in sunflower is considered as polygenic and characterized by additive 

gene effects (Vear & Tourvieille 1988; Van Becelaere & Miller 2004). Unfortunately, some 

wild sunflower species are meanwhile listed as endangered and threatened (Jan & Seiler 2007). 

The major aims of this study were to provide an analysis to detect changes in gene expression 

in wild sunflower and to screen for differentially expressed transcripts in two resistant wild 

sunflower accessions after infection with the pathogen S. sclerotiorum. 


Inoculation method 

The artificial infection of sunflower leaves was carried out with one isolate of the pathogen S. 

sclerotiorum (SS01) in accordance to Bertrand & Tourvieille (1987) and Achbani et al. (1994). 

Mycelial agar plugs (Ø 1 cm) from a 7-day-old culture were placed onto young fully grown 

leaves of 8-week old sunflower plants. At the site of inoculation the tissue was covered with 

parafilm and moisturized by spraying with sterile water. All inoculation experiments were 

performed in the greenhouse for 7 days at 23°C / 20°C (day / night) and with 90-100% rLF. 

The time-point of infection and the lesion lengths were observed at 1, 2, 3, 4, 5 and 6 days post 

inoculum (DPI). 

310

Isolation of poly(A) + -RNA and Differential Display RT-PCR 

To screen for differentially expressed genes during Helianthus-Sclerotinia-interaction, leaf 

discs (Ø 2.5 cm, ~100 mg) were harvested from infected and non-infected leaf tissue directly 

bordering the extending lesion. The leaf discs were ground in liquid nitrogen and subsequently 

used for RNA-extraction by means of the RNeasy Plant Mini Kit (Qiagen). 6 µg of isolated 

RNA were used for isolation of poly(A) + -RNA (Oligotex mRNA Mini Kit, Qiagen). 6.9 µL of 

poly(A) + -RNA, 3.3 µL of modified Oligo-dT17-(A,C,G)-Primer, 2 μL dNTP´s (1mM), 2 μL 

reaction buffer (10x), 4 μL MgCl2 (25 mM), 1 μL RNase-Inhibitor (50 units) and 0,8 μL AMV 

reverse transkriptase (20 units) were mixed for reverse transcription in a 20 µL reaction 

volume. The cycling parameters for PCR reaction were as follows: 25°C for 10 min, 42°C for 

60 min, 99 °C for 5 min. 1 µL aliquots of the reverse transcription-reaction were used as 

templates for Random-PCR and mixed each with 2 µL Taq-buffer (10x), 1.5 µL MgCl2 

(25mM), 0.4 µL dNTP´s (10 mM), 0.2 µL Taq-Polymerase (2.5 u, Fermentas), 1.4 µL of a 

Cy5-labeled Oligo-dT-Primer (10 pmol/L) and 1.4 µL Arbitrary Primer (10 pmol/µL) in a 20 

µL PCR-reaction. In total, 20 different modified Random-Primer combinations were performed 

with a PCR-reaction as follows: 92°C for 5 min, 10 cycles of 92°C for 30 s, 33°C for 1 min 

and 72°C for 1 min, 30 cycles of 95°C for 30 s, 64°C for 1 min and 72°C for 1 min, and a final 

extension at 72°C for 10 min. 

Separation and detection of cDNA-fragments 

The products of the Random-PCR amplification were used to detect differentially expressed 

cDNA-fragments by means of an 8-capillary gel electrophoresis system (CEQ 8800, Beckman- 

Coulter). 1 µL of the amplified cDNA were mixed with 40 µL Sample Loading Solution and 

0.5 µL 600bp size standard. For sequence analysis of candidate fragments, samples were 

additionally separated in a 2% agarose gel stained with ethidium bromide for visualization of 

amplification products. Bands of interest were preparatively cut out of the agarose gel and 

subsequently eluted, reamplified, cloned and sequenced. 

Statistical analysis of gene expression data 

The fragment data generated by capillary gel electrophoresis was exported as ASCII-file and 

evaluated with the JmpGenomics 3 software (SAS). Statistical analysis to select differences of 

expression rates between infected and control tissue samples were performed by analysis of 

Variance (ANOVA, α < 0,05). To control the false-positive rate (error type I) by adjusting pvalues, 

the False Discovery Rate according to Benjamini & Hochberg (1995) for multiple 

testing was applied. 

Analyis of soluble phenolic compounds 

Freshly harvested leaves were ground in liquid nitrogen and lyophilized prior to phenolic 

311

extraction. 50 mg (DW) of pulverized leaf material was mixed with 1 mL methanol/water (1:1) 

at RT for 90 s and centrifugated at 4°C and 15,000 rpm for 30 min. The supernatant was used 

as phenolic extract for analysis of soluble phenolics. The total phenolic content of non-infected 

and infected leaf material was measured according to the colorimetrical assay by Singleton & 

Rossi (1965). 20 µL of phenolic extract were mixed with 1.59 mL aqua bidest. and, 

subsequently, 100 µL Folin-Ciocalteau reagent were added. After 1 min 300 µL of saturated 

sodium carbonate solution were added and mixed thoroughly. The absorption of the samples 

was measured photometrically at 765 nm after 2 h of incubation. Total phenolic contents were 

specified as gallic acid equivalent (GAE). 

The separation and analysis of single phenolic substances was carried out via High 

Performance Liquid Chromatography (HPLC) equipped with a UV-VIS photodiode array 

detector (DAD). 20 µL of leaf extract were manually injected and chromatographic separation 

took place with a Synergi Polar-RP-column (Phenomenex, 150 x 3.0 mm) and a pre-column 

(Phenomenex 4.0 x 2.0). Phenolic compounds were separated with a flow rate of 0.3 mL/min, 

constant column temperature (Tk = 30°C) and using a binary gradient of 

water:acetonitrile:acetic acid (1000:20:5; A) and acetonitrile:acetic acid (1000:5; B) as follows: 

0 min 4% B, 0-90 min 33 % B, 90-95 min 33 % B, 95-100 min 100 % B, 100-105 min 4 % B. 

Compounds were compared according to their UV-spectra and maximum of absorption (λ 

max) and specified as chlorogenic acid equivalent (CAE). 

RESULTS 

Phenotypic reaction of sunflower leaf tissue after Sclerotinia-infection 

The first symptoms were observed 24 h after inoculation. These included light brown necrotic 

spots that extended during colonization of the pathogen. 62 h after inoculation, 99 % of all 

inoculated leaves showed these type of symptoms irrespective of genotype. Significantly 

higher rates of lesion length were examined in the infected leaves of cv. Albena at time-points 

DPI1 to DPI4 compared to the wild sunflower genotypes (Fig. 1). The wild sunflower AC 7 

indicated the significantly lowest rates of lesion length after inoculation with the pathogen at 

every time-point examined. 

Isolation and characterization of differentially expressed cDNA-fragments 

In regard to the inoculation results, the gene expression studies comprised examination timepoints 

48 h (DPI 2, early time-point), 62 h (DPI 3, mid-time-point) and 86 h (DPI 4, late timepoint) 

after inoculation. The results of the ANOVA revealed that in total a high number of 

differentially expressed cDNA fragments were observed in wild sunflower AC 7 and in the 

annual sunflower cv. Albena, whereas a comparable low number of cDNA fragments was 

detected in wild sunflower AC M (Table 1). In AC 7 the highest number of significant 

transcripts was observed at the early time-point DPI 2. 

312

Figure 1. Mean values of lesion length in leaves of two resistant sunflower accessions 

(AC7, ACM) and one susceptible H. annuus (cv. Albena) inoculated with S. 

sclerotiorum measured at Days Post Inoculum (DPI) 1 – 6. 

Significant at p < 0.01 = ** and p < 0.001 = *** (Tukey-Kramer, HSD). 

Table 1. Number of significant differentially expressed transcripts in total found in 

resistant varieties (AC7 and ACM) and in a susceptible variety (cv. Albena) 

after inoculation with S. sclerotiorum. 

∑ 

AC 7 15 

AC M 4 

Albena 8 

DPI 2 DPI 3 DPI 4 

up-regulated (+) 

down-regulated (-) 

15 (+) 

0 (–) 

4 (+) 

0 (–) 

4 (+) 

4 (–) 

∑ 

7 

4 

5 



7 (+) 

0 (–) 

4 (+) 

0 (–) 

2 (+) 

3 (–) 

∑ 

3 

1 

12 



3 (+) 

0 (–) 

1 (+) 

0 (–) 

11 (+) 

1 (–) 

313

However, in the susceptible cv. Albena the highest number of differentially expressed 

transcripts was observed 62 h after inoculation. In Sclerotinia-infected leaves of ACM all of 

the examined time-points showed a comparable low number of differentially expressed cDNAfragments. 

In total, 9 differentially regulated cDNA-fragments were isolated, cloned and sequenced. The 

sequences of these transcripts were blasted (NCBI). Two of the differential expressed cDNAtranscripts 

originated from the pathogen S. sclerotiorum. Two more transcripts revealed no 

distinct function. However, 3 differentially up-regulated transcripts originating from leaves of 

the wild sunflower AC 7 were characterized with one transcript isolated at DPI 3 showing high 

homology to a cysteine protease and another transcript isolated at DPI 2 was highly homolog to 

4-coumarat-CoA-ligase, a key enzyme of the plant phenylpropanoid pathway. A further 

transcript showed low homology to a heat shock protein originating from Arabidopsis thaliana. 

Two more differentially regulated transcripts were characterized that were isolated from 

infected leaves of cv. Albena. One of these transcripts with high homology to a sequence 

coding for the S-adenosyl-methionine-synthetase in Cucumis sativus was up-regulated at DPI 

3. The other transcript was post-infectionally down-regulated 48 h after inoculation with S. 

sclerotiorum and revealed the highest identity to a chlorophyll a/b-binding protein originating 

from Brassica juncea. 

314 

AC 7 

Table 2. Total phenol content in healthy and Sclerotinia-infected leaves of two resistant 

wild sunflowers (AC 7, AC M) and one susceptible sunflower cultivar 

(Albena) at different time-points (Days Post Inoculum (DPI) 1-6). Not 

significant (n.s.) and significant (*) at p < 0.05 (Tukey-Kramer, HSD). 

AC M 

Albena 

Total phenol content 

[mg GAE / 100 mg DW] 

control DPI 1 DPI 2 DPI 3 DPI 4 DPI 6 

54.6 ± 

18.9 

118.7 ± 

44.9 

36.4 ± 

17.1 

76.5 n.s. ± 

14.2 

84.8 n.s. ± 

36.0 

27.9 n.s. ± 

9.4 

86.6 n.s. ± 

17.6 

128.6 n.s. ± 

38.6 

30.7 n.s. ± 

2.0 

DW: dry weight, GAE: Gallic acid equivalent 

61.9 n.s. ± 

26.8 

131.6 n.s. ± 

4.7 

45.1 n.s. ± 

13.2 

Phenolic content in Sclerotinia-infected sunflower leaves 

100.6 n.s. ± 

58.5 

152.6 n.s. ± 

32.2 

33.6 n.s. ± 

8.3 

112.8* ± 

48.2 

119.5 n.s. ± 

29.1 

56.8* ± 

19.1 

Total 

mean 

74.6 

118.8 

The results shown in Table 2 reveal that in comparison to AC7 and cv. Albena, leaves of the 

wild sunflower ACM contained high levels of phenolics in healthy leaves and in Sclerotiniainfected 

leaves. AC7 and cv. Albena showed a significantly induced phenolic content in 

42.7

infected leaves 6 days after inoculation and low concentrations of phenolics in healthy leaves. 

In AC7 the phenolic content of Sclerotinia-infected leaves at DPI 6 reached levels similar to 

leaves of ACM at DPI 6. Moreover, the chromatographic separation of phenolic substances via 

HPLC revealed that in both wild sunflower species a high number of compounds can be 

characterized as hydroxycinnamic acids. Whereas, in healthy and Sclerotinia-infected leaves of 

the susceptible variety Albena none of these compounds was detected. Furthermore, it was 

possible to spectroscopically differentiate between substances belonging to caffeoylquinic 

acids and those belonging to coumaroylquinic acids (Fig. 2). In both wild sunflower accessions 

the portion of caffeoylquinic acids prevailed in both non-infected and infected leaves. 

Moreover, in both wild sunflower genotypes the portion of compounds belonging to the group 

of caffeoyl quinic acids was induced after inoculation with the pathogen S. sclerotiorum. 

Figure 2. Content of coumaroyl- and caffeoylquinic acids in healthy and Sclerotiniainfected 

leaves at Days Post Inoculum (DPI) 6 according to UV-spectrum and 

absorption maxima of chromatographically separated phenolic compounds in 

AC7 and ACM. 

DISCUSSION 

In comparison with the annual cv. Albena, the wild genotypes AC7 and ACM showed reduced 

lesion lengths and very similar phenotypic reaction after infection with S. sclerotiorum. This 

indicates that the dissemination of the pathogen inside the host leaf tissue was potentially 

inhibited in both resistant genotypes. Furthermore, the results of the gene expression analysis 

315

evealed an early up-regulation of distinct fragments in AC7 compared with a late reaction in 

leaves of the susceptible genotype which is similar to observations of other host-funguspathosystems 

(Dixon et al. 1994; Li et al. 2006). The transcriptomic response at an early stage 

of pathogenesis is an indication for early initiated defense mechanisms. Accordingly, these 

mechanisms possibly elucidate the reduction of lesion lengths in infected leaf tissue of wild 

sunflower. 

Four differentially expressed cDNA-fragments were successfully characterized and revealed 

high homologies to known genes in other plants originating from different plant pathways. 

Two of these transcripts with homology to a chlorophyll a/b-binding protein and with 

homology to a cysteine protease give the potential evidence of a pathogen-induced oxidative 

stress. S-adenosyl-methionine-synthetase is a key enzyme of the methionine pathway and most 

likely secondarily involved in the methylation processes of phenolic substances. The 

significant up-regulation of a transcript with high homology to 4-coumarat-CoA-ligase 

indicates that the induction of the phenylpropanoid pathway also plays a crucial role for 

pathogen defense in wild sunflower as previously reported in cultivated sunflower (Bazzalo et 

al. 1985; Tourvieille de Labrouhe et al. 1997). 4-coumarat-CoA-ligase catalyses the formation 

of all CoA-ester of hydroxycinnamic acids and the induction by external stimuli has been 

demonstrated for other plants (Kuhn et al. 1984; Soltani et al. 2006). 

The results of the phenolic content of sunflower leaves underline the potential role of phenolic 

compounds of the phenylpropanoid pathway synthesized during the interaction of S. 

sclerotiorum and the resistant wild genotypes. However, both wild varieties showed 

differences in phenolic content. The synthesis of phenolic compounds was either induced as for 

AC7 or constitutively present as for ACM. As the number of differentially expressed 

transcripts was not noticeably high in ACM at any examined time-point in comparison to AC7, 

and comparable high total phenol content was present in all examined leaves, it can be assumed 

that ACM features a different kind of resistance type. 

In this study it was also shown that caffeoylquinic acids are predominantly found in resistant 

wild sunflower. Mono- and di-caffeoylquinic acids possess antioxidative and antimicrobial 

effect and are known to be involved in numerous defense processes against pathogens 

(Baranowski & Nagel 1982; Takahama 1998). In spite of the potential antifungal effect, a 

specific effect on the pathogen S. sclerotiorum can be predicted, but has to be investigated in 

further studies. Most likely the induction of 4-coumarat-CoA-ligase is associated with the 

increased concentrations of caffeoylquinic acids 6 days after inoculation as this enzyme is 

involved in the synthesis of precursors that specifically lead to formation of mono- and dicaffeoylquinic 

acids in plants (Hahlbrock & Scheel 1989). 

316

REFERENCES 

Achbani E H; Tourvieille D; Vear F (1994). Methods of determining the reaction of sunflower 

genotypes to terminal bud attack by Sclerotinia sclerotiorum. Agronomie 14, 195-203. 

Baranowski J D; Nagel C W (1982). Inhibition of Pseudomonas fluorescens by 

hydroxycinnamic acids and their alkyl esters. Journal of Food Science 47, 1587-1589. 

Bazzalo M E; Heber E M; Del Pero Martinez M A; Caso O H (1985). Phenolic coumpounds in 

stems of sunflower plants inoculated with Sclerotinia sclerotiorum and their inhibitory 

effects on the fungus. Phytopathologische Zeitschrift 112, 322-332. 

Benjamini Y; Hochberg Y (1995). Controlling the false discovery rate: A practical and 

powerful approach to multiple testing. Journal of the Royal Statistical Society, Series 

B57, 289-300. 

Bertrand F; Tourvieille, D (1987). Phomopsis sur tournesol: tests de sélection. Information 

Techniques CETIOM 98, 12-18. 

Boland G J; Hall R (1994). Index of plant hosts of Sclerotinia sclerotiorum. Canadian Journal 

of Plant Pathology 16, 93-108. 

Dixon R A; Harrison M J; Lamb C J (1994). Early events in the activation of plant defense 

responses. Annual Review of Phytopathology 32, 479-501. 

Godoy G; Steadman J R; Dickman M B; Dam R (1990). Use of mutants to demonstrate the 

role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. 

Physiological and Molecular Plant Pathology 37, 179-191. 

Hahlbrock K; Scheel D (1989). Physiology and molecular biology of phenylpropanoid 

metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 40, 

347- 369. 

Jan C-C; Seiler G (2007). Sunflower. In: Genetic Resources, chromosome engineering, and 

crop improvement, Chapter 5, ed R J Singh, pp.103-119. CRC Press, Taylor & Francis 

Group: Boca Rotan – London - New York. 

Kuhn D N; Chappell J; Boudet A; Hahlbrock K (1984). Induction of phenylalanine ammonialyase 

and 4-coumarate:CoA ligase mRNA´s in cultured plant cells by UV light of 

fungal elicitor. Proceedings of the National Academy of Science 81, 1102-1106. 

Li C; Bai Y; Jacobsen E; Visser R; Lindhout P; Bonnema G (2006). Tomato defense to the 

powdery mildew fungus: Differences in expression of genes in susceptible, monogenic- 

and polygenic resistance responses are mainly in timing. Plant Molecular Biology 62, 

127-140. 

Masirevic S; Gulya T J (1992). Sclerotinia and Phomopsis – two devastating sunflower 

pathogens. Field Crops Research 30, 271-300. 

Purdy L H (1979). Sclerotinia sclerotiorum: History, diseases and symptomatology, host range, 

geographic distribution, and impact. Phytopathology 69, 875-880. 

Riou C; Freyssinet G; Fèvre M (1991). Production of cell wall-degrading enzymes by the 

phytopathogenic fungus Sclerotinia sclerotiorum. Applied and Environmental 

Microbiology 57, 1478-1484. 

Schuster W H (1993). Die Züchtung der Sonnenblume (Helianthus annuus L.). In: Fortschritte 

der Pflanzenzüchtung, Heft 14. Paul Parey Verlag: Berlin – Hamburg. 

Seiler G J (1992). Utilization of wild sunflower species for the improvement of cultivated 

sunflower. Field Crop Research 30, 195-230. 

317

Singleton V L; Rossi J A (1965). Colorimetry of total phenolics with phosphomolybdicphosphotungstic 

acid reagents. American Journal of Enology and Viticulture 16, 

144-58. 

Skoric D (1987). FAO subnetwork report 1984-1986. In: Genetic evaluation and use of 

Helianthus wild species and their use in breeding programs. FAO, Rome, Italy. 

Skoric D (1993). Wild species use in sunflower breeding – results and future directions. Plant 

Genetic Resource Letters 93, 17-23. 

Soltani B; Ehlting J; Hamberger B; Douglas C (2006). Multiple cis-regulatory elements 

regulate distinct and complex patterns of developmental and wound-induced expression 

of Arabidopsis thaliana 4CL gene family members. Planta 23, 1239-1240. 

Takahama U (1998). Ascorbic acid-dependent regulation of redox levels of chlorogenic acis 

and its isomers in the apoplast of leaves of Nicotinia tabacum L. Plant Cell Physiology 

39, 681-689. 

Tourvieille de Labrouhe D; Mondolot-Cosson L; Walser P; Andary C; Serieys H (1997). 

Relation entre teneurs en dérives caffeoylquiniques des feuilles et la résistance de 

Helianthus spp. a Sclerotinia sclerotiorum. Helia 20, 39-50. 

Van Becelaere G; Miller J F (2004). Combining ability for resistance to Sclerotinia head rot in 

sunflower. Crop Science 44, 1542-1545. 

Vear F; Tourvieille D (1988). Heredity of resistance to Sclerotinia sclerotiorum in sunflower. 

II. Study of capitulum resistance to natural and artificial ascospores infections. 

Agronomie 8, 503-508. 

318

Konietzki S, Socquet-Juglard D, Diederichsen E: Mapping of genes controlling development and resistance to 

Verticillium longisporum in Brassica alboglabra. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to 



5-5 Mapping of genes controlling development and resistance to 

Verticillium longisporum in Brassica alboglabra 

Konietzki S, Socquet-Juglard D, Diederichsen E 

Institut für Biologie - Angewandte Genetik, Albrecht-Thaer-Weg 6, 14195 Berlin 

Email: loni.cera@web.de 

Abstract 

Verticillium longisporum is a plant pathogenic fungus affecting cruciferous plants 

such as Brassica napus, B. oleracea or Arabidopsis. Its relevance in the middle and 

northern European growing areas of oilseed rape has strongly increased in the last 

decades. Stunting and growth abnormalities are the most common symptoms in 

greenhouse assays, whereas in the field the fungus affects yield and seed size by 

precocious maturation. Whereas B. napus shows only little variation for resistance, 

resistance sources have been identified in its two ancestors, B. oleracea and B. rapa. 

Our aim is to identify the genetic basis of these resistance sources and to support 

resistance breeding in Brassica by developing molecular markers. We identified two 

accessions of strongly contrasting disease reactions in B. alboglabra, a close relative 

of B. oleracea, and used these to generate an F2/F3- mapping population. This 

population was studied in greenhouse assays for resistance and using PCR-based 

markers. Different disease parameters were analysed: AUDPC, % colonisation, and 

fresh weight. Both parents vary also for their flowering time. As we observed 

resistance to be correlated to slow flowering behaviour, both parameters are studied 

in our seggregating population. 

319

Mascher F, Marcello Z, Celeste L: Dynamics of adaptation of powdery mildew to triticale. In: Feldmann F, Alford 



5-6 Dynamics of adaptation of powdery mildew to triticale 

Mascher F 1 , Marcello Z 2 , Celeste L 3 

1 

Agroscope Changins-Wädenswil research station ACW, route de Duillier, 1260 Nyon, 

Switzerland 

2 

ETH Zürich, Universitätsstrasse 2, 8096 Zürich, Switzerland 

3 Australian National University, Canberra ACT 0200, Australia 

Email: fabio.mascher@acw.admin.ch 

320 

Abstract 

Powdery mildew of triticale (Blumeria graminis) is a new emerging disease. It has 

been observed for the first time on commercial triticale cultivars in Europe at the 

end of the last century. In 2005, a first pan-European epidemic occurred. Mainly 

cultivars, until then considered immune, were infected. In heavily affected stands, 

the infections resulted in high yield losses and serious deterioration of the yield 

quality.

Kopahnke D, Brunsbach G, Miedaner T, Lind V, Rode J, Schliephake E, Orden F: Screening of Triticum 

Monococcum and T. Dicoccum to Identify New Sources of Resistance to Fusarium Head Blight. In: Feldmann F, 



5-7 Screening of Triticum Monococcum and T. Dicoccum to Identify New 

Sources of Resistance to Fusarium Head Blight 

Kopahnke D 1 , Brunsbach G 2 , Miedaner T 2 , Lind V 1 , Rode J 1 , Schliephake E 1 , Orden F 1 

1 

Julius Kuehn-Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for 

Resistance Research and Stress Tolerance, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany, 

email: doris.kopahnke@jki.bund.de 

2 State Plant Breeding Institute, Universitaet Hohenheim, Fruwirthstr. 21, 70593 Stuttgart, 

Germany 

ABSTRACT 

Improving Fusarium head blight (FHB) into adapted cultivars is the best long-term 

approach to prevent wheat from yield losses and mycotoxin contamination. In order 

to broaden the genetic base of resistance to FHB, a total of 257 accessions of winter 

Triticum monococcum, 32 accessions of winter T. dicoccum, 27 accessions of 

winter T. turgidum and five accessions of winter T. boeticum were analysed for 

resistance to Fusarium culmorum in field trials at two locations in the growing 

seasons from 2005 to 2008. Those genotypes performing significantly better in field 

tests than the resistant T. aestivum check cultivar were analysed in detail in growthchamber 

experiments. Applying this approach, seven accessions of T. monococcum 

and six accessions of T. dicoccum were identified showing a high level of FHB 

resistance in both, field trials and growth chamber tests. To test the independency of 

our FHB resistant germplasm from already known QTLs on chromosomes 3BS and 

5A, the respective genomic regions were haplotyped for 34 T. monococcum and 

nine T. dicoccum accessions. First results concerning a QTL located on 

chromosome 5A based on three simple-sequence repeat (SSR) markers showed that 

at least three T. monococcum accessions have a haplotype different from T. 

aestivum accessions carrying the resistance allele at this QTL. These potentially 

new sources are now crossed to susceptible T. monococcum accessions to map the 

Fusarium resistance on the diploid level. 

321

INTRODUCTION 

Fusarium head blight (FHB) has become one of the most important diseases of wheat (Triticum 

spp.) in the EU and North America. Crop rotation, soil tillage including the burying of crop 

debris, growing of less susceptible cultivars and good crop husbandry help to reduce the risk of 

infection, but breeding for durable resistance to FHB in wheat is up to now the most 

economical and effective method to reduce yield losses and mycotoxin contamination. 

Although considerable breeding progress has been achieved in the last decade, improving 

resistance to FHB is still an ongoing and important task. However, highly efficient and 

environmentally stable sources of resistance in Triticum aestivum, such as cv. Sumai 3, are 

quite limited. Ancestors of hexaploid wheat, i.e. Triticum monococcum and T. diccocum turned 

out to be highly resistant to Puccinia triticina, Pyrenophora tritici-repentis and Blumeria 

graminis (Lind 2006) and resistance to FHB has been identified e.g. in wild emmer wheat 

(Oliver et al. 2007). A comprehensive meta analysis showed that 176 QTL has been described 

in literature with each chromosome of hexaploid wheat associated with FHB resistance (Löffler 

et al. 2009). QTLs on chromosomes 3BS and 5AS are among the most prevalent and are 

identified in many Chinese lines (Sumai-3 and its progenies) (Yu 2007). Our objectives were 

(1) to screen gene bank accessions of T. monococcum, T. dicoccum, T. turgidum and T. 

boeticum for their head blight resistance to Fusarium culmorum and (2) to analyse the 

independency of identified resistance donors from already known QTL on chromosomes 3BS 

and 5A. The overall aim is to evaluate whether these diploid and tetraploid FHB rsistance 

sources are suited to broaden the genetic basis of resistance in cultivated wheat. 


A total of 257 accessions of winter T. monococcum, 32 accessions of winter T. dicoccum, 27 

accessions of winter T. turgidum and 5 accessions of winter T. boeticum were analysed for 

resistance to Fusarium culmorum in field trials at two locations in the growing seasons from 

2005 to 2008 (at Quedlinburg, Saxony-Anhalt and at Stuttgart-Hohenheim, Baden- 

Württemberg, Germany). Seeds of these accessions originated from different gene banks and 

other sources. Nothing was known on the degree of FHB resistance of the accessions prior to 

the experiments. Trials were sown in double rows using a randomized block design with two 

replications. The released German cvs Toras and Solitär were included as resistant standards 

and cvs Ritmo and Reaper as susceptible standards. At its full flowering, each accession was 

spray inoculated with a freshly prepared spore suspension at a final concentration of 1 x 10 6 

conidia/ml. Inoculations were repeated after 4 to 8 days depending on the weather to account 

for the within-plot variation of anthesis. In all trials, the highly aggressive, DON producing F. 

culmorum isolate FC46 was used. Plants were evaluated for plant height, heading date and 

FHB resistance. FHB resistance was scored plot-wise on a percentage scale (0-100%) several 

times. The area under the disease progress curve (AUDPC) and the mean FHB rating were 

calculated across four observations. It should be mentioned that the same intervals between 

322

inoculation and rating date were used for each genotype to account for the highly varying 

flowering dates of the accessions. DON content was analysed by a commercially available 

immunotest (Biopharm FAST DON ® , Darmstadt) in the field-grown grain of selected 

accessions. Analysis of variance was conducted using the Statistical Analysis System (SAS, 

Version 9.1). 

Twenty-five T. monococcum accessions exhibiting significantly less severe disease symptoms 

than ‘Toras’ in the growing season 2006/07 as well as nine T. dicoccum and nine T. 

monococcum accessions from the season 2005/06 were retested in growth chamber 

experiments for resistance to primary infection - type I (Mesterhazy 1995). The heads were 

spray inoculated with a spore suspension of the same virulent isolate FC46 than used in the 

field. The final concentration was adjusted to 300,000 conidia/ml. After inoculation, plants 

were covered with plastic bags for 48 h. Resistance was evaluated by counting the infected and 

total number of spikelets of inoculated spikes on the 7th, 14th and 21st day after inoculation. 

Mean FHB ratings based on the percentage of infected spikelets (0-100%) were compared 

statistically by the Dunnett test at P

m ean FHB r ating 

324 

441* 

347* 

322* 

316* 449* 

313* 448* 

310* 438* 

348* 457* 

412* 365* 

352* 450* 

436* 319* 

455* 395* 

456* 350 

339 382* 

337* 451 

439* 437* 

366* 466* 

468* 416* 

392* 357 

454* 469* 

368* 453 

358 345 

399 415 

278 467* 

371 359 

332 406 

353 362 

346 470* 

363 432 

464 459 

369 452 

400 351* 

354 364 

460 458 

373 330 

321 340 

334 360 

5 

1 32* 

349 45* 

31 

43* 

39 8* 

6* 

22* 5* 

2* 21* 

17* 13 

25* 19 

15 7 

18 20 

1 9 

4 27 

14 24 

16 3 

12 23 

10 

26 Solitär 

Ritmo* 

Reaper 

Toras 

Quedlinburg 

Hohenheim 

T. dicoccum 

60 

T. turgidum 

T. monococcum 

50 

40 

30 

20 

10 

0 

Figure 1. Mean FHB rating of 73 T. monococcum, 5 T. dicoccum and 27 T. turgidum tested at Hohenheim and Quedlinburg in 2007/08. 

Light-coloured columns represent the cvs Toras, Solitär (resistant standards), Reaper and Ritmo (susceptible standards), 

respectively. Accessions with an * were significantly better than the resistant check cv. Toras.

Results obtained in 2006/2007 cannot be directly compared to those obtained in 2007/2008 

because accessions were different. However, similar proportions of resistant genotypes were 

achieved in both years and locations, e.g. in 2006/2007 in Hohenheim 24 accessions showed an 

FHB rating higher than 40% while no accessions were detected in Quedlinburg revealing a 

disease severity higher than 40%. On the other hand in 2007/2008, no accession revealed a 

disease severity higher than 40% at both locations. The most resistant accessions will be 

retested in 2008/2009 at Quedlinburg and Hohenheim to confirm their resistance reactions. 

Generally, the more resistant accessions had less DON in their kernels (Fig. 2). Because T. 

monococcum and T. dicoccon accessions are hulled, we additionally analysed the DON content 

in the chaff. In all accessions, the chaff contained several times more DON than the kernels. 

No considerable difference in this proportion was found compared with the free-threshing T. 

aestivum cultivars. Four accessions, however, had low DON contents also in the chaff. 

The results of the growth-chamber experiments of the field-selected accessions are shown in 

Figures 3 and 4. Based on Dunnett‘s test most of the accessions were significantly better 

performing than the susceptible check cvs Bobwhite and Remus. Especially the T. 

monococcum accessions # 93, 149, 273 and the T. dicoccum accessions # 5, 13, 23, 25 and 14 

revealed a very high level of FHB resistance in the field and also in the growth chamber. As in 

many breeding programs the focus is on type II resistance, which turned out to be more durable 

than the type I resistance (Bai & Shaner 2004), these accessions will now be tested for pure 

type II resistance by single-spikelet inoculation. 

mg DON kg -1 

200 

150 

100 

50 

0 

274 

266 

96 

248 

99 

262 

217 

108 

151 

5 

28 

15 

4 

9 

34 

20 

24 

13 

18 

Toras 

Solitär 

Reaper 

Ritmo 

T. monococcum T. dicoccon T. aestivum 

260.2 

Chaff 

Kernels 

Figure 2. DON content of kernels and chaff from nine T. monococcum, ten T. dicoccum accessions 

and four T. aestivum standards inoculated by F. culmorum at Hohenheim and Quedlinburg 

in 2006/07. 

325

120 

100 

80 

60 

40 

20 

0 

120 

100 

80 

60 

40 

20 

0 

326 

infested spikelets % 

7 dpi 

14 dpi 

21 dpi 

273* 

93* 

247* 

149* 

104* 

140* 

139* 

274* 

75* 

275* 

277* 

266* 

238* 

162 

98 

Alsen 

Bobwhite 

Remus 

Figure 3. Infected spikelets per ear (%) of T. monococcum accessions in growth 

chamber test measured at three dates after inoculation (dpi). Accessions with * 

are significantly (P

Preliminary results on type II resistance illustrate, however, that no accession reached the 

resistance level of the US spring wheat cv. Alsen, a line incorporating FHB resistance type II 

from the Chinese resistance source Sumai-3 or its derivates (Mergoum et al. 2005). 

Preliminary molecular data showed that at least 3 T. monococcum accessions have a different 

haplotype compared to T. aestivum accessions carrying the FHB QTL on chromosome 5A. 

They are now crossed to susceptible accessions to map their FHB resistance. Nine resistant T. 

diccocum accessions were additionally analysed by four SSR markers linked to the FHB QTL 

on chromosome 3BS. All accessions showed the same haplotype which is already known from 

resistant T. aestivum accessions with a Sumai 3 background. In future, allele-specific markers 

for additional QTL selected from the literature will be analysed in our T. monococcum and T. 

diccocum accessions in order to detect highly effective, new resistance sources. 


We thank the Bundesministerium für Bildung und Forschung (BMBF, Bonn) for financial 

support of this study within the project GABI-CANADA “Reducing Fusarium Toxins in 

Wheat Through Genomics-Guided Strategies” grant ID: 0313711A 

REFERENCES 

Bai G H; Shaner G E (2004). Management and resistance in wheat and barley to Fusarium 

head blight. Annual Review Phytopathology 42, 135-161. 

Buerstmayr H; Steiner B; Hartl L; Griesser M; Angerer N; Lengauer D; Miedaner T; Schneider 

B; Lemmens M (2003). Molecular mapping of QTLs for Fusarium head blight 

resistance in spring wheat. II. Resistance to fungal penetration and spread. Theoretical 

and Applied Genetics 107, 503-508. 

Kopahnke D; Brunsbach G; Miedaner T; Lind V; Rode J; Schliephake E; Ordon F (2008). 

Screening of Triticum monococcum and T. dicoccum for resistance to Fusarium 

culmorum. Cereal Research Communications 36, 109-111. 

Lind V (2006). Triticum monococcum als Resistenzquelle für den Weizen: Prähaustorielle 

Resistenz gegen Braunrost, Puccinia triticina. Vorträge Pflanzenzüchtung 68, 7. 

Löffler M; Schön C C; Miedaner T ( 2009). Revealing the genetic architecture of FHB 

resistance in hexaploid wheat (Triticum aestivum L.) by QTL meta-analysis. Molecular 

Breeding 23, 473–488. 

Mergoum M; Frohberg R C; Miller J D; Rasmussen J B; Stack R W (2005). Registration of 

spring wheat germplasm ND 744 resistant to Fusarium head blight, leaf, and stem rusts. 

Crop Science 45, 430-431. 

Mesterhazy A (1995). Types and components of resistance to Fusarium head blight of wheat. 

Plant Breeding 114, 377-386. 

Oliver R E; Stack R W; Miller J D; Cai X (2007). Reaction of wild emmer wheat accessions to 

Fusarium head blight. Crop Science 47, 893-899. 

Yu J B (2007). Identification of new sources and mapping of QTL for FHB resistance in Asian 

wheat germplasm. PhD thesis, Kansas State University. 103 pp 

327

Glavendekić M, Roques A: Invasive species following new crops. In: Feldmann F, Alford D V, Furk C: Crop 



6-1 Invasive Species Following New Crops 

Glavendekić M 1 , Roques A 2 

1 University of Belgrade, Kneza Viseslava 1, SR-11030 Belgrade, Serbia 

2 INRA, UR633 Zoologie Forestière, F-45166 Olivet, France 

Email: milka.glavendekic@nadlanu.com 

INTRODUCTION 

Biological invasions by alien species, as a component of global environmental change, can 

have significant negative impacts on biological diversity and functions of invaded ecosystems. 

They can cause a significant loss in economic value in agriculture, forestry and horticulture, as 

well as threat to human and animal health. Research on biological invasions in Europe has a 

long tradition. The most significant contribution to the knowledge of alien species present in 

Europe was achieved by a consortium of leading researchers on biological invasions in Europe 

working within the project DAISIE (Delivering Alien Invasive Species Inventories for 

Europe), which was supported by the European Commission from 2005 to 2008 under the 

Sixth Framework Programme. Large-scale environmental risks for biodiversity in EU 

including both biotic and abiotic factors were also studied recently. Biological invasions are 

dynamic and large-scale phenomena and inventory accounts and distribution maps of alien 

species provide an up-to-date view of the current status and distribution of alien taxa in Europe. 

A major output of DAISIE, the Handbook of Alien Species in Europe (DAISIE 2009) 

illustrates that, for most taxa, an ever- increasing number of non-native species are 

continuously introduced from other continents, especially Asia followed by North America 

(Hulme et al. 2009). For example, an average of 19 terrestrial invertebrates (Roques et al. 

2009), 16 plants (Pyšek et al. 2009) and one mammal (Genovesi et al. 2009) are arriving every 

year into one or more parts of Europe. This process largely results from the development of 

global trade with merchandises continuously moved throughout the world and at an everincreasing 

speed. The most affected ecosystems are those under strong human influence and 

unstable ecosystems. Thus, alien plants and terrestrial invertebrates are more frequent in urban 

than in semi-natural habitats (Pyšek et al. 2009, Roques et al. 2009), whilst birds and 

amphibians (Kark et al 2009), as well as mammals (Genovesi et al. 2009), are most frequently 

recorded from arable lands, gardens and parks. The current appreciation of the impacts of 

invasive species on biodiversity in Europe seems underestimated in comparison to North 

America, e.g. for plants (Levine et al. 2003) and terrestrial invertebrates (Roques et al. 2009). 

However, the data gathered during the DAISIE project evidenced on ecological effect on 

328

iodiversity for 5% for plants, about 15% for invertebrates and marine taxa, and 30% for 

mammals (Vila et al., in press). 

The precise effects of climate change on the diseases and pests in agriculture, forestry and 

ornamental horticulture remain little evaluated. However, they are likely to influence the hostparasite 

systems and stimulate the change in cultivars in order to be better adapted to changed 

abiotic factors (Verreet & Klink 2008). It was found that impact of invasive species vary within 

various cultivars of the same species. For example, the South African geranium bronze 

butterfly (Cacyreus marshalli) (Lepidoptera, Lycenidae), was firstly observed in 1991 in 

Europe, and it is at present threatening very popular ornamentals Geranium spp. and 

Pelargonium spp. Tests of susceptibility carried out in Italy revealed large differences between 

16 commercial cultivars of Pelargonium (Lupi & Jucker 2005). Similarly, tolerance of host 

plants to Horse chestnut leaf miner, Cameraria ohridella, differed between Asian Aesculus 

species and cultivated hybrids originating from North American species (Straw & Tilbury 

2006). In horticulture industry the creation of Ulmus cultivars e.g. ’Clusius’, 

’Commelin’,’Homestead’ and ’Dodoens’ tolerant to Dutch elm disease is especially looked for. 

DEFINITIONS 

We used the following terms in acceptance with those used in DAISIE and by the Convention 

on Biological Diversity (CBD 2001). ‘Alien’ refers to an organism occurring outside its 

natural past or present range and dispersal potential, whose presence and dispersal is due to 

intentional or unintentional human action. ‘Native’ refers to an organism that has originated in 

a given area without human involvement or that has arrived there without intentional or 

unintentional intervention of humans. Introduction / introduced refers to a direct or indirect 

movement by human agency, of an organism outside its past or present natural range. 

Establishment / Naturalization: refers to aliens that form free-living, self-sustaining 

(reproducing) and durable populations persisting in the wild. Invasion / invasive refers to 

established alien organisms that are rapidly extending their range in the new region. This is 

usually associated, although not necessarily for an organism to qualify as invasive, with 

causing significant harm to biological diversity and ecosystem functioning in invaded regions). 


Invasive Fungi of Europe 

fungi are a major component of biodiversity worldwide as the second largest group of 

Eukaryotes, after insects. They have been so far less studied and their invasion ecology is 

poorly represented with few species in invasive alien databases. Fungal taxonomy has been 

evolving rapidly over recent years due to the use of molecular tools and phylogenetic analysis. 

Many fungal species previously defined on the ground of morphology have been shown to be a 

complex of several cryptic species differing in their ecology and geographic range (Pringle et 

al. 2005). In the compiled European list of 688 alien species, there are 77% of plant pathogens; 

329

other symbiotic fungi represent 6% and saprobes 17% (Desprez-Loustau 2008). A majority of 

species originate from North America, but the proportion of species coming from Asia is 

higher in the last 30 years. Ophiostoma novo-ulmi, Phytophthora cinnamomi, Seiridium 

cardinale are recognized as the most invasive terrestrial fungi and they are chosen among 100 

of the worst invasive alien species. Cryphonectria parasitica is highly important in orchards 

and forestry, causing chestnut blight or canker. One of the new invaders is Ceratocystis 

platani. The European populations recorded from France, Italy, Switzerland, Greece, Belgium, 

Spain, and Serbia, probably resulted from a single introduction from the USA to Naples in 

World War II (Soulioti et al. 2008). The eradication and mitigation of pathogenic fungi 

requires knowledge-based measures. There is a lack of baseline ecological data on fungal 

communities, which especially applies to non-pathogenic fungi. The more fungal ecology in 

general is understood, the better the prevention will be (Desprez-Loustau 2009). From the 

beginning of 2000, Phytophthora ramorum deserved much concern in Europe and North 

America, as the causal agent of sudden oak death. It caused high level of local destructions in 

native habitats. Its high prevalence in nurseries increases the potential of spread to new areas. 

Invasive Ophiostom spp. affect cultivated elm trees and natural stands. Hybridization between 

North American and European species resulted in the establishment of hybrid (Ulmus x 

holandica) tolerant to Dutch elm disease The genetic improvement has developed numerous 

clones of hybrid elm resistant to the vascular wilt disease caused by Ophiostoma ulmi. 

Micropropagation of clones resistant to Dutch elm disease is one of appropriate methods for 

vegetative propagation of resistant cultivars (Anna et al. 1998). Nursery production support 

mass production of resistant cultivars for ornamental purposes. Some of the most popular 

resistant elm cultivars are ’Clusius’, ’Commelin’, ’Dodoens’,’Homestead’ and ’Vegeta’. 

Invasive vascular plants of Europe 

Plants could be introduced accidentally (e.g. as seed contaminating the other traded products), 

but most of them are introduced intentionally for production (crops for food, fodder, fuel, etc.), 

protection (windbreaks, soil conservation, erosion control), or social value (ornamental trees, 

shrubs and flowers). The DAISIE database contains records of 5,789 alien plant species in 

Europe, of which 2,843 are alien to Europe, i. e. of extra-European origin (Pyšek et al. 2009). It 

was found that the most common European alien species Conyza canadensis, native to North 

America, occurred in 47 countries/regions (94%). Datura stramonium, Helianthus tuberosus, 

Robinia pseudoacacia (all native to North America) are recorded on more than 80% of the 

studied regions. The most important woody invaders are black locust (Robinia pseudoacacia) 

and tree of heaven (Ailanthus altissima). Among invasive plants in Europe are noxious weeds 

as Japanese knotweed (Fallopia japonica), annual ragweed (Ambrosia artemisiifolia), 

Himalayan balsam (Impatiens glandulifera), Japanese rose (Rosa rugosa), giant hogweed 

(Heracleum mantegazzianum), and ice plant (Carpobrotus edulis), and they were found in 

more than 20 European countries. The pathways to Europe are intentional introductions (63%) 

and unintentional ones (37%). The most important are the escapes of species cultivated for 

330

ornamental and horticultural purposes, 58% of the total. The contaminants of seed mineral 

materials and other commodities are responsible for the introduction of 17% of all plant species 

(Pyšek et al. 2009). For 10% of introduced species, it was found that they arrived as directly 

associated with human transport but arriving independently of commodity. The most invaded 

habitats are manmade habitats (industrial habitats and arable land, parks and gardens). 64% of 

naturalized alien species occur in industrial habitats. 58% of established aliens were found on 

arable land, in parks and gardens. Grasslands are highly invaded with 37% of aliens and 

woodlands with 31% (Pyšek et al. 2009). In USA, horticultural industry is responsible for 85% 

of woody invasive species, which were introduced for ornamental purposes by landscape 

industry (Reichard & Hamilton 1997). Rapid changes are occurring in the global production of 

cut flowers. Fast growth of production in Latin America and Africa, as well as increased 

production in some Asian countries, increase the volume of floricultural products (mainly cut 

flowers) reaching the world market. New flower crops are demanded, as well as the 

specialization and intensification of production (Shillo 2000). Exotic plants and especially 

tropical trees, such as eucalyptus and palm trees, are vectors for the introduction and 

distribution of alien invasive insects and according to Roques (2007), there is more than 50 

insects related to them. The most naturalized alien plants are recorded from the United 

Kingdom. 

Ornamental cultivars of Pelargonium vary in susceptibility to invasive pests. Due to the 

investigations of Lupi & Jucker (2005), it was found that zonale and iviy-leafed pelargoniums 

are susceptible to Cacyreus marchalli. Regal and scented-leafed cultivars were attacked later 

and at the lower level of infestation. Scented-leafed Pelargonium cultivars: ’Abrotanifolium’, 

’Concolor lice’, ’Denticulatum’, Fair ellen’, ’Filicifolium’, ’Odoratissimum’, ’Purple unique’, 

’Prince of orange’, ’Royal oak’ and ’Wayward angel’ were not attacked or damaged by C. 

marchalli (Lupi & Jucker 2005). 

Invasive terrestrial invertebrates of Europe 

The first continental inventory of the alien species established in Europe based on the results of 

the project DAISIE, showed that terrestrial invertebrates are one of the most numerous groups 

of introduced organisms in Europe with 1517 species of alien origin. Among them, insects 

represent more than 85% (1315 spp.) followed by mites, spiders, and nematodes the other taxa 

being more anectodical (Roques et al. 2009). In addition, there are 964 species of European 

origin which are considered to have been introduced from one to another European region. 

Thus, the Iberian slug Arion vulgaris (= lusitanicus), several species of Deroceras and snails 

such as Milax gagates and Cryptomophalus asperses were unintentionally translocated within 

Europe (Wittenberg 2006). A precise region of origin is known for 79% of the alien 

invertebrates, while for 7% it is only known that they are native in tropical and subtropical 

regions and 14% are qualified as ’cryptogenic’ because of their unknown origin as it is the case 

for a number of nowadays cosmopolitan species infesting stored products. For example, the 

horse-chestnut leaf miner Cameraria ohridella is illustrative of the difficulty in identifying the 

331

native range of such species. For a long time, it was qualified as cryptogenic, and only recently 

genetic studies tended to ascertain a balkanic origin (Augustin et al., in press). Most of the 

alien invertebrates in Europe originate from Asia (29%). The rate of established alien 

invertebrate species in Europe since 1492 is shown in Figure 1. Significant increase is to be 

seen from the second half of 20 th century. 

332 

Figure 1. Rate of established alien invertebrate species in Europe since 1492 as mean 

number of alien invertebrates recorded per year (Roques et al. 2009) 

Among the 794 alien species which present a phytophagous regime, there are 463 species 

related to trees and shrubs of which 2 nematodes, 49 mites and 412 insects (Roques 2007). The 

rate of establishment of these species related to woody plants exponentially increased during 

the second half of the 20 th century. The mean number of species arriving per year during the 

period 2000-2007, 7.9, is nearly twice as large as the one recorded in the period 1950-1975 

(4.2). Due to the increased trade with Asia, this continent became the major source of alien 

arrival (>30%) far beyond North America. The trade of ornamental plants (e.g., plants for 

planting, cut flowers, pot plants, seeds, bonsais, …) was observed to be the dominant way of 

introduction of such alien species related to trees and shrubs whilst the trade of forestry 

products had only a limited contribution. Broadleaved trees, fruit trees and conifers are the 

most colonized woody species for the moment. However, trees of tropical and subtropical 

origin planted in Europe, especially palms, eucalyptus and acacia, appeared comparatively 

more colonized than the others since the late 1990s, probably in relation with global warming. 

Alien insect species predominantly belong to the orders Hemiptera and Coleoptera. Families 

Aphididae (aphids) and Diaspididae (scales) are the families with the most important number 

of invaders related to trees and shrubs. Only few of them were intercepted and included in the 

quarantine lists in Europe. Most of these aliens have an Asian origin, especially these 

belonging to Hymenoptera (38%), Lepidoptera (35%) and Hemiptera (33%) (Roques et al. 

2009). It was expected because their host plants, mostly ornamental plants, were imported from 

that region. By contrast, Diptera predominantly arrived from North America (30%). An 

example of these new invasive Diptera is the black locust midge, Obolodiplosis robiniae, a

cediomyiid which was found for the first time in 2003 in the Veneto region of north-eastern 

Italy (Duso & Skuhravá 2004). In less than 5 years, it has spread throughout Europe in all 

directions and it is at present well established in almost the whole Western, Central and South- 

East Europe. However, its specific larval parasitoid, Platygaster robiniae, was also 

unintentionally imported together with the host midge, and it is quickly spreading throughout 

Europe favoring biological control (Glavendekic et al., in press). 

The chestnut gall wasp (Dryocosmus kuriphilus; Cynipidae) was accidentally imported from 

China at the end of the 20 th century, probably in the middle of 1990s, together with cuttings for 

grafting of cultivars tolerant to Cryphonectria parasitica. It was for the first time recorded in 

2002 from chestnut orchards in the Cuneo province in Italy (Brussino et al. 2002). Three years 

later D. kuriphilus was introduced into Slovenia with nursery stock imported from Italy (Seljak 

2008). During the last few years, the gall wasp populations increased significantly, and 

chestnut fruit production decreased by 40- 70% in the highly infested orchards in Italy. From 

2005 to 2008, new spots were reported from all of the provinces of Piedmont together with a 

large expansion of the wasp all over Italy (Graziosi & Santi 2008) as well as in Slovenia 

(Seljak 2008). Thus, Dryocosmus kuriphilus is considered to be one of the most harmful pests 

on Castanea worldwide and classified by EPPO as a quarantine organism. The females lay 

their eggs into buds. Larvae feed within the galls disrupting twig growth and causing severe 

plant decline and yield reduction. Chestnut gall wasp originates from China and attacks native 

European chestnut Castanea sativa, Chinese species Castanea crenata and Castanea 

mollissima, American chestnut Castanea dentata as well as hybrids of Castanea sativa x 

crenata. The evaluations of susceptibility of Castanea cultivars to D. kuriphilus were carried 

out in July 2004 2005, and 2006 on young plants of 41 cultivars, including 7 inter-specific 

hybrids. They were infested inside insect-proof screen houses using a controlled number of 

cynipids. In the following spring the number, size and position of galls were observed and 

recorded. Results obtained so far indicate that all tested C. sativa cultivars are susceptible to 

the gall wasp. Among euro-japanese hybrids, cv ’Bouche de Bétizac’ and ’Marsol’ showed 

opposite reaction to the insect: no gall development was observed in cv ’Bouche de Bétizac’ 

while the highest levels of infestation were observed in cv ’Marsol’. In 3 years of observations 

complete resistance to D. kuriphilus was thus found only in ’Bouche de Bètizac’ (Sartor et al. 

2009). 

There are also large variations in the susceptibility of Aesculus species and hybrids to horse 

chestnut leaf miner, Cameraria ohridella. The probable original host, A. hippocastanum 

originating from the Balkan Peninsula is highly susceptible such as the Japanese horse chestnut 

(A. turbinata) whilst other Asian species (A. assamica, A. chinensis and A. indica) seem more 

resistant and are usually not damaged. North American species (A. californica, A. flava, A. 

glabra, A. parviflora, A. pavia and A. sylvatica) show an intermediate level of susceptibility 

between the European and Asian species. The most common hybrid, the red-flowering horsechestnut 

A. x carnea, a cross between A. hippocastanum and A. pavia, is highly resistant to C. 

ohridella. Eggs can be laid on its leaves, and larvae capable of hatching, but these larvae die 

333

during the first or the second instars. This A. x carnea hybrid rarely suffers any significant 

damage from C. ohridella (Straw & Tilbury 2006). 

The western corn rootworm Diabrotica virgifera virgifera, originating from North America, 

has been observed in Europe since 1992 and it is presently spreading in Central and Western 

Europe (Baufeld & Unger 2008). Another lepidopteran pest, Diaphania perspectalis 

(Pyralidae) was recently introduced in 2007 along with ornamental common box, Buxus spp., 

in Germany, France, Netherlands, and Switzerland. It originates from China, Japan and South 

Korea, where it is also related to Ilex purpurea, Euonymus japonicus and E. alatus (Baufeld 

2008). The above mentioned plants are common plants in gardens, parks and other categories 

of urban green, and it is expected that some ornamental shrubs in Europe could also be 

threatened. 

The highest number of alien invertebrate species is observed in Italy (652 spp.), followed by 

France (626 spp.). These high numbers appeared correlated to the volume of merchandise 

imports, especially of agriculture imports (Roques et al. 2009). Alien insects and mites often 

invade Italy as the first part of Europe, owing to its intensive nursery production, commercial 

exchanges of plants and goods and constantly increasing tourist traffic. From 1945 to 2004 

there were 162 exotic pests introduced to Italy. Most of them are pests of ornamentals (79 

species) and woody plants (38 species). Citrus (16 pests), horticultural crops (15 species), fruits 

and grapevine (14 species). Over the whole 60-year period, it was found that majority species 

originated from America (37%). But in the period from 1999 to 2004, there were 46.43% of 

alien insects accidentally introduced from Asia and 42.86% from both North and South 

America. Only 3.57% originated from Australia. A great majority of alien insects are recorded 

as pests of ornamentals (64.29%), from which 17.86% are pests in greenhouses on ornamental 

and horticultural crops (Pellizzari et al. 2005). 

Pathways of introduction for invertebrates are not easy to estimate but can be inferred from the 

species biology and from interceptions by quarantine services. Most introductions appears to 

be unintentional, species having arrived as contaminants or hitchhikers. The dominant pathway 

seems to correspond to ornamental trade (>30%) followed by stored products (ca. 16%). There 

are also evidences that some biological control agents intentionally released for biological 

control in glasshouses further escaped and became established in the field. However, such 

cases are very limited and represent less than 10% of the exotic species having established in 

Europe (Roques et al. 2009). However, they include some emblematic species such as the 

multicolored Asian ladybeetle Harmonia axyridis, which has spread throughout western and 

central Europe, reaching Southeast Europe in autumn 2008 (Thalji & Stojanovic 2009). 

Multicolored Asian ladybeetle has become a human nuisance, a grape and vine pest and a 

threat to native biodiversity. This was the reason why scientific research was undertaken in 

order to develop management strategies against H. axyridis (Kenis et al. 2008). 

Alien invertebrates and fish in the European inland waters are also very important in ecological 

as well as economic senses. The European inland waters contribute significantly to annual 

global value of entire biosphere and they are also vulnerable and threatened by alien organisms 

334

(Gherardi et al. 2009). Alien marine biota, alien birds, amphibians and reptiles and mammals 

of Europe are also the part of DAISIE databases. 

CONCLUSIONS 

A number of alien invasive organisms are affecting production of agriculture crops, forest 

production as well as ecosystem functioning. Dryocosmus kuriphilus is one of alien species 

highly significant for chestnut orchards, the native European chestnut, Castanea sativa being 

very susceptible whereas the Euro-Japanese hybrid ’Bouche de Bétizac’ showed high 

resistance to the insect with no gall development. Spontaneous hybrids and various Ulmus 

cultivars also show a significant level of tolerance to Dutch elm disease. Ornamental trees, 

shrubs, flowers, grasses and potential new industrial crops are explored in Europe. Some of the 

invasive species are also on the list of fast-growing cultivars for biomass production and 

recommended for the commercial cultivation. Their benefits should be taken into consideration 

together with the threats to ecosystem, human and animal health. The scientific results aimed to 

support decision making and specific EU policy could be favorable for European economy and 

environment. 


This work was partly supported by the projects ALARM (Assessing Large-scale environmental 

Risks for biodiversity with tested Methods; GOCE-CT-2003-506675; 

http://www.alarmproject.net(, and DAISIE (SSPI-CT-2003-511202) funded by the European 

Commission under the Sixth Framework Programme We also thank Dr Falko Feldman and 

reviewer for useful comments on the text and for support. 

REFERENCES 

Anna M; Mariella L; Lorenzo M (1998). Elm tissue culture: micropropagation of clones 

resistant to Dutch elm disease. Acta Hort. (ISHS) 457, 235-242 

http://www.actahort.org/books/457/457_29.htm 

Augustin S; Kenis M; Valade R; G M; R A; Lopez-Vaamonde C (in press): A stowaway 

species probably arriving from the Balkans, the horse chestnut leafminer (Cameraria 

ohridella). In: Atlas of Biodiversity Risks - from Europe to the globe, from stories to 

maps. Eds J. Settele et al, in press. Pensoft, Sofia & Moscow, 

(http://www.pensoftonline.net/alarm-atlas-info). 

Baufeld P, Unger J-G (2008). Der Westliche Maiswurzelbohrer (Diabrotica virgifera virgifera) 

– aktuelle Situation and Hintergründe. 56. Deutsche Pflanzenschutztagung in Kiel, 

Mitteillungen aus dem Julius Kühn-Institute, 233. 

Baufeld P (2008). Beanstandete und neue, eingeschlepptee nichtendemische Schadinsekten in 

Deutschland. 56. Deutsche Pflanzenschutztagung in Kiel, Mitteillungen aus dem Julius 

Kühn-Institute, 229. 

Brussino G; Bosio G; Baudino M; Giordano R; Ramello F; Melika G (2002). A dangerous 

exotic insect threatening European chestnut. Informatore Agrario, 58 (37), 59-61. 

335

CBD 2001. Convention on Biological Diversity SBSTTA 6, Recommendation VI/4 

www.cbd.int/recommendations/sbstta/view.shtml?id=7035 (date of access 20.03.2009) 

Desprez-Loustau M-L (2009). Alien Fungi of Europe. In: DAISIE, Handbook of Alien Species 

in Europe, pp 15-28. Springer: Berlin. 

Duso C, Skuhravá M (2004). First record of Obolodiplosis robiniae (Haldeman) (Diptera: 

Cecidomyiidae) galling leaves of Robinia pseudoacacia L. (Fabaceae) in Italy and 

Europe. Frustula entomologica, XXV, 117-122. 

Genovesi P; Bacher S; Kobelt M; Pascal M; Scalera R (2009). Alien Mammals of Europe. In: 

DAISIE, Handbook of Alien Species in Europe, 119-128. Springer: Berlin. 

Gherardi F; Gollasch S; Minchin D; Olenin S (2009). Alien Invertebrates and Fish in European 

Inland Waters. In: DAISIE, Handbook of Alien Species in Europe, 81-92. Springer: 

Berlin. 

Glavendekić M; Roques A; Mihajlović L (2009). An ALARM Case study: The rapid 

colonization of an introduced tree, black locust by an invasive North-American midge 

and its parasitoid, in press. Pensoft: Sofia, Moscow. 

(http://www.pensoftonline.net/alarm-atlas-info). 

Hulme P E; Roy D B; Cunha T; Larson T-B (2009). A pan-European Inventory of Alien 

Species: Rationale, Implementation and Implication for Managing Biological Invasions. 

In: DAISIE, Handbook of Alien Species in Europe, 1-14. Springer: Berlin. 

Kark S; Solarz W; Chiron F; Clergeau P; Shirley S (2009). Alien Birds, Amphibians and 

Reptiles of Europe. In: DAISIE, Handbook of Alien Species in Europe, 105-118. 

Springer: Berlin. 

Kenis M; Roy H E; Yindel R; Majerus M e N (2008). Current and potential management 

strategies against Harmonia axyridis. BioControl 53 (1), 235-252. 

Levine J M; D’Antonio C M; Dukes J S; Grigulus K; Lavorel S; Vila M (2003). Mechanisms 

underlying the impacts of exotic plant invasions. Proceedings of the Royal Society 270, 

775-781. 

Lupi D; Jucker C (2005). The butterfly Cacyreus marshalli in northern Italy, and susceptibility 

of commercial cultivars of Pelargonium. BCBP Symposium Proceedings No. 81: Plant 

protection and Plant Health in Europe. Introduction and Spread of Invasive Species, 

pp 249-250. 

Pellizzari G; Montá L D; Vaccante V (2005). Alien insect and mite pests introduced to Italy in 

sixty years (1945-2004). BCPC Symposium Proceedings No. 81: Plant protection and 

Plant Health in Europe. Introduction and spread of Invasive Species, pp 275-276. 

Pyšek P; Lambdon P W; Arianoutsou M; Kühn I; Pino J; Winter M (2009). Alien Vascular 

Plants of Europe. In: DAISIE, Handbook of Alien Species in Europe, 43-62. Springer: 

Berlin. 

Reichard H S; Hamilton C W (1997). Predicting invasions of woody plants introduced into 

North America. Conservation Biology 11, 193-203. 

Roques A (2007). Invasive insect species in Europe: First Results of the DAISIE Program. 

USDA Interagency Research Forum- GTR-NRS-P-28, p.62 

Roques A; Rabitsch W; Rasplus J-I; Lopez-Vaamonde C; Nentwig W; Kenis M (2009). Alien 

Terrestrial Invertebrate of Europe. In: DAISIE, Handbook of Alien Species in Europe, 

63-79. Springer: Berlin. 

Sartor C; Botta R; Mellano M G; Beccaro G L; Bounous G; Torello Marinoni D; Quacchia A; 

Alma A (2009). Evaluation of susceptibility to Dryocosmus kuriphiluS Yasumatsu 

336

(Hymenoptera: Cynipidae) in Castanea seativa Miller and in hybrid cultivars. Acta 

Hort. (ISHS) 815, 289-298. http://www.actahort.org/books/815/815_38.htm (date of 

access 02.04.2009). 

Seljak G (2008). Chestnut gall wasp Dryocosmus kuriphiluS Yasumatsu. Report – 

Phytosanitary Administration of the Republic of Slovenia 

http://www.furs.si/svn/zvr/kost_siskarica.asp (date of access 02.04.2009). 

Shillo R (2000). The Importance of new Crops for Israeli Floriculture. 

www.actahort.org/members/showpdf?booknrarnr=543_31 (date of access 20.03.2009). 

Soulioti N; Tsopelas P; Woodward S (2008). Ceratocystis platani: An invasive fungal 

pathogen threatening natural populations of oriental plane in Greece. IUFRO Unit 

7.03.12: Alien invasive species and international trade, Meeting, NCTC, 

Shepherdstown. Abstracts of papers, p.48. 

Straw N A; Tilbury C (2006). Host plants of the horse-chestnut leaf-miner (Cameraria 

ohridella), and the rapid spread of the moth in the UK 2002-2005. Arboricultural 

Journal 29, 83-99. 

Thalji R; Stojanovic D (2009). [First sighting of the invasive ladybird Harmonia axyridis 

Pallas (Coleoptera, Coccinellidae) in Serbia]. Biljni lekar/Plant Doctor XXXVI, 6, 

389-393 (in Serbian) 

Verreet J-A; Klink H (2008). Auswirkungen des Klimawandels auf Krankheitserreger und 

Pflanzenschutz in landwirtschaftlichen Kulturen Norddeutschlands. In: 56.Deutsche 

Pflanzenschutztagung in Kiel. Pflanzenproduktion im Wandel – Wandel im 

Pflanzenschutz? Mitteilungen aus dem Julius Kühn-Institut, No. 417:87 

Vilà M; Basnou C; Pyšek P (in press). How well do we understand the impacts of alien species 

on ecosystem services? A pan-European cross-taxa assessment. Frontiers in Ecology 

and the environment (in press). 

Wittenberg R (2006). Invasive alien species in Switzerland. An inventory of alien species and 

their threat to biodiversity and economy in Switzerland. Environmental Studies 29, 

Federal Office for the Environment, Bern. 

337

Hummel H E, Bertossa M, Deuker A: The Current Status of Diabrotica virgifera virgifera in Selected European 

Countries and Emerging Options for its Pest Management. In: Feldmann F, Alford D V, Furk C: Crop Plant 



6-2 The Current Status of Diabrotica virgifera virgifera in Selected 

European Countries and Emerging Options for its Pest Management 

Hummel H E 1,2 , Bertossa M 3 , Deuker A 1 

1) 

Justus-Liebig-University, Organic Agriculture, Karl-Gloeckner-Strasse 21 C, D-35394 

Giessen, Germany; e-mail: hans.e.hummel@agrar.uni-giessen.de 

2) 

Illinois Natural History Survey, Biodiversity and Ecological Entomology, Champaign, 

Illinois 61820-6960, USA 

3) 

Agroscope Changins-Wädenswil ACW, Centro Cadenazzo, CH-6594 Contone, Switzerland 

338 

Abstract 

Globalized traffic activities, monocultural production practices and generally 

increasing demand patterns for food and feed all favour the spread of alien invasive 

species. Among them, the western corn rootworm Diabrotica virgifera virgifera 

(Col.:Chrysomelidae) ranks high within the top dozen agricultural pests worldwide. 

Over reliance on non-sustainable control approaches with pesticides and genetically 

modified plant varieties may have raised false hopes of eventual WCR eradication 

throughout Europe. However, WCR is now so firmly established in SE Europe that 

eradication is a hopeless dream. Nevertheless, the present situation in Central 

Europe including Germany with a narrowly spaced network of monitoring and 

survey efforts at least offers the option of succeeding in WCR management and 

containment. As the special example of Switzerland shows, exclusive use of crop 

rotation and phytosanitation indeed conserves the "status quo" at minimal cost and 

risk for the environment. However, this approach may not be directly applicable to 

the conditions of large countries with vastly different economic, ecological and 

geographic structures. Thus, the search for practical IPM approaches suitable 

suitable thoughout Europe will continue and will require a high degree of 

intraeuropean cooperation. Some current successful approaches employing 

sustainable biotechnical methods such as monitoring and trapping with attractants 

are highlighted.

INTRODUCTION 

High yield maize hybrids currently in use are bred for intensive agriculture and large-scale 

animal and human consumption. In this selection process, natural genes of the wild maize 

ancestors coding for natural resistance against insect attack have been largely lost. Thus, a 

majority of high yielding cultivars available today both in North America and Europe are quite 

susceptible to the vicious attacks of both the larvae and adult western corn rootworm (WCR). 

This species ranks high among the top dozen agricultural insect pests, both in economic and 

ecological terms (Metcalf 1986, Hummel 2007, Baufeld 2008). Economically, WCR and its 

close relatives cause annual yield losses and expenses for pest control measures approaching 

one billion US dollars (Metcalf 1986). The ecological impact of WCR is more difficult to 

quantify being a hidden consequence of the biased priorities within our western-style farming 

practices with their disregard for the limits of natural resources. Present-day farming systems 

favour monocultural production in a globalized world whose members are connected by a 

multinational network of traffic by air, water, rail and roads. The farming community tends to 

over-rely on chemical pesticides. Combined with the selection pressure towards resistant insect 

ecotypes (Metcalf 1983) there is a powerful trend toward mass buildups and spread of WCR in 

all areas where Zea mays is a major economic factor. It is hard to break out of this vicious 

circle clearly recognized and identified by R. L. Metcalf (1916-1998) in his series of seminal 

books and papers initiated 3 decades ago (Metcalf & Luckmann 1975, Metcalf 1983, Metcalf 

1986, Metcalf & Metcalf 1992, Metcalf & Lampman 1997) and ending with his death in 1998. 

Unfortunately, monocultural European farm practices now tend to duplicate experiences made 

by American entomologists during the late 20 th century. It is high time for a remodelling of our 

agricultural approaches toward sustainability. Fortunately, Metcalf's self-imposed role as 

environmental Kassandra also identified sustainable pest management approaches using the 

tools of chemical ecology, such as kairomones and pheromones, including trapping systems for 

the monitoring of WCR (Levine & Metcalf 1988). In this paper, we briefly report on some of 

our varied experiences over the last few years applying Metcalf's tools and management 

approaches in 4 geographically distinct areas of Europe: Germany, Southern Switzerland, 

Slovenia and Romania. 


Traps were mainly of the omnidirectional, symmetrical, inverted Metcalf sticky cup type first 

described by Levine & Metcalf (1988) and Hummel (1989) using 0.5 litre plastic cups and 

polyisobutene as an adhesive. Occasionally other trap models like the Csalomon ® trap were 

used for comparison. Generally the Metcalf cup trap was superior to other models both in 

sensitivity and low price. Often, it could detect beetles a few days sooner than other trap types 

which is a great help in early pinpointing new infestations. It therefore has been used 

extensively for the last 20 years (Hummel 1989, 2007; Hummel et al. 2005, 2006, 2008, 2009; 

Bertossa & Hummel 2008). The female sex pheromone lure 8-methyl-decane-2-ol propanoate 

was developed by Guss et al. (1982) as a sensitive and highly specific attractant for males, 

339

while kairomonal lures of the 4-methoxycinnamaldehyde (MCA) type were discovered and 

developed by Metcalf & Metcalf (1992) and Metcalf & Lampman (1997). This latter plant 

kairomone mimetic is highly specific for WCR males and females. It is an excellent population 

indicator later in the maize growing season when there are more female than male beetles in 

the field. All of these synthetic lures are separately dispensed, in amounts of 0.1 (pheromone) 

to 10 mg (kairomone) onto heavy duty chromatography paper squares, attached to the trap top, 

and exchanged once a day to once every 4 days depending on the prevailing temperature and 

weather conditions. Traps have a minimal distance of 20 m in order to avoid trap competition. 

If high population densities are to be monitored, high capacity traps are needed (Dinnesen et al. 

2009, e.g. the roofed VARIO or OMNI or UNIVERSAL trap models as depicted in Hummel 

2007). 


Systematic trapping and surveying with pheromone and kairomone attractants is an 

indispensable help for diagnosing the present status of WCR. Results obtained in Germany by 

various authors are summarized in Table 1. The last column lists increases and decreases from 

2007 to 2008. Thus, the effectiveness of treatments including successes and failures become 

transparent. Judged by the reduction of beetles in Lake Constance county, the treatment of 

2007 was a full success. Less favorable are the results in other locations of the state. At 

Orthenau county an increase of population by a factor of 12.6 was recorded in connection with 

a significant expansion of the invaded area. In spite of treatments following the same set of EU 

rules, eradication attempts in this area failed. In Bavaria, the situation is mixed: while the small 

infestation of 2007 near Munich international airport vanished, the WCR population at Passau 

township was reduced to a factor of 0.12. Passau county, however, experienced a major 

population increase by a factor of 85.5. Also worrisome is the expansion of WCR to the 

counties of Deggendorf and Straubing / Bogen. Thus, in absolute terms, the number of WCR 

trapped appears stagnant. However, the area under active observation in 2008 decidedly and 

significantly increased (Glas 2008, Baufeld 2008, Bögel 2008). Both the effort and cost of 

monitoring multiplied considerably. WCR did not yet arrive in 2008 in Rhineland-Palatine or 

Hesse. But both states are the next natural targets of the beetle in its future drive northward. 

In Switzerland’s Ticino canton, a continuting series of yearly trapping experiments has been 

undertaken beginning in 2003. Their aim was to produce quantitative data on the influence of 

crop rotation on overall WCR population density and population distribution over time. Results 

of 2001 to 2007 mirror the typical steady state (or slight reduction) of WCR population level 

achieved after areawide crop rotation was initiated in 2003 (Bertossa & Hummel 2008). WCR 

distributions summed up over 10 selected sites located in the northern part of the Ticino are 

depicted in Fig. 1. The flight maximum monitored by 3 different trap models peak during week 

33 (Aug. 11-18, 2008). Noteworthy are some sites (Gudo, Lumino, Gordola and Cugnasco) 

showing consistently lower WCR numbers than the other sites (Agroscope Research Farm, San 

Vittore, Camorino, Contone and Lostallo in the adjacent canton Grisons). It is tempting to 

340

Table 1. Monitoring results in Germany 2007 und 2008 (Baufeld 2008, Bögel 2008, 

Hummel et al. 2009) 

Location State 2007 2008 

Total 

number 

Total 

number 

Increase-, 

decrease factor 

Δ 2008 / 2007 

1. Salem (Lake Constance county) 348 0 0 

2. Lahr (Orthenau county) 6 76 76 / 6 = 12.6 

3. Altmannshofen A 96 

Baden- 

Württemberg 

- 1 

1 / 0 random 

event? 

1. Airport Munich 1 - 0 / 1 

2. City of Passau 236 28 28 / 236= 0.12 

3. Passau county 

Bavaria 1 

2 

(3 1 ) 

171 

(7 1 ) 

171 / 2= 85.5 

4. Deggendorf county - 16 16 / 0 

5. Straubing Bogen county 

- 4 4 / 0 

Rhineland- 

Palatine 

0 2 

0 3 

Hesse 0 0 4 

Total 593 296 296 / 593 =0.49 

1 The numbers in brackets represent beetles caught in Austria near the frontier to Bavaria 

2 own monitoring at Schweighofen / Palatine, August 24-27, 2007 

3 own monitoring at Schweighofen and several sites near motorways A6, A61, A65 from August 10 to 

October 4, 2008 

4 own monitoring in Aumenau and Elkerhausen / Hesse from August 12 to October 23, 2008 

assume that the latter 5 sites owe their higher WCR populations to their closer proximity to 

roads and railroads. But other factors not investigated in closer detail may play an even more 

important role. The 2 WCR beetles caught in 2008 at Mt. Ceneri pass road where no maize is 

being grown provide a key for a likely explanation. Why should WCR fly there at all? 

Considering the topography and meteorology of the Ticino, Mt. Ceneri pass with its 550 m 

elevation is considered an important portal connecting the southern and northern Ticino. It 

probably is a major transit route for migrant beetles moving from Lombardy in Italy actively or 

passively to zones A, B and C (all in canton Ticino as defined by Bertossa et al. 2001). Zone C 

is the Magadino plain and adjoining side valleys where the majority of WCR are found. Those 

sites situated closer to prevailing wind currents may receive more WCR than their average 

share because migrants WCR may settle down there preferentially. A combination of passive 

(by wind currents) and active transportation (by trucks, cars and trains) may be responsible for 

this observed spatial distribution pattern. 

0 

0 

341

342 

Figure 1. Diabrotica captures in Ticino Canton according to the type of lure and trap used 

Monitoring in East Slovenia from 2003 to 2007 produced evidence for WCR immigration 

across the border from Croatia and Hungary during July and August of 2004. In 2005, at the 

village of Pince in the easternmost appendix of Slovenia, a most intensive trapping effort 

produced a total of 78 WCR within 37 days. Now, 3 years later, Dinnesen et al. (2009) found 

4039 WCR at the same location within 43 days trapped in late July and August of 2008. This is 

an alarming increase by a factor of 52. These data corroborate further observations by Urek & 

Modic 2004, Modic et al. 2006, Hummel et al. 2005, Hummel et al. 2007, Ulrichs et al. 2008. - 

Countrywide survey counts collected by the Agricultural Research Institute of Slovenia (2008) 

strongly support these findings. A small beetle population in 2005 thus dramatically had risen 

by 2007 to 401-644 WCR / trap near Pince in E.Slovenia, to 101-200 WCR / trap both at Novo 

Mesto and Domzale in Central Slovenia and 16-30 WCR / trap at Nova Gorica in Western 

Slovenia, respectively. Thus, both the total number of WCR and also the infested area 

increased steeply between 2003 and today, which makes treatments in E. Slovenia necessary. 

Predictions by Wudtke et al. (2005) of impending human and natural transport of WCR within 

Europe proved to be valid. 

In Romania, several authors found a strong increase of WCR populations and WCR spreading 

eastward between 1998 and 2008 (Vonika 1998, Vilsan & Vonika 2002, Grozea et al. 2008, 

Dinnesen et al. 2009) and attribute this increase mainly to monocultural practices in maize 

production. Grozea et al. (2008) argue the best approach to WCR control in Romania will be 

by biological means because all other options seem to be impractical or economically 

unfeasible under Romanian conditions.

Strategy 

Table 2. Experiences of Diabrotica management in Southern Germany and Switzerland 

chemical 

insecticides and 

crop rotation 

chemical 

insecticides only 

chemical 

insecticides and 

partly crop 

rotation 

crop rotation only 

since 2003 

Country 

or State 

Baden- 

Württemberg 

Baden- 

Württemberg 

Bavaria 

Region results observed tendency 

Lake 

Constance 

area 

Orthenau 

area 

Passau 

Pocking 

Switzerland Ticino 

no further beetle 

captures 2008 

in Rhine valley 

increase of beetles 

by factor of 12.6 

decrease of 

absolute beetle 

numbers, large 

increase of area 

infested 

stagnant WCR 

population, 

reaching stable 

equilibrium? 

full success 2008 

Significant expansion of 

pest 

slight decrease in numbers, 

strong increase in area 

(19 % of Bavarian maize 

cropping) 

exclusive crop rotation 

since 2003: stabilization of 

population below 

economic threshold 

Figure 2. Spreading in Europe of WCR from 1992 to 2008. (Edwards & Kiss 2008, 

modified) 

343

SYNOPSIS 

Attempting a synopsis from a central European perspective, experiences of WCR management 

in Southern Germany and Switzerland are compared in Table 2. Only Switzerland could show 

conclusively that crop rotation without pesticide application works over a 9-year period and 

can produce a stabilized WCR population never reaching the economic threshold. In contrast, 

results from different regions of Germany vary considerably from full success 2008 in the Lake 

Constance area to control failures in Bavaria and the upper Rhine valley, in spite of using the 

same standard protocol approaches prescribed by EU regulations. At this point entomologists 

are at a loss how to explain these variables conclusively. WCR, as the past showed, is a species 

with a rich set of genetic and behavioral resources (Metcalf 1983, Hummel 2003, Miller et al. 

2009) that do not match well with human predictions. Noteworthy is the juxtaposition of 

infestation risk by WCR as calculated by Baufeld & Enzian (2005) and Baufeld (2008) (Fig. 3) 

344 

Figure 3. WCR infestation risk in central-western-Europe due to intensive maize 

cropping, modified after data of Baufeld & Enzian (2005) and Baufeld (2008) 

versus actually observed WCR infestations (Fig. 2). The latter map compiled by Edwards & 

Kiss (2008) includes actual WCR distribution data of all major countries situated in Central 

and SE Europe and shows remarkably close parallels to the calculated data. Evidently, the 

single WCR introduction (Baca 1993) and subsequent ones cited by Miller et al. (2005) and 

Ciosi et al. (2008) within 15 years developed into a contiguous WCR infestation “block”, with

an infestation "belt" projecting westward far into Northern Italy. North of the Alps, a second 

belt is going to establish itself through Austria and Southeastern Germany. The bridgeheads are 

forming right now. A number of WCR advances and spot infestations cover Southern Germany 

and Eastern France and also the Paris area. In the East, the Ukraine, Romania, Bulgaria list 

similar spot advances, as does Central Italy in the South. Eastern Europe is specifically at risk 

(Hummel 2007). 

CONCLUSIONS 

Most Diabrotica experts agree that WCR is now a new and unwanted but permanent member 

of the European insect fauna. 

Current monocultural farming practices invite WCR to proceed and expand. 

In response, a unified action plan of WCR diagnosis for determining its actual status is in 

place, while an action plan for WCR therapy is sorely needed, specifically for Eastern Europe. 

Really unsolved remains the task how to manage WCR on a short, medium and long term basis 

in a sensible, but also economical and sustainable manner (Hummel 2007). 

WCR in Europe apparently is no lighter challenge than in the US. It will require all available 

resources including entomological man-and mindpower for curbing its progress and to arrive at 

a level of constant pest management efforts where neither side, bug or man, in the words of 

S. Forbes (1915), "can claim a final victory". 


Schwarz Foundation kindly supported the experiments conducted between 2004 and 2008. 

Dipl. Ing. agr. L. Colombi, Ticino Phytosanitary Service, Bellinzona, kindly made available 

some of his monitoring data of 2008. 

B. sc. S. Dinnesen and cand. B. sc. T. Nedelev, Berlin, commendably performed field 

experiments in Slovenia 2006 and 2008, and in Romania in 2008. 

Prof. Dr. I. Grozea, Banat's University, Timisoara, kindly provided field space and student 

accomodation facilities in 2008. 

REFERENCES 

Agricultural Research Institute of Slovenia - Fitosanitarna Uprava RS (2008). Zadrževalni 

program koruznega hrošča Diabrotica virgifera Le Conte 2007-2008 URL: 

http://www.furs.si/Diabrotica/Index.asp (date of access 06.02.2009). 

Baca F (1993). New member of the harmful entomofauna of Yugoslavia Diabrotica virgifera 

virgifera LeConte (Coleoptera: Chrysomelidae). IWGO Newsletter XIII (1-2), 21-22 

Baufeld P (2008). Westlicher Maiswurzelbohrer (Diabrotica virgifera virgifera) – Aktuelle 

Situation und Hintergründe. 

http://www.jki.bund.de/nn_1171854/DE/Home/pflanzengesundheit/schadorganismen/a 

ktuelleProbleme/datenblaetter/diabvi__datenblatt.html. (date of access 20.01.2009) 

345

Baufeld P; Enzian S (2005). Maize growing, maize high-risk areas and potential yield losses 

due to Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) damage in 

selected European countries. In: Western Corn Rootworm: Ecology and Management, 

eds S Vidal; U Kuhlmann & C R Edwards, pp. 285-302. CABI Publishing:Wallingford, 

ISBN 0-85199-817-8 

Bertossa M; Derron J; Colombi L; Brunetti R (2001). Update of monitoring data of Diabrotica 

virgifera virgifera LeConte in Switzerland in 2001. In: Proceedings of the VIIIth 

Diabrotica subgroup meeting, ed Veneto Agricoltura XXI IWGO Conference, pp. 

169 -173, Padova, Italy 

Bertossa M; Hummel H E (2008). Experiences with population dynamics of Diabrotica 

virgifera virgifera LeConte in the Swiss canton of Ticino up to 2007. Communications 

in Agricultural and Applied Biologicol Sciences 73 (3), 421-428. 

Bögel C (2008). LfL - Westlicher Maiswurzelbohrer - Eingrenzungsprogramm in 

Niederbayern. http://www.lfl.bayern.de/ips/pflanzengesundheit/32396/index.php (date 

of access 20.01.2009) 

Ciosi M; Miller N J; Kim K S; Giordano R; Estoup A; Guillemaud T (2008). Invasion of 

Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple 

transatlantic introductions with various reductions of genetic diversity. Molecular 

Ecology 17 (August), 3614-3627. doi:10.1111/j.1365-294X.2008.03866.x. 

Dinnesen S; Ulrichs C; Hummel H E; Grozea I; Carabet A; Stef R (2009). Monitoring 

Diabrotica virgifera virgifera LeConte (Col.: Chysomelidae) with different lures and 

traps. (this volume) 

Edwards C R; Kiss J (2008). Diabrotica virgifera virgifera LeConte in Europe 2008 

htttp://www.entm.purdue.edu/WCR/ (date of access 09.02.2009) 

Forbes S A (1915). The Insect, the farmer, the teacher, the citizen and the state. Bull. Ill. State 

Lab., Natural History 

Glas M (2008). First Occurence of Diabrotica virgifera virgifera in Baden-Wurttemberg 

(Germany) - Part 1: Overview of the Measures in 2007 and 2008. Mitteilungen- Julius 

Kühn Institut, 417, 487. 

Grozea I; Carabet A;. Chirita R; Badea A M (2008). Natural enemies in control of invasive 

species Diabrotica v. virgifera from maize crops. Communications in Agricultural and 

Applied Biological Sciences. 73 (3): 501-508. ISSN: 1379-1176 

Guss P L; Tumlinson J H; Sonnet P E; Proveaux A T (1982). Identification of a femaleproduced 

sex pheromone of the western corn rootworm. Journal of Chemical Ecology 8 

(2), 545-556. 

Hummel H E (1989). Natural products as biotechnical weapons towards the future pest 

management of Diabrotica beetles. Mededelingen Faculteit Landbouwwetenschapen 

Rijksuniversiteit Gent 54 (3a), 945-954. 

Hummel H E (2003). Introduction of Diabrotica virgifera virgifera into the old world and its 

consequences: a recently acquired invasive alien pest species on Zea mays from North 

America. Communications in Agricultural and Applied Biologicol Sciences 68 (4a), 

45-57. 

Hummel H E (2007). Diabrotica virgifera virgifera LeConte: Inconspicuous leaf beetle- 

Formidable Challenge to Agriculture. Communications in Agricultural and Applied 

Biologicol Sciences 73 (2), 7-32. 

346

Hummel H E; Bertossa M; Hein D F; Wudtke A; Urek G; Modic S; Ulrichs Ch (2005a). The 

western corn rootworm Diabrotica virgifera virgifera en route to Germany. 

Communications in Agricultural and Applied Biologicol Sciences 70 (4), 677-686. 

Hummel H E; Shaw J T; Hein D F (2005b). A promising biotechnical approach to pest 

management of Diabrotica virgifera virgifera in Illinois maize fields under kairomonal 

shielding with the new MSD technique. Communications in Agricultural and Applied 

Biologicol Sciences 70 (4), 625-32. 

Hummel H E; Urek G; Modic S; Hein D F (2005c). Monitoring presence and advance of the 

alien invasive western corn rootworm beetle in eastern Slovenia with highly sensitive 

Metcalf traps. Communications in Agricultural and Applied Biologicol Sciences 70 (4), 

687. 

Hummel H E; Modic S; Urek G (2006). Diabrotica virgifera virgifera in eastern Slovenia: 

increasing population trend 2005. Communications in Agricultural and Applied 

Biologicol Sciences 71 (2b), 571-577. 

Hummel H E; Urek G; Modic S; Dinnesen S; Nedelev T; Ulrichs C (2007). Diabrotica 

virgifera virgifera in confrontation mood: current geographical and host spectrum 

expansion in Europe. In: Best Practice in Disease, Pest and Weed Management. 

Symposium Proc., eds D V Alford, F Feldmann, J Hasler & A von Tiedemann: Berlin 

Hummel H E; Dinnesen S; Nedelev T; Modic S; Urek G; Ulrichs C (2008). Diabrotica 

virgifera virgifera LeConte in confrontation mood: simultaneous geographical and host 

spectrum expansion in southeastern Slovenia. Mitteilungen Deutsche Gesellschaft für 

Allgemeine Angewandte Entomologie 15: 127-130. 

Hummel H E; Deuker A; Leithold G (2009). The leaf beetle Diabrotica virgifera virgifera 

LeConte: a merciless entomological challenge for agriculture. International 

Organisation Biological Control /wprs Bulletin 41, 103-110. 

Levine E; Metcalf R L (1988). Sticky attractant traps for monitoring corn rootworm beetles. 

The Illinois Natural History Survey Reports 279, 1-2. 

Metcalf R L (1986). Foreword. In: Methods for the study of pest Diabrotica. eds J L Krysan & 

T A Miller, pp. vii-xv. Springer: New York. 

Metcalf R L; Lampman R L (1997). Plant kairomones and kairomone mimetics in basic and 

applied research with diabroticite rootworms. Trends in Entomology 1, 49-62. 

Metcalf R L (1983). Implications and prognosis of resistance to insecticides. In: Pest 

Resistance to Pesticides, eds G P Georghiou & T Saito, pp. 703–733. Plenum Press: 

New York. 

Metcalf R L; Metcalf E R (1992). Plant Kairomones in Insect Ecology and Control. Springer: 

New York. 

Miller N; Estoup A; Toepfer S; Bourguet D; Lapchin L; Derridj S; Kim K S; Reynaud P; Furlan 

L; Guillemaud T (2005). Multiple Transatlantic Introductions of the Western Corn 

Rootworm. Science 310 (5750) (November 11), 992. doi:10.1126/science.1115871. 

Miller N; Guillemaud T; Giordano R; Siegfried B D; Gray M E; Meinke L J; Sappington T W 

(2009) Genes, geneflow and adaptation of Diabrotica virgifera virgifera. Agricultural 

Forest Entomology 11, 47-60 

Modic S; Knapic M; Cergan Z; Urek G (2006). Spread and population dynamics of western 

corn rootworm Diabrotica v. virgifera LeConte, in Slovenia. In: Proc. IWGO 22nd 

Conference Vienna, Austria. November 5. 

347

Ulrichs C; Dinnesen S; Nedelev T; Hummel H E; Modic S; Urek G (2008). Monitoring 

Diabrotica v. virgifera (Col.: Chrysomelidae) in southern Slovenia: increasing 

population trend and host spectrum expansion. Communications in Agricultural and 

Applied Biological Sciences 73 (3), 493-499 

Urek G; Modic S (2004). Occurrence of the western Corn Rootworm (Diabrotica virgifera 

virgifera Le Conte) in Slovenia. Acta Agriculturae Slovenica 83 (1), 5-13. 

Vilsan D; Vonica I (2002). 1998 Results of Monitoring Diabrotica virgifera virgifera LeConte 

in Romania. Acta Phytopathologica et Entomologica Hungarica 37 (1), 175-182. 

Vonica I (1998). Auftreten und Verbreitung von Diabrotica virgifera virgifera Le Conte in 

Rumanien (The occurrence and dissemination of Diabrotica virgifera virgifera LeConte 

populations in Romania). Pflanzenschutzberichte 57, 3-14 

Wudtke A; Hummel H E; Ulrichs C (2005). Der Westliche Maiswurzelbohrer Diabrotica 

virgifera virgifera LeConte (Col.: Chrysomelidae) auf dem Weg nach Deutschland. 

Gesunde Pflanzen 57 (4), 73-80. 

348

Gaafar N, Cöster H, Volkmar C: Evaluation OF Ear Infestation by Thrips And Wheat Blossom Midges in Winter 

Wheat Cultivars. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

6-3 Evaluation of Ear Infestation by Thrips and Wheat Blossom Midges in 

Winter Wheat Cultivars 

Gaafar N 1 , Cöster H 2 , Volkmar C 1 

1 

Institute of Agric. & Nutritional Sciences, Martin-Luther-University Halle-Wittenberg 

2 RAGT 2n, Steinesche 5a, D-38855 Silstedt 

ABSTRACT 

Infestations of thrips and wheat blossom midges (WBM) in the ears of wheat were 

studied in two research fields (Halle & Silstedt) in central Germany in 2008. Ninety 

cultivars were evaluated at the university of Halle-Wittenberg, and 20 cultivars at 

the plant breeding station in Silstedt, including some resistant cultivars against 

WBM. Infestation levels were studied in early milk stage (GS 73). The infestation 

percentages of thrips and WBM were investigated in every single-spikelet in sample 

of 10 ears in the studied cultivars and sites. 

There were significant differences in thrips and WBM among cultivars in both 

Halle and Silstedt. Numbers of thrips were higher in cultivars Türkis and Welford in 

Halle and Türkis and Anthus in Silstedt, while thrips were the lowest in cultivars 

Potenzial and Boomer in Halle and Robigus and Potenzial in Silstedt. WBM 

numbers were higher in cultivars Tommi and Potenzial in Halle and Türkis and 

Dekan in Silstedt, while the least WBM numbers were observed in cultivars Anthus, 

Welford and Robigus in both Halle and Silstedt. The ears infested were 

significantly positively correlated with wheat midge numbers among cultivars and 

in both sites. Finally, the results give a first indication for choosing the best 

cultivar(s) as an efficient method of integrated plant protection. 

INTRODUCTION 

Wheat (Triticum spp.) is a worldwide cultivated cereal crop over the world. Globally, wheat is 

most produced food among the cereal crops (Cauvain & Cauvain 2003). Wheat grain is a staple 

food used to make flour for leavened, flat and steamed breads, pasta, noodles and couscous. 

Wheat is planted to a limited extent as a forage crop for livestock, and the straw can be used as 

fodder for livestock (USDA 2007). Wheat is attcked by wheat midges and thrips. 

349

The orange wheat blossom midge Sitodiplosis mosellana is a significant pest of wheat in 

Europe, North America, Russia, Japan, and China (Lamb et al. 2003). In Europe, it coexists 

with another gall midge that attacks the wheat head, the lemon wheat blossom midge, 

Contarinia tritici (Berzonsky et al. 2002; Harris et al. 2003). The wheat midge is a periodic 

pest of wheat crop and occasionally inflicts severe damage, particularly where a sequence of 

seasons favouring the midges triggers an outbreak. Severe outbreaks occur occasionally over 

the world; in Canada (Barker et al. 1995), China (Gao 1995), Finland (Kurppa 1989), Japan 

(Katayama et al. 1987), Germany (Basedow 1975, 1977; Volkmar & Wetzel 1989; Volkmar et 

al. 2008) and UK (Oakley 1981 1994a). Consequently, there have been few opportunities for 

research on this pest during outbreak years, especially as the initiation of an outbreak period is 

difficult to forecast because of the pest's life history. The midge larvae hibernate in the soil and 

each spring a proportion develop and pupate. Each year’s cohort of midge larvae pupate and 

produce adults over a spread of years, the proportion doing so in any given year depending on 

soil temperatures and moisture (Lamb et al. 2003). Wheat midge host selection occurs when 

the mated female (Pivnick & Labbé 1993). Oviposition typically occurs in the days preceeding 

anthesis as the wheat head emerges from the flag leaf and before pollination occurs. Eggs are 

laid 1 to 2 at a time, about 60–80 eggs per female, often on the inner surfaces of the leaf-like 

glumes that enclose the florets (Waiters 1993; Lunn et al. 1995; Oakley et al. 1998; Smith & 

Lamb 2001; Olfert et al. 2009). 

The thrips fauna on wheat crop caused a serious damage, and methods of control are not 

sufficiently investigated. Thrips species Haplothrips tritici, H. aculeatus, Limothrips 

denticornis, Frankliniella tenuicornis and Thrips angusticeps were recorded on different wheat 

cultivars (Andjus 1996; Moritz 2006). Thrips feeding on the ovaries of tender wheat leads to 

distortions, degenerations, grains sometimes aborting. This has considerable consequences on 

yield as well as on the baking quality of flour (Kucharzyk 1998). 

During the severe infestation of thrips and orange wheat blossom midge experienced in the UK 

in 1993, half of the national wheat crop suffered physical damage to more than 5% of the 

harvested grain, 21% of crops were damaged to such an extent that a spray treatment to control 

wheat blossom midge would have been cost effective, had the problem been identified in time 

for application (Oakley 1994b). Wheat thrips and WBM may decrease wheat yield and grain 

quality (Olfert et al. 1985; Oakley et al. 1993; Oakley 1994a) but the widespread use of broad 

spectrum insecticides used to control midge numbers in winter wheat has also highlighted 

potentially damaging environmental impacts and natural enemies (Dexter et al. 1987; Elliott & 

Mann 1997; Elliott 1998). Attack by the wheat midge is associated with a reduced proportion 

of well-formed wheat seeds (Glen 2000; Lamb et al. 2003; Doane & Olfert 2008). As well as 

yield losses, thrips or wheat midge adversely affects grain quality and important agronomic 

characters (Helenius & Kurppa 1989). 

Due to the potentially high economic and environmental costs of an inappropriate control 

strategy being adopted in response to the outbreak, the development of evaluating ear insects in 

wheat fields were conducted. The subsequent effect on wheat thrips and midge populations and 

350

grain quality were determined by dissecting wheat ears during the susceptible growth stages 

(GS 73) in two fields (research fields (Halle and Silstedt)) in 2008. 


Monitoring sites 

Two sites were selected for detailed study in 2008. The sites were chosen to cover the infested 

area of central Germany. At each site a crop of wheat was monitored to establish the actual 

level of damage caused by wheat midges (S. mosellana and C. tritici) and wheat thrips. Twenty 

cultivars were sown in plant breeding station in Silstedt (sandy loam), while ninety wheat 

cultivars were sown in Julius Kühn field in Halle (sandy loam); each plot was 8m x 1.5m 

(12m 2 ). Eight cultivars were chosen for comparison between Halle and Silstedt. These cultivars 

are Tommi, Türkis, Anthus, Potenzial, Dekan, Boomer Welford and Robigus. The later two are 

resistant cultivars. 

Ear insect’s evaluation 

Numbers of thrips species (Limothrips denticornis and Thrips angusticeps), and wheat midge 

larvae (S. mosellana and C. tritici) were assessed by collecting 10 ears per plot in June at 

approximately GS 73 (Tottman 1987) in 2008, before any of the larvae had matured and left 

the ears. These samples were frozen at -20°C; then thrips and midges were counted after field 

work had finished. The ears were dissected under a low power microscope in the laboratory 

recording the number of grains and the numbers of larvae present on each and number of grains 

infested per ear was recorded from both fields. Data were analyzed by linear model (analysis of 

variance (ANOVA)) using Statistix 8 (Thomas & Maurice 2008), then following with Tukey 

test to compare means of cultivars. Significant differences were noted at P < 0.05 for all trials. 

Effects of thrips and midge larvae were recorded for shrivel, crack, and deform on kernels in 

ears. The relationship between among numbers of thrips and midge larvae per ear was 

correlated with infested kernels in both sites and different cultivars using linear model 

(Correlation coefficient (Pearson)) by Statistix 8 program. Test produces a value that ranges 

from -1 for total disagreement between rankings to 1 for total concordance. 

RESULTS 

Halle site 

There was significant difference (p < 0.046) in the number of thrips adults per ear among 

cultivars. Tommi and Welford cultivars had the highest numbers of thrips adults 7.6 and 8.2/ 

ear, respectively, then followed by Türkis (6/ ear), Anthus, Robigus, Potenzial and Boomer 

(4.8, 4.8 and 3.4/ ear), lastly, Dekan (1.8/ ear) (Fig. 1). 

There was significant difference (p < 0.049) in the number of thrips larvae per ear among 

cultivars. Türkis and Welford cultivars had the highest numbers of thrips larvae 28.0 and 26.4/ 

351

ear, respectively, then followed by Tommi (20.6/ ear), Anthus, Robigus, Dekan and Boomer 

(18.2, 18.6, 18.6 and 17.4/ ear), lastly, Potenzial (12.6/ ear) (Fig. 1). 

There was significant difference (p < 0.027) in the number of total thrips per ear among 

cultivars. Türkis and Welford cultivars had the highest numbers of total thrips 32.0 and 34.6/ 

ear, respectively, then followed by Tommi (28.4/ ear), Anthus, Robigus, Dekan and Boomer 

(23.0, 23.4, 20.4 and 20.8/ ear), lastly, Potenzial (17.4/ ear) (Fig. 1). 

There was significant difference (p < 0.045) in the number of wheat midges larvae per ear 

among cultivars. Tommi and Potenzial cultivars had the highest numbers of WBM larvae 1.4 

and 1.2/ ear, respectively, then followed by Dekan (0.8/ ear), Robigus, and Boomer (0.2/ ear) 

lastly, Turkis, Anthus and Welford (0/ ear) (Fig 1). 

There was significant difference (p < 0.032) in the infested kernels resulted from thrips or 

wheat midges among cultivars. Tommi and Potenzial cultivars had the highest infested kernels 

0.4 and 1.0/ ear, respectively, then followed by Dekan, Robigus, and Boomer (0.2/ ear) lastly, 

Turkis, Anthus and Welford (0/ ear) (Fig. 1). There is a correlation between WBM infestation 

and infested kernels (R= +0.79), while no correlation between thrips and infested kernels (R= 

+0.02, -0.26 and -0.22) in adults, larvae and total thrips, respectively (Table 1). 

352 

Table 1. Correlation coefficient between ear insects (thrips & wheat midges) and 

infested kernels in Halle and Silstedt 

Sites Thrips adults Thrips larvae Total thrips Wheat midges 

Halle +0.02 -0.26 -0.22 +0.79 * 

Silstedt +0.12 +0.17 +0.16 +0.94 ** 

Silstedt site 

* Significant differences 

There was significant difference (p < 0.009) in the number of thrips adults per ear among 

cultivars. Türkis and Anthus cultivars had the highest numbers of thrips adults 5.5 and 5.1/ ear, 

respectively, then followed by Boomer, Tommi and Dekan (3.9, 2.6 and 2.4/ ear), lastly, 

Welford, Robigus and Potenzial (2.0, 1.9 and 1.8/ ear) (Fig. 2). 

There was significant difference (p < 0.0019) in the number of thrips larvae per ear among 

cultivars. Türkis and Anthus cultivars had the highest numbers of thrips larvae 12.2 and 12.1/ 

ear, respectively, then followed by Tommi and Boomer (6.7 and 6.4/ ear), Dekan, Welford and 

Potenzial (4.6, 4.0 and 3.5/ ear), lastly, Robigus (1.3/ ear) (Fig. 2). 

There was significant difference (p < 0.0003) in the number of total thrips per ear among 

cultivars. Türkis and Anthus cultivars had the highest numbers of total thrips 17.7 and 17.2/ 

ear, respectively, then followed by Tommi and Boomer (11.1 and 10.3/ ear), Dekan, Welford 

and Potenzial (7.0, 6.0 and 5.3/ ear), lastly, Robigus (3.2/ ear) (Fig. 2).

Thrips adults 

Thrips larvae 

Total thrips 

Wheat midges larvae 

10 

8 

6 

4 

2 

A 

B 

A 

C C C 

D 

Thrips adults 

0 

30 A 

A 

Thrips larvae 

25 

20 

B 

B 

B B 

B 

15 

10 

5 

0 

C 

40 

30 

20 

10 

0 

2.0 

1.5 

1.0 

0.5 

0.0 

Tommi 

AB 

A 

Türkis 

A 

Anthus 

B 

A 

WBM larvae 

Infested kernels 

Welford 

Robigus 

B 

D 

C 

C 

Total thrips 

Infested kernals 

Figure 1. Mean of thrips adults, larvae, and total thrips, wheat midge larvae and the 

relation to infested kernels in different winter wheat cultivars (growth stage 

73) in Halle 2008 (Different letters and colors indicate significant differences. 

C 

A 

Potenzial 

Dekan 

B 

Boomer 

C 

C 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 



353

There was significant difference (p < 0.000) in the number of wheat midges larvae per ear 

among cultivars. Türkis and Dekan cultivars had the highest numbers of WBM larvae 15.5 and 

16.0/ ear, respectively, then followed by Tommi, Potenzial and Boomer (12.4, 10.7& 10.4/ 

ear), Anthus (4.3/ ear), lastly, Welford and Robigus (2.3& 1.5/ ear). The later are 2 resistant 

cultivars (Fig. 2). 

There was significant difference (p < 0.000) in the infested kernels resulted from thrips or 

wheat midges among cultivars. Türkis, Tommi and Dekan cultivars had the highest infested 

kernels 8.6, 7.8 and 7.8/ ear, respectively, then followed by Potenzial and Boomer (6.6 and 6.7/ 

ear), Anthus (3.2 ear/ ear) lastly, Welford and Robigus (0.9 and 1.0/ ear) (Fig. 2). There is a 

correlation between WBM infestation and infested kernels (R= +0.94), while no correlation 

between thrips and infested kernels (R= +0.12, +0.17 and +0.16) in adults, larvae and total 

thrips, respectively (Table 1). 

Comparison between Halle and Silstedt 

There were significant differences in thrips and WBM among cultivars in both sites Halle and 

Silstedt. Numbers of thrips adults were higher significantly (P < 0.000) in cultivars Tommi and 

Welford in Halle and Türkis and Anthus in Silstedt, while the least thrips' adults numbers were 

recorded in cultivars Dekan and Boomer in Halle and Robigus and Potenzial in Silstedt. Thrips 

larvae and total thrips were significantly higher (P < 0.000) in cultivars Türkis and Welford in 

Halle and Türkis and Anthus in Silstedt. The least thrips populations were recorded in cultivars 

Potenzial and Boomer in Halle and Robigus and Potenzial in Silstedt (Fig. 3). 

There were significantly differences in the midge larvae. Their numbers were higher 

significantly (P < 0.000) in cultivars Tommi and Potenzial in Halle and Türkis and Dekan in 

Silstedt, while the least WBM numbers were observed in cultivars Anthus, Welford and 

Robigus in Halle and Silstedt. Welford and Robigus cultivars are resistant cultivars in both 

sites (Fig. 3). 

The proportion of ears infested with wheat midges also differed significantly (P < 0.000) 

among cultivars and between both fields. The number of midge larvae per ear was significantly 

positively correlated (P < 0.005, r 2 = 0.96) with the percentage of ears infested. There was a 

not significant correlation between the number of thrips and infested kernels (Fig. 3). 

DISCUSSION 

There were significant differences in thrips and WBM among cultivars in both Halle and 

Silstedt. Numbers of thrips were higher in cultivars Türkis and Welford in Halle and Türkis 

and Anthus in Silstedt. The least thrips populations were recorded in cultivars Potenzial and 

Boomer in Halle and Robigus and Potenzial in Silstedt. This result is similar with Basedow 

(1977) and Volkmar et al. (2008), who studied some wheat cultivar in Germany for their 

susceptibility of wheat midges. 

354

Thrips adults 

Thrips larvae 

Total thrips 


6 

4 

2 

0 

14 

12 

10 

8 

6 

4 

2 

0 

20 

15 

10 

5 

0 

20 

15 

10 

5 

0 

Tommi 

B 

B 

B 

Türkis 

A 

Anthus 

A 

A A 

A 

A A 

C 

Welford 

Robigus 

Thrips adults 

C C C 

WBM larvae 


D D 

Thrips larvae 

B B 

C 

Total thrips 


C C 

Figure 2. Mean of thrips adults, larvae, and total thrips, wheat midge larvae and the 

relation to infested kernels in different winter wheat cultivars (growth stage 

73) in Silstedt 2008 (Different letters and colors indicate significant 

differences). 

D 

D 

C 

B 

Potenzial 

Dekan 

C 

C 

C 

A 

Boomer 

B 

B 

B 

10 

8 

6 

4 

2 

0 

10 

8 

6 

4 

2 

0 



355

Thrips adults 

Thrips larvae 

Total thrips 


356 

10 

0 

30 

25 

20 

15 

10 

0 

40 

30 

20 

10 

20 

15 

10 

5 

0 

8 

6 

4 

2 

5 

0 

A 

AB 

AB 

C 

Tommi 

C 

C 

CD 

AB 

A 

A 

Türkis 

AB AB AB AB 

BC 

C 

A 

AB 

B 

Anthus 

BC 

C 

BC 

A 

A 

A 

Halle 

C 

DE 

DE 

B 

AB 

B 

C 

E 

E 

WBM larvae Halle 

WBM larvae Silstedt 

Infested kernels Halle 

Infested kernels Silstedt 

Welford 

Robigus 

B 

B 

BC 

E 

Potenzial 

Silstedt 

C C C 

E 

AB 

B 

Dekan 

DE 

DE 

BC BC 

B 

B 

Boomer 

CD 

D 

AB AB 

C C 

C C C C C C C 

Figure 3 Comparison between Halle and Silsted in mean of thrips adults, larvae, and 

total thrips, WBM larvae and the relation to infested kernels in different winter 

wheat cultivars (growth stage 73) in 2008 (Different letters and colors indicate 

significant differences). 

A 

8 

6 

4 

2 

0 

10 

8 

6 

4 

2 

0 

10 


infested kernels

There were significantly differences in the midge larvae. Their numbers were higher in 

cultivars Tommi and Potenzial in Halle and Türkis and Dekan in Silstedt, while the least WBM 

numbers were observed in cultivars Anthus, Welford and Robigus in both Halle and Silstedt. 

The ears infested were significantly positively correlated with wheat midge’s numbers among 

cultivars and between both sites. A strong correlation was found between ears infested and 

number of midge per ear, same results were also reported by Olfert et al. (1985) and Smith & 

Lamb (2001), who mentioned that such a strong correlation was expected because midges not 

prefer to oviposit in wheat ears that are already infested. 

In conclusion, to minimize the economic and ecological impact of S. mosellana and thrips, 

wheat producers must be aware with monitoring tools. There were more thrips or midges and 

the infested kernels in some cultivars than others in two sites. Some wheat cultivars also have 

evolved a defense mechanism that deters oviposition by the wheat midge as mentioned by 

Berzonsky et al. (2002). These discrepancies may have been a result of speed ripening time as 

reported by Elliott et al. (2000). The wheat midge has evolved preferences for ovipositing at 

particular developmental stages of its host. This may have been sufficient to make some 

cultivars less favourable for oviposition, such these cultivars are recommended to cultivate in 

next year. If a lower degree of infestation is predicted, producers may stick to their plans to 

grow wheat, but may choose a less susceptible wheat cultivar and plant early to avoid large 

populations of midges during heading. 

AKNOWLEDGEMENT 

We thank Dr. Nabil El-Wakeil for his help in statistical analysis and useful comments on this 

manuscript. 

REFERENCES 

Andjus L (1996). The research into the Thrips fauna and significance of the plants of 

spontaneous flora for the survival of pest species. Ph.D Thesis, Belgrade University. 

Barker P S; McKenzie R I H; Czarnecki E (1995). Incidence of damage to spring wheat by the 

orange wheat blossom midge in Manitoba during 1993. Proceedings of Entomological 

Society of Manitoba 51, 12-20. 

Basedow T (1975). Predaceous arthropods in agriculture, their influence upon the insect pests 

and how to spare them while using insecticides. Semaine d'etude Agriculture et Hygiene 

des Plantes 1975, 311-323. 

Basedow T (1977). The susceptibility of some spring wheat varieties to the attack by the two 

wheat blossom midge species. Anz. Schadlingskde., Pflanzenschutz Umweltschutz 50, 

129-131. 

Berzonsky W; Shanower T; Lamb R; McKenzie R; Ding H (2002). Breeding wheat for 

resistance to insects. Plant Breeding Review 22, 221–297. 

Cauvain S P; Cauvain C P (2003). Bread Making. CRC Press. p. 540. ISBN 1-85573-553-9. 

Doane J F; Olfert O (2008). Seasonal development of wheat midge, Sitodiplosis mosellana, in 

Saskatchewan, Canada. Crop Protection 27, 951–958. 

357

Doane J F; Mukerji M K; Olfert O (2000). Sampling distribution and sequential sampling for 

subterranean stages of orange wheat blossom midge, Sitodiplosis mosellana (Géhin) in 

spring wheat. Crop Protection 19, 427-434. 

Dexter J E; Preston K R; Cooke L A; Morgan B C; Kruger J E; Kilborn R H; Elliott R H 

(1987). The influence of orange wheat blossom midge Sitodiplosis mosellana (Géhin) 

damage on hard red spring wheat quality and the effectiveness of insecticide treatments. 

Canadian Journal Plant Science 67, 697-712. 

Elliott R H (1998). Evaluation of insecticides for protection of wheat against damage by the 

wheat midge, Sitodiplosis mosellana (Géhin). Canadian Entomologist 120, 615–626. 

Elliott R H; Mann L M (1997). Control of wheat midge, Sitodiplosis mosellana (Géhin), at 

lower chemical rates with small capacity sprayer nozzles. Crop Protection 16, 235-242. 

Elliott R H; Mann L; Olfert O (2000). Susceptibility of hard red spring wheats to damage by 

high populations of wheat midge. SRC-Saskatoon Research Letter 2000–09, 3 pp. 

Glen D M (2000). The effects of cultural measures on cereal pests and their role in integrated 

pest management. Integrated Pest Management Reviews 5, 25–40. 

Gao Y J (1995). Chemical control of the wheat blossom midge Sitodiplosis mosellana Géhin. 

Bulletin of Agricultural Sciences and Technology 8, 22. 

Harris M O; Stuart J J; Mohan M; Nair S; Lamb R J; Rohfritsch O (2003). Grasses and 

gallmidges: Plant defense and insect adaptation Annual Review Entomology 48, 

549-577. 

Helenius J; Kurppa S (1989). Quality losses in wheat caused by orange wheat blossom midge 

Sitodiplosis mosellana. Annals Applied Biology 114, 409-417. 

Katayama J; Fukui M; Sasaki H (1987). Seasonal prevalence of adult occurrence and 

infestation of the wheat blossom midge Sitodiplosis mosellana Géhin in Kyoto 

Prefecture. Japanese Journal of Applied Entomology and Zoology 31, 46-50. 

Kucharzyk H (1998). Thysanoptera and other insects collected in differently coloured traps in 

eastern Poland. Proceedings Sixth International Symposium on Thysanoptera. PP, 

81-87. 

Kurppa S L A (1989). Wheat blossom midges, Sitodiplosis mosellana Géhin and Contarinia 

tritici (Kirby) in Finland during 1981 -87. Annales Agriculture Fenniae 28, 87-96. 

Lamb R J; Sridhar P; Smith M A H; Wise I L (2003). Oviposition preference and offspring 

performance of a wheat midge Sitodiplosis mosellana on defended and less well 

defended wheat plants. Environmental Entomology 32, 414–420. 

Lunn G D; Scott R K; Kettlewell P S; Major B J (1995). Effects of orange wheat blossom 

midge (Sitodiplosis mosellana) infection on pre-maturity sprouting and Hagberg falling 

number of wheat. Aspects of Applied Biology 42, 355-358. 

Moritz G (2006). Thripse. Die Neue Brehm- Bücherei, Bd. 663, p. 384. 

Oakley J N (1981). Wheat Blossom Midges. Leaflet 788. London: Ministry of Agriculture, 

Fisheries and Food. 

Oakley J N (1994a). Impact of CAP reforms on decision making for pest control in cereals. 

Aspects of Applied Biology 40, Arable Farming under CAP Reform, pp. 183-192. 

Oakley J N (1994b). Orange wheat blossom midge - a literature review and survey of the 1993 

outbreak. Research Review No. 28, Home-Grown Cereals Authority London, 51 pp. 

Oakley J N; Ellis S A; Walters K F A; Watling M (1993). The effects of cereal aphid feeding 

on wheat quality. Aspects of Applied Biology 36, Cereal Quality III, pp. 383-390. 

358

Oakley J N; Cumbleton P C; Corbett S J; Saunders P; Green D I; Young J E B; Rodgers R 

(1998). Prediction of orange wheat blossom midge activity and risk of damage. Crop 

Protection 17, 145-149. 

Olfert O; Elliott R H; Hartley S (2009). Non-native insects in agriculture: strategies to manage 

the economic and environmental impact of wheat midge, Sitodiplosis mosellana, in 

Saskatchewan. Biological Invasions 11, 127–133. 

Olfert O O; Mukerji M K; Doane J F (1985). Relationship between infestation levels and yield 

loss caused by wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), 

in spring wheat in Saskatchewan. Canadian Entomologist 117, 593-598. 

Pivnick K A; Labbé E (1993). Daily patterns of activity of females of the orange wheat 

blossom midge Sitodiplosis mosellana Géhin. Canadian Entomologist 125, 125-736. 

Smith M; Lamb R (2001). Factors influencing oviposition by Sitodiplosis mosellana (Diptera: 

Cecidomyiidae) on wheat spikes. Canadian Entomologist 133, 533–548. 

Thomas C R; Maurice S C (2008). Statistix 8, Ninth Edition, Managerial Economics McGraw- 

Hill/Irwin. (ISBN: 0073402818). More information at http://www.statistix.com 

Tottman D R (1987). The decimal code for the growth stages of cereals, with illustrations. 

Annals Applied Biology 110, 441-454. 

USDA – United States Department of Agriculture (2007). Annual World Production Summary, 

Grains, http://www.usda.gov/, retrieved on 4 September 2007. 

Volkmar C; Wetzel T (1989). Zum Auftreten und zur Bekämpfung von Ährenschädlingen des 

Winterweizens unter Praxisbedingungen. Nachrichtenblatt Pflanzenschutz DDR 43, 

14-17. 

Volkmar C; Werner C; Matthes P (2008). On the occurrence and crop damage of wheat 

blossom midges (Contarinia tritici (Kby.) and Sitodiplosis mosellana (Geh.) in Saxony- 

Anhalt. Mitteilungen Deutsche Gesellschaft Allgemeine & Angewandte Entomologie 

16, 305-308. 

Waiters K F A (1993). Orange wheat blossom midge: the scourge of 1993? Biologist 40, 215. 

359

Mewis I, Rohr F, Tokuhisa J G, Gershenzon J, Ulrichs C: Varying Glucosinolate Profiles in Arabidopsis Influence 

Plant Defense Against Generalist and Specialist Caterpillars Differently. In: Feldmann F, Alford D V, Furk C: 



6-4 Varying Glucosinolate Profiles in Arabidopsis Influence Plant Defense 

Against Generalist and Specialist Caterpillars Differently 

Mewis I 1 , Rohr F 1 , Tokuhisa J G 2 , Gershenzon J 3 , Ulrichs C 1 

1 

Humboldt Universität zu Berlin, Institute for Horticultural Science, Urban Plant Ecophysiology, 

Lentzeallee 55, 14195 Berlin, Germany 

2 

Virginia Tech University, Horticulture Department, Blacksburg, VA 24061, USA 

3 Max Planck Institut for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany 

360 

ABSTRACT 

Glucosinolates (GS) are characteristic secondary defense compounds in 

Brassicaceae and other families in the order Brassicales. To date more than 120 

different GS are described sharing a common chemical core structure with a varying 

side chain. Depending on the chemical nature of the side chain they are classified as 

aliphatic, aromatic or indolyl GS. The GS-myrosinase system is an effective 

defense system especially against generalist insects, pathogens, and herbivores 

The aliphatic GS profiles of the model plant Arabidopsis thaliana and other 

members of Brassicaceae are highly variable, but indolyl GS are widely distributed 

in this plant family. Indeed studies are missing to discover the function of GS 

classes and different GS side chains within the plant resistance against insects. To 

study the effect of GS classes on different insects, the host plant suitability of two 

A. thaliana mutants with altered GS profile compared with Columbia wild type (Col 

WT) for three Lepidoptera species was tested. The performance of the generalist 

caterpillar Spodoptera exigua was better on mam3 + , lower aliphatic GS content, 

followed by cyp79B2 - cyp79B3 - , absence of indolyl GS, when compared with Col 

WT. No significant differences within weight gain of larvae on genotypes was 

found for the specialist lepidopteran Pieris rapae and P. brassicae The impact of 

different GS within plant defense response is discussed.

INTRODUCTION 

Glucosinolates (GS) are characteristic secondary metabolites present in the plant family 

Brassicaceae and other families of the order Brassicales (Halkier & Gershenzon 2006). To date 

more than 120 different GS are described, which share a common core structure with variable 

side chain (Fahey et al. 2001). Three major classes of GS are distinguished: aliphatic GS which 

derive principally from methionine, indolyl GS which derive from tryptophan, and aromatic 

GS which mostly derive from phenylalanine. All GS containing plants store in different 

compartments and cells GS hydrolyzing enzymes, so called myrosinases (Koroleva et al. 

2000). After tissue damage, e. g. after herbivory, the actual biologically active compounds such 

as isothiocyanates and nitriles are released (Rask et al. 2000). The GS-myrosinase system 

comprises an efficient defense especially against generalist insects, pathogens, and bacteria 

(Halkier & Gershenzon 2006). But many specialists are using these compounds in host plant 

recognition (Renwick 2002). 

The molecular model plant Arabidopsis thaliana belongs to the Brassicaceae and contains GS 

as defense compounds against herbivory. Aliphatic GS profiles of A. thaliana and Brassica 

species are highly variable, but indolyl GS are widely distributed in this plants (Kliebenstein 

2001; Li & Quiros 2002). Indeed studies are missing to discover the function of GS classes and 

different GS side chains within the plant resistance against insects. To study the effect of GS 

classes on insects with different host plant specialization, the host plant suitability of two 

A. thaliana mutants with altered GS profile, reduced aliphatic GS or absence of indolyl GS, 

was compared with Columbia wild type (WT). Three Lepidoptera species were used in this 

study, two crucifer specialist pests: Pieris rapae and Pieris brassicae and one generalist pest 

Spodoptera exigua. 


Arabidopsis genotypes 

Lines (three each) of two different A. thaliana mutants with modified GS profiles and the 

corresponding Columbia wild-type (WT) were used for the bioassays with caterpillars. The 

first mutant was characterized by the double knock-out of CYP79B2 and CYP79B3 (cyp79B2 - 

/cyp79B3 - , a gift from J. Chory, The Salk Institute for Biological Studies, California). The 

construction of the mutant can be reviewed in Zhao et al. (2002). The second plant mutant lines 

are characterized by over expression of MAM3 (mam3 + ) and were generated as described in 

Textor et al. (2007). 

Insect bioassays 

To study the impact of different GS profiles in A. thaliana genotypes within the plant defense 

against different specialist insect herbivores, 3 rd instars of the lepidopterans P. rapae, 

P. brassicae and S. exigua were used. Larvae were pre adapted on the respective genotype 

(cyp79B2 - /cyp79B3 - , mam3 + , and Col WT) and were forced to feed on the plants for four days. 

361

Initial larval weight was determined before release on the plants, one larva per plant (ten 

replications per genotype and caterpillar species). Pots were covered with small cages made 

from transparent plastic cylinders and fine mesh gaze. Weight increase was determined after 

three days feeding on the three genotypes. The experimental plants were kept in a climate 

chamber at 22 ± 1°C, with a 12 hours light period and at 200 µmol m -2 s -1 light intensity. 

Chemical analysis 

At the end of the experiments plants were about 40 days old. Whole plants were cut and flashfrozen 

in liquid nitrogen Plant samples were freeze-dried and 20 mg were extracted in 70% 

methanol following the procedure described by Mewis et al. (2005). Extraction was done in 

five replications for each treatment and genotype. To quantify GS content, an internal standard 

p-hydroxybenzyl GS was added initially to the first methanol extract. The GS of extracts were 

analyzed as desulfo GS and for this purpose the extracts were desulfated on DEAE Sephadex 

A-25 mini columns with aryl sulfatase solution (Mewis et al. 2005). The GS amount was 

calculated from HPLC peak areas using response factors of desulfo GS at 229 nm. 


Data from chemical analysis and bioassays were analyzed by using variance analysis with 

following mean comparison test with SYSTAT 11.0. Furthermore, linear regression analysis of 

bioassay and GS data was performed. 

RESULTS 

Constitutive GS level in Col WT was 24.5 µmol aliphatic GS and 6.1 µmol indolyl GS per g 

dry weight. A different constitutive GS profile was detected for the mam3 + lines, whereas the 

proportion of aliphatics decreased about 15% compared with Col WT. The indolyl GS did not 

change significantly compared with Col WT. The double knock out mutant cyp79B2 - cyp79B3 - 

was characterized by the absence of indolyl GS with aliphatic levels like Col WT. The GS 

phenotype of plants from the bioassay with insects corresponded to the GS genotype (GS 

induction data not presented). 

Percentage weight increase of caterpillars in the force feeding bioassays on each of the 

genotypes was different and species dependent (Fig. 1 A-C). After three days feeding on 

mam3 + and cyp79B2 - cyp79B3 significant higher weight increase were observed for the 

generalist S. exigua when compared with Col WT (Fig. 1 C). The highest weight increase with 

12 mg was found on mam3 + , which is twice as high than the larval weight increase observed 

on Col WT. Larvae weight increase within three days was not significant different on the three 

genotypes in the specialist species, P. rapae (Fig. 1 A) and P. brassicae (Fig. 1 B). 

362

Weight gain of P. rapae [mg] 

A 

Weight gain of P. brassicae [mg] 

B 

Weight gain of S. exigua [mg] 

C 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

120 

100 

80 

60 

40 

20 

14 

12 

10 

8 

6 

4 

2 

0 

0 

a 

a 

Col WT CYP79B2-/3- 

Genotype 

MAM3+ 

a 

a 


Genotype 

MAM3+ 

b 


Genotype 

MAM3+ 

Figure 1: Caterpillar (L3) weight increase of Pieris rapae (A), Pieris brassicae (B), and 

Spodoptera exigua (C) within three days on the mutants compared with 

Columbia WT. (different letter indicate significant differences among 

genoypes, Tukey’s HSD-test: p ≤ 0.05) 

a 

a 

b 

a 

363

Simple correlation of the larval weight increase to constitutive and induced total GS contents 

were performed to explain the impact of content and different types of GS on lepidopteran 

performance. We found that there was no correlation with R = - 0.08 between larval weight 

increase of S. exigua and increasing levels of constitutive GS among all genotypes. But for this 

species a weak negative correlation with R = - 0.44 was found between weight gain and 

induced GS levels. The contrary was true for the specialist P. rapae. Here the correlation 

results of larval weight gain to total GS were R = - 0.42 for constitutive GS levels and R = - 

0.08 for induced GS levels. Larval weight gain in S. exigua was comparable low on Col WT at 

similar GS levels like the mutants, data points were mostly below the regression line. 

Differently in P. rapae, the weight gain was lower on cyp79B2 - cyp79B3 at similar GS levels 

with most data points below the regression line. 

DISCUSSION 

The characteristic defense system GS of Brassicaceae, the GS and their corresponding 

hydrolysis products, is proved to be efficient against generalist insects (Halkier & Gershenzon 

2006). However, many crucifer specialists use these compounds in host recognition. Different 

compounds within a chemical group, like the GS, can have effects on specialized herbivorous 

insects as well (Bartlett et al. 1999). Our current study revealed a negative correlation of 

weight increase to induced GS levels in S. exigua like we reported in previous studies (Mewis 

et al. 2005). Furthermore, lower contents of aliphatic as well as indolyl GS in mam3 + and 

cyp79B2 - cyp79B3 respectively, compared with Col WT influenced positively the host plant 

suitability of genotypes for the generalist S. exigua. The correlation results showed that both 

GS classes equal influence the insect performance of this caterpillar species. 

The bioassay results revealed a different host plant suitability of genotypes for the three 

lepidopteran species. Contrary to the generalist S. exigua, the performance of the specialist 

species P. rapae and P. brassicae was not different on the genotypes. That specialists are less 

strong influenced by changes of characteristic secondary metabolite in their host plant is 

accepted (Schonhoven et al. 1998; Renwick 2002). However, that GS and their corresponding 

hydrolysis products can have an effect on specialists as well is reported by Agrawal & 

Kurashige (2003) and Mewis et al. (2006) for P. rapae. Corresponding in the present study a 

weak negative correlation of weight gain to constitutive GS levels but not to induced GS levels 

was found. Although the weight gain on genotypes was not found to be different in the Pieris 

species, the correlation results of bioassay data to GS contents indicate a different effect of GS 

classes on P. rapae performance. The weight gain of P. rapae was lower on cyp79B2 - cyp79B3 

(absence indolyl GS) at similar GS levels compared with mam3+ and Col WT. This indicates a 

higher plant defense activity of aliphatic GS compared with indolyl GS in P. rapae. The 

different host plant suitability of mutants for the generalist and specialist lepidoperan could 

also be attributed to a different induction of GS which is distinct from Col WT. Further feeding 

studies with more A. thaliana mutants with different GS profiles are on the way and extensive 

correlation studies will be performed. 

364

REFERENCES 

Agrawal A A; Kurashige N S (2003). A role for isothiocyanates in plant resistance against the 

specialist herbivore Pieris rapae. Journal of Chemical Ecology 29 (6), 1403-1415. 

Bartlet E.; Kiddle G; Williams I; Wallsgrove R M (1999). Wound-induced increases in the 

glucosinolate content of oilseed rape and their effect on subsequent herbivory by a 

crucifer specialist. Entomologia Experimentalis et Applicata 91 (1), 163-167. 

Fahey J W; Zalcmann A T; Talalay P (2001). The chemical diversity and distribution of 

glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5-51. 

Halkier B A; Gershenzon J (2006). Biology and biochemistry of glucosinolates. Annual Review 

Plant Biology 57, 303-333. 

Kliebenstein D J; Kroymann J; Brown P; Figuth A; Pedersen D; Gershenzon J; Mitchell-Olds 

T (2001). Genetic control of natural variation in Arabidopsis glucosinolate 

accumulation. Plant Physiology 126, 811-825. 

Koroleva O A; Davies A; Deeken R; Thorpe M R; Tomos A D; Hedrich R (2000). Different 

myrosinase and ideoblast distribution in Arabidopsis and Brassica napus. Plant 

Physiology 127, 1750-1763. 

Li G; Quiros C F (2002). Genetic analysis, expression and molecular characterization of 

BoGSL-ELONG, a major gene involved in the aliphatic glucosinolate pathway of 

Brassica species. Genetics 162, 1937-1943. 

Mewis I; Appel H M; Hom A; Raina R; Schultz J C (2005). Major signaling pathways 

modulate Arabidopsis thaliana (L.) glucosinolate accumulation and response to both 

phloem feeding and chewing insects. Plant Physiology 138 (2), 1149-1162. 

Mewis I; Tokuhisa J G; Schultz J C; Appel H M; Ulrichs C; Gershenzon J (2006). Gene 

expression and glucosinolate accumulation in Arabidopsis thaliana signaling mutants in 

response to generalist and specialist herbivores of different feeding guilds and the role 

of defense signaling pathways. Phytochemistry 67, 2450-2462. 

Rask L; Andréasson E; Ekbom B; Eriksson S; Pontoppidan B; Meijer J (2000). Myrosinase: 

gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology 

42, 93-113. 

Renwick J A A (2002). The chemical world of crucivores: lures, treats and traps. Entomologia 

Experimetalis et Applicata 104, 35-42. 

Schoonhoven L M; Jermy T; van Loon J J A (1998). Insect-Plant Biology: From Physiology to 

Evolution. Chapman & Hall: London. 

Textor S; Kraker de J-W; Hause B; Gershenzon J; Tokuhisa J G (2007). MAM3 catalyzes the 

formation of all aliphatic glucosinolate chain lengths in Arabidopsis. Plant Physiology 

144, 60-71. 

Zhao Y; Hull A K; Gupta N R; Goss K A; Alonso J; Ecker J R; Normanly J; Chory J; Celenza 

J L (2002). Trp-dependent auxin biosynthesis in Arabidopsis: involvement of 

cytochrome P450s CYP79B2 and CYP79B3. Genes and Development 16, 3100-3112. 

365

El-Wakeil N E, Volkmar C, Sallam A A: Promising Acquired Resistance Against Some Wheat Insects by 

Jasmonate Application. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

6-5 Promising Acquired Resistance Against Some Wheat Insects by 

Jasmonate Application 

El-Wakeil N E 1 , Volkmar C 2 2, 3 

, Sallam A A 

1 

Pests & Plant Protection Dept. National Research Center, Dokki, Cairo, Egypt 

2 Institute of Agric. & Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Germany 

3 

Plant Protection Dept. Faculty of Agriculture, Sohag University, Sohag, Egypt 

Email: n_emara@islamway.net (Nabil El-Wakeil) 

366 

ABSTRACT 

Wheat plants are attacked by many insects (e.g. aphids, thrips and wheat blossom 

midge (WBM)) during different growth stages (GS). Insect damage induces 

chemical changes in plants, and frequently these changes are part of a defensive 

response to the insect injury. In this study, induced resistance was activated in 

winter wheat using a foliar application of synthetic Jasmonic acid (JA). Field trials 

were conducted in Julius Kühn field in Halle University in 2008 to observe effects 

of jasmonate application on some wheat insects. Two wheat cultivars (Cubus and 

Tommi) were sprayed twice at GS 41 and 59 with two concentrations of jasmonate 

in addition to control plots which were sprayed with water. Wheat aphids and thrips 

were surveyed by direct counts 1 day before spraying and 1, 3, 7 and 15 days post 

spray. Wheat midges (Sitodiplosis mosellana (Géhin) and Contarinia tritici (Kirby)) 

were the most devastating insect pests of winter wheat production in central 

Germany. Thrips and WBM were counted at milky stage (GS 73) in each treatment 

by dissecting 10 ears using binocular microscopes. Wheat midge larvae were also 

monitored using white traps in treated and untreated jasmonate plots. Wheat yield 

was also assessed in treated and untreated plots. There was a significant difference 

in the number of thrips and midges among treatments in both cultivars. Plants in 

control plots had higher numbers of thrips and midges than in treated plots. There 

were higher numbers of thrips in the Tommi cultivar than the Cubus cultivar, while 

the latter had higher WBM larvae numbers than Tommi cultivar. Tommi was less 

affected than Cubus in infested kernels. There was a positive correlation between 

WBM numbers and infested kernels in both cultivars. This study also indicated that

jasmonate application enhances the wheat yield in sprayed plots compared to 

control plots. It is possible that some of the yield responses may have been due to 

reduced wheat insect damage. 

INTRODUCTION 

Wheat (Triticum aestivum) is the most important cereal crop to the bread industry. As the 

world population increases, there is an ever-increasing pressure for more efficient agricultural 

production (Cauvain & Cauvain 2003). Coupled with this pressure is the demand for 

minimizing insect damage as well as for the decreasing use of insecticides. Wheat is very prone 

to insect attacks. The key damaging pests are aphids, thrips and wheat blossom midges. 

Aphids feed by sucking sap from their hosts. The common cereal aphids are Rhopalosiphum 

padi (L.), Sitobion avenae (F.) and Metopolophium dirhodum (Wlk.). When aphid populations 

are large, aphids feeding can cause plants to become deformed and the leaves curled and 

shriveled (Carter et al. 1980; Dewar & Carter 1984). Extensive damage can occur when aphid 

populations are large throughout the crop, therefore settling of aphids needs to be reduced from 

the beginning of the crop development (Dixon 1998). 

The thrips fauna on wheat crops can cause serious damage, and methods of control are not 

sufficiently investigated. Thrips species Haplothrips tritici, H. aculeatus, Limothrips 

denticornis, Frankliniella tenuicornis and Thrips angusticeps were recorded on different wheat 

cultivars (Andjus 1996; Kucharzyk 1998; Moritz 2006). Thrips feeding on the ovaries of tender 

wheat leads to distortion, degeneration and sometimes abortion of grains. This has considerable 

consequences on yield as well as on the baking quality of flour (Holt et al. 1984). 

Infestation of wheat midges (Sitodiplosis mosellana, Contarinia tritici) can reduce crop yields 

and lower the quality of the harvested grain. Midges may exist at low population levels for 

several years before they become a significant problem. But if conditions become favourable, 

populations can reach epidemic proportions quickly. Producers inexperienced with wheat 

midge infestations often mistake the symptoms of damage and report that frost or drought was 

responsible for reduced wheat yields or grain quality (Gries et al. 2000; Birkett et al. 2004). 

Plants are known to produce jasmonic acid following herbivore damage, which results in 

increased production of compounds involved in resistance against herbivores (Thaler et al. 

1996). Jasmonic acid is derived from linolenic acid via the octodecanoid pathway and activates 

defensive genes that initiate induced systemic resistance against insect herbivores and the 

release of volatile compounds that attract natural enemies to herbivore-infested plants 

(Agrawal et al. 1999; Karban et al. 2000; Pickett et al. 2006). Application of jasmonic acid 

results in induced production of proteinase inhibitors and polyphenol oxidases and a decrease 

in the preference, performance, and abundance of herbivores in fields of tomato (Thaler et al. 

1996, 1999 a, b) and cotton (El-Wakeil 2003 a, b, 2009). 

367

Leaves normally release small quantities of volatile chemicals, but when a plant is damaged by 

herbivorous insects, many more volatiles are released. The chemical identity of the volatile 

compounds varies with the plant species and with the herbivorous insect species. These 

volatiles attract both parasitic and predatory insects that are natural enemies of the herbivores. 

They may also induce defense responses in neighboring plants. For example, wheat seedlings 

without herbivore damage attract aphids, whereas odors released from wheat seedlings with a 

high density of aphids repel other aphids (Quiroz et al. 1997). 

For conventional control of wheat insect, large quantities of the most toxic pesticides have 

been used (Cramer 1998). Control of wheat insects requires careful monitoring and integration 

of cultural practices and biological controls. The intensive use of toxic pesticides in wheat has 

caused serious health and environmental impacts. This background requires the need for the 

development of alternative protection strategies for aphids, thrips and wheat blossom midges 

due to their selectivity and safety (Bruce et al. 2003). 

We aimed to increase wheat production by focusing on minimizing insect damage using 

jasmonic acid to control wheat insects by increasing the induced resistance of wheat plant and 

host plant resistance, as the major components for integrated pest management programs. 

Finally, we could provide an IPM program of wheat insects. 


Winter wheat field 

The experiments were conducted in Julius Kühn field (sandy loam soil) in Halle University in 

2008. Two winter wheat cultivars (Tommi and Cubus) were chosen for these experiments. Two 

rates of jasmonic acid plus an untreated control were used. The experimental plots were 

designed as a randomized completed block experiment (four blocks); each treatment was 

replicated four times in each block (the experimental unit was 1.5 x 3m as plot). 

Jasmonic acid preparation and treatments 

The jasmonate plots were sprayed with jasmonic acid (Sigma-Aldrich, Germany) twice on May 

14 th and 29 th 2008 based on insect populations (Thaler et al. 1996). Jasmonic acid was applied 

at two rates of 5ml/ 5L (jasmonate 1) and 2.5ml/ 5L (jasmonate 2) (100 and 200ml jasmonic 

acid/ ha −1 ) dissolved in 10 ml acetone, using a hand-held hydraulic sprayer. Thus, three 

treatments were evaluated: (i) untreated (control); (ii) treated with low concentrations and (iii) 

with high concentrations of jasmonic acid (treatment codes are in Table 1). Each assessment 

was replicated four times. 

Direct counts of aphids and thrips 

Wheat aphids and thrips were directly counted about one day prior to treatment, and about 1, 3, 

7 and 15 days post jasmonic acid treatment as shown in Table (1). Cereal aphid, S. avenae, 

368

ose-grain aphid, M. dirhodum and the bird-cherry oat aphid, R. padi were counted. Five 

randomly selected tillers were inspected from each replicate, totaling 20 tillers for each 

treatment (Birkett et al. 2000). Thrips larvae or adults were mainly collected by the method of 

shaking plants by using white sheets (Jenser 1993). 

Evaluation of thrips and midges in wheat ears 

Ten ears were sampled randomly from each plot in the treated and untreated plots. Wheat 

midge larvae and thrips larvae and adults were counted at milky stage (GS 73) by dissecting 10 

ears under a binocular microscope. 

Table 1. Dates of jasmonic acid application and observations on different wheat growth 

stages 

Treatments Dates 

Wheat cultivars 

Tommi Cubus 

Treatment codes 

TJ1 Tommi Jasmonate 1 

1st application 14-05-08 (GS*) 41-43 (GS) 43 TJ2 Tommi Jasmonate 2 

1 st day after application 15-05-08 (GS) 43 (GS) 45 TC Tommi Control 

5 th day after application 19-05-08 (GS) 45 (GS) 51 CJ1 Cubus Jasmonate 1 

8 th day after application 22-05-08 (GS) 49-51 (GS) 55 CJ2 Cubus Jasmonate 2 

15 th day after application 29-05-08 (GS) 55-59 (GS) 

59-61 

CC Cubus Control 

2 nd application 29-05-08 (GS) 55-59 (GS)59-61 

1 st day after application 30-05-08 (GS) 59 (GS) 61 

3 rd day after application 02-06-08 (GS) 65 (GS) 65 

7 th day after application 05-06-08 (GS) 69 (GS) 69 

15 th day after application 12-06-08 (GS) 73 (GS) 73 

* GS: Growth Stage of wheat 

Wheat midge larvae in white traps 

White traps were used to sample wheat midge larvae in the treated and untreated wheat plots 

(setup at 17 June till 14 July 2008). The traps consisted of plastic white dishes; 12.5cm 

diameter and 6.5cm deep placed on the ground among wheat plants in each plot and were 

partly filled with water with a small quantity of detergent. Those traps were observed twice a 

week in the field; and the caught larvae were counted using a magnifying glass in field or a 

binocular microscope in the laboratory. 

369

Wheat yield 

Yields of jasmonic acid-treated– wheat plants were compared to the control. The wheat yield 

was measured as dry matter in 10 ears per plot (total 40 ears/ treatment), to verify treatment 

effectiveness on wheat yield by studying kernels numbers, ear weights and thousand grain 

weights. The mature kernels were weighed to estimate the plot production and yield of each 

treatment. Finally, this was converted to yield in kilograms per hectare. 


The differences in insect infestations in jasmonate-treated and control plots were analyzed by 

linear model (analysis of variance (ANOVA)) using Statistix 8 (Thomas & Maurice 2008). 

Tukey tests were used to compare means of cultivars. Significant differences were noted at P < 

0.05 for all trials. 

RESULTS 

Direct count of aphids and thrips 

Wheat aphid and thrip populations were not recorded after the first application because of 

weather conditions. Aphid and thrip numbers decreased post jasmonate application often for 15 

days; populations were slow to recover. Populations of cereal aphids and thrips were 

consistently lower on the plots sprayed with jasmonate. In this experiment, the predominant 

aphid species were M. dirhodum, S. avenae and R. padi. Thrips species were L. denticornis and 

T. angusticeps. The aphids and thrips populations were almost halved in the jasmonate-treated 

plots compared to control. There was a significant difference in cumulative aphid and thrip 

numbers in jasmonate and control plots as assessed by ANOVA (P < 0.037) (Table 2). 

Evaluation of thrips and midges in wheat ears 

There was a significant difference (P< 0.048) in the number of thrips adults among treatments 

in both cultivars. Both cultivars in control plots had the highest numbers of thrips adults 3.7 

and 3.1/ ear, respectively, while these numbers ranged from 1.8 to 2.4/ ear in jasmonate plots 

(Fig.1). There was a significant difference (P< 0.000) in the number of thrips larvae among 

jasmonate treatments. Tommi cultivar in control plot had the highest numbers of thrips larvae 

15.2/ ear, while thrips larvae in treated plots ranged from 4.2 to 8.5/ ear (Fig. 1). The same 

trend was observed in total thrips; there was also significant difference (p < 0.000) among 

treatments. Total thrips were higher in control plots in both cultivars than in treated plots, while 

total thrips in treated plots ranged from 6.4 to 10.3/ ear (Fig. 1). 

There was significant difference (P< 0.047) in the number of wheat midges larvae per ear 

among treatments. Control plots had the highest numbers compared to treated plots in both 

cultivars. Cubus had WBM larvae higher than Tommi cultivar (Fig. 1). There was a significant 

difference (P< 0.045) in the number of thrips and wheat midge in infested kernels resulting 

370

from treatments to both cultivars. Treated plots had lower infested kernels than control plots in 

both cultivars. Tommi was less affected than Cubus in infested kernels (Fig. 1). There was a 

correlation between WBM numbers and infested kernels (R= +0.87 and +0.64) in Tommi and 

Cubus cultivars respectively; while no correlation between thrips adults, larvae & total thrips 

and infested kernels among treatments in both cultivars as shown in Table (3). 

Table 2. Mean of wheat thrips and aphids by direct counts method on two winter wheat 

cultivars in treated and untreated plots 

Treatments 

Thrips 

Aphids Treatments 

Thrips 

Adults Larvae 

Adults Larvae 

Before application 29-05-08 (GS 55-59) 

Aphids 

TJ1 7 5 3 CJ1 6 4 2 

TJ2 8 4 4 CJ2 7 5 3 

TC 7 5 4 CC 7 5 3 

1 st day after application 30-05-08 (GS 59-61) 

TJ1 3 3 1 CJ1 3 3 1 

TJ2 4 3 2 CJ2 5 4 2 

TC 7 5 4 CC 7 5 3 

3 rd day after application 02-06-08 (GS 65) 

TJ1 3 3 2 CJ1 3 3 1 

TJ2 4 4 4 CJ2 5 4 3 

TC 8 5 6 CC 9 5 4 

7 th day after application 05-06-08 (GS 69) 

TJ1 4 4 4 CJ1 4 2 2 

TJ2 5 4 5 CJ2 5 3 3 

TC 8 5 8 CC 9 5 5 

15 th day after application 12-06-08 (GS 73) 

TJ1 5 2 8 CJ1 6 0 4 

TJ2 6 3 11 CJ2 7 1 10 

TC 9 2 12 CC 9 4 12 

371

372 

Table 3. Correlation coefficient between ear insects (thrips & wheat midges) and 

infested kernels in both wheat cultivars (* Significant differences) 

Sites Thrips adults Thrips larvae Total thrips Wheat midges 

Tommi -0.25 +0.20 +0.09 +0.87 * 

Cubus +0.29 +0.29 +0.38 +0.64 ** 

Thrips adults 

Thrips larvae 

Total thrips 


3 

2 

1 

0 

20 

15 

10 

5 

0 

20 

15 

10 

4 A 

5 

0 

6 

5 

4 

3 

2 

1 

0 

B B 

C 

C 

C 

C 

C 

C 

C 

B 

B 

B 

C 

DE 

DE 

WBM larvae Tommi 

WBM larvae Cubus 

Infested kernels Tommi 

Infested kernels Cubus 

C 

Tommi Cubus 

C 

C 

AB 

High Jasmonate Low Jasmonate Control 

Figure 1. Effects of jasmonate application on mean of thrips adults, larvae and total 

thrips, WBM larvae and the relation to infested kernels in both wheat cultivars 

(Growth stage 73) (Different letters indicate significant differences) 

C 

C 

C 

E 

A 

A 

E 

BC 

A 

B 

B 

A 

2.0 

1.5 

1.0 

0.5 

0.0 

-0.5 

2.0 

1.5 

1.0 

0.5 

0.0 


infested kernels

Wheat midge larvae population in white traps 

Populations of wheat midge larvae (S. mosellana and C. tritici) were monitored using white 

traps. Generally populations of WBM larvae were higher on control wheat plots than 

jasmonate- treated plots. The caught larvae were lower in plots of high rate of jasmonate, than 

in plots of low rate of jasmonate (Fig. 2). Population density was significantly higher (P < 

0.021) on two sampling occasions (26 th June and 7 th July 2008) than other dates. These 

numbers were 24, 33 and 59 WBM larvae on 26 th June in high jasmonate rate, low jasmonate 

rate and control, respectively. The corresponding records were 21, 32 and 55 on 7 th July. 

Analyses of the cumulative data using ANOVA to compare total WBM larvae numbers in 

jasmonate and control plots showed that there was a significant difference (P = 0.034). The last 

WBM larvae were caught on 11 th July (Fig 3). 

Caught WBM larvae 

Caught WBM larvae 

70 

60 

50 

40 

30 

20 

10 

40 

30 

20 

10 

0 

High rate of Jasmoante 

Low rate of Jasmoante 

Control 

C 

B 

A 

C 

B 

A 

B 

C 

A 

B 

C 

19 June 23 June 26 June 30 June 3 July 7 July 11 July 14 July 

A 

Investigated dates 

Figure 2. Orange wheat midge larvae were caught in white traps in treated and untreated 

jasmonate (Different letters inidcated significant differences) 

0 

TJ1 

CJ1 

TJ2 

CJ2 

TC 

CC 

19 June 23 June 26 June 30 June 3 July 7 July 11 July 14 July 

Investigated dates 

Figure 3. Orange wheat midge larvae caught in white traps in treated and in treated and 

untreated jasmonate plots 

C 

B 

A 

C 

A 

B 

373

Wheat yield 

Comparison of yields of both wheat cultivars (Tommi and Cubus) indicated that the Cubus 

cultivar outyielded Tommi cultivar. The results indicated that the yield index was higher in the 

Cubus cultivar (55.67 kernels, 50.85 g, 3.69 Kg and 8200.00 Kg) than in the Tommi cultivar 

(51.53 kernels, 49.49 g, 3.40 Kg and 7548.15 Kg) in kernel numbers / ear, weight of 1000 

kernels, weight of grain / plot and weight of grain / ha, respectively (Table 4). 

The analysis of yield data suggests that jasmonate treatments enhance yield relative to the 

control showing significant differences (P< 0.0239) between treatments. Within Cubus 

cultivar, the highest yield was recorded in CJ1 treatment (8688.89 Kg/ha), then CJ2 (8222.22 

Kg/ ha), while control plots had the least value (7688.89 Kg/ ha). Within Tommi cultivar, the 

highest yield was gained in TJ1 treatment (8044.44 Kg/ha), then TJ2 (7577.78 Kg/ ha); while 

control plots had the least yield (7022.22 Kg/ ha). Data are shown in Table (4). 

374 

Table 4. Mean of yield index in winter wheat treated with jasmonate (kernel numbers, 

weight of one ear, weight of 1000 kernels (TKM), weight of grains/ plot and 

weight of grains/ ha) 

Jasmonate 

treatments 

Kernel 

numbers in 

one ear 

Weight of 

one ear 

Weight of 

1000 

kernels 

Weight of 

grains /plot 

(Kg) 

Weight of grains/ 

ha (Kg) 

TJ1 58.00 2.90 49.98 3.62 a 8044.44 a* 

TJ2 52.70 2.58 49.03 3.41 ab 7577.78 ab 

TC 43.90 2.17 49.45 3.16 c 7022.22 c 

Mean 51.53 2.55 49.49 3.40 7548.15 B 

CJ1 61.50 3.19 51.87 3.91 A 8688.89 A 

CJ2 54.70 2.75 50.22 3.70 B 8222.22 AB 

CC 50.80 2.56 50.47 3.46 C 7688.89 C 

Mean 55.67 2.83 50.85 3.69 8200.00 A 

* Different letters indicated significant differences 

DISCUSSION 

Generally, wheat insect populations were lower in treated plots than untreated plots which may 

be due to induced wheat plants after jasmonate spray causing repellence for these insects. 

Repellency of some insects due to induced plant volatiles has been established in many studies 

such as Quiroz et al. (1997), Birkett et al. (2000) and Bruce et al. (2003). They found that 

induced plant volatile jasmonate repelled aphids from plants in field trials. Foliar application of 

jasmonate on wheat also reduced insect populations and their activities as reported by Bruce et 

al. (2003) for increased repellency of the insects following jasmonate sprays on S. avenae in 

wheat and also by Cooper & Goggin (2005) for reduced potato aphid populations in tomatoes.

There was a significant difference in the number of thrips and midge larvae among treatments 

in both cultivars. Control plots had thrips and midges higher than the treated plots. Tommi had 

thrips numbers higher than Cubus cultivar, while the latter had WBM larvae numbers higher 

than Tommi cultivar. Tommi was less affected than Cubus in infested kernels. There was a 

correlation between WBM numbers and infested kernels. 

The significant reduction of wheat insect populations in field trials after treatment with 

jasmonate may be due to a combination of reduced settling and slower population 

development. Because induced wheat plants are more resistant to wheat insects, it is possible 

that jasmonate acts as a phytopheromone, alerting plants to an attack by insects (Chamberlain 

et al. 2000). These results correspond well with data reported by other authors; Pettersson et al. 

(1996) and Slesak et al. (2001) found that volatiles released by aphid-infected cereals or thrips 

could induce antixenotic effects in neighboring plants, which cause non-preference in some 

wheat insects. Nevertheless, it is clear that jasmonate is biologically active and is a 

semiochemical signal which increases the resistance of young wheat plants to attack by thrips, 

midges or aphids. 

The experiments indicated that jasmonate application affected yield of both wheat cultivars. It 

is possible that some of the yield responses may have been due to jasmonate treatment due to 

reduce wheat insect damage. This result is similar to those obtained by Thaler (1999a) who 

mentioned that jasmonate applications as well as resistant cultivars improve the crop yield. 

The results indicate that jasmonate induced wheat plants and may act as resistance mechanisms 

of wheat against insect herbivores. The analysis of yield data suggests that both jasmonate 

applications enhance yield relative to the control. The results recommend that using jasmonic 

acid in insect-management programs will help farmers to increase wheat yields and to 

minimize insecticides use. 

REFERENCES 

Agrawal A A; Kobayashi C; Thaler J S (1999). Influence of prey availability and induced hostplant 

Resistance on omnivory by western flower thrips. Ecology 80, 518–523. 

Andjus L (1996). The research into the Thrips fauna and significance of the plants of 

spontaneous flora for the survival of pest species. Ph.D Thesis, Belgrade University. 

Birkett M A; Bruce T J A; Martin J L; Smart L E; Oakley J; Wadhams L J (2004). Responses 

of female orange wheat blossom midge, Sitodiplosis mosellana, to wheat panicle 

volatiles. Journal of Chemical Ecology 30, 1319-1328. 

Birkett M A; Campbell C A M; Chamberlain K; Guerrieri E; Hick A J; Martin J L; Matthes M; 

et al. (2000). New roles for cis-jasmone as an insect semiochemical and in plant 

defense. Proceedings National Academic Sciences (PNAS) USA 16, 9329-9334. 

Bruce T J A; Martin J L; Pickett J A; Pye B J; Smart L E; Wadhams L J (2003). cis-Jasmone 

treatment induces resistance in wheat plants against the grain aphid, Sitobion avenae 

(Fabricius) (Homoptera: Aphididae). Pest Management Sciences 59, 1031-1036. 

Carter N; Mclean I F G; Watt A D; Dixon A F G (1980). Cereal aphids: a case study and 

review. Applied Biology 5: 271-348. 

375

Cauvain S P; Cauvain C P (2003). Bread Making. CRC Press. p. 540. ISBN 1-85573-553-9. 

Chamberlain K; Pickett J A; Woodcock C M (2000). Plant signaling and induced defense in 

insect attack. Molecular Plant Pathology 1, 67–72. 

Cooper W R; Goggin F L (2005). Effects of jasmonate-induced defenses in tomato on the 

potato aphid, Macrosiphum euphorbiae. Entomologia Experimentalis et Applicata 115, 

107–115. 

Cramer J (1998). Environmental Management: from Fit to Stretch. Business Strategy and the 


Dewar A M; Carter N (1984). Decision trees to assess the risk of cereal aphid outbreaks in 

summer in England. Bulletin of Entomological Research 74, 387-398. 

Dixon A F G (1998). Aphid ecology- an optimization approach, 2 nd ed. Chapman & Hall: 

London. 

El-Wakeil N E (2003a). New aspects of biological control of Helicoverpa armigera in organic 

cotton production. Ph.D Thesis in Goettingen University, Germany. 

El-Wakeil N E; Bernal J; Vidal S (2003b). Effects of jasmonate applications on pest and 

natural enemy recruitment in cotton fields. Proceedings of the World Cotton Research 

Conference-3 (9 to 13 March 2003). Cape Town, South Africa, 1239-1248. 

El-Wakeil N E; Bernal J; Vidal S (2009). Study affects of Jasmonic acid (Jasmonate) on 

resistant-plant and attractive-natural enemies in BT and Non-BT cotton Fields. Journal 

of Applied Entomology (In Press). 

Gries R; Gries G; Khaskin G; King S; Olfert O; Kaminski L A; Lamb R; Bennett R (2000). 

Sex pheromone of orange wheat blossom midge, Sitodiplosis mosellana. 

Naturwissenschaften 87, 450-454. 

Holt J; Griffiths E; Wratten S D (1984). The influence of wheat growth stage on yield 

reductions caused by Metopolophium dirhodum. Annals of Applied Biology 105, 7-14. 

Jenser G (1993). Studies on the vertical distribution of some Thysanoptera species in an oak 

forest. Zoology 4, 233-238. 

Karban R; Baldwin I T; Baxter K J (2000). Communication between plants: induced resistance 

in wild tobacco plants following clipping of neighboring sagebrush. Oecologia 125, 

66-71. 

Kucharzyk H (1998). Thysanoptera and other insects collected in differently coloured traps in 

eastern Poland. Proceedings Sixth International Symposium on Thysanoptera, 81-87. 

Moritz G (2006). Thripse. Die Neue Brehm- Bücherei Bd. 663. Westarp Wissenschaften: 

Hohenwarsleben. 

Pettersson J; Quiroz A; Fahad A E (1996). Aphid antixenosis mediated by volatiles in cereals. 

Acta Agriculturae Scandinavica, Section B, Soil Plant Science 46, 135–140. 

Pickett J A; Bruce T J A; Chamberlain K; Hassanali A; Khan Z R; Matthes M C; Napier J A; 

Smart L E; Wadhams L J; Woodcock C M (2006). Plant volatiles yielding new ways to 

exploit plant defence. In: Chemical ecology: from gene to ecosystem, eds M Dicke & 

W Takken, pp. 161-173. Wageningen (Netherlands): Springer. 

Quiroz A; Pettersson J; Pickett J A; Wadhams L J; Niemeyer H M (1997). Semiochemicals 

mediating spacing behavior of bird cherry-oat aphid, Rhopalosiphum padi, feeding on 

cereals. Journal of Chemical Ecology 23, 2599–2607. 

376

Slesak E; Slesak M; Gabrys B (2001). Effect of methyl jasmonate on hydroxamic acid content, 

protease activity, and bird cherry–oat aphid Rhopalosiphum padi (L.) probing behavior. 

Journal of Chemical Ecology 27, 2529-2543. 

Thaler J S (1999a). Induced resistance in agricultural crops: Effects of jasmonic acid on 

herbivory and yield in tomato plants. Environmental Entomology 28, 30-37. 

Thaler J S (1999b). Jasmonate-inducible plant defenses cause increased parasitism of 

herbivores. Nature 399, 686-688. 

Thaler J S; Stout M J; Karban R; Duffey S S (1996). Exogenous jasmonates simulate insect 

wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field. 

Journal of Chemical Ecology 22, 1767-1781. 

Thomas C R; Maurice S C (2008). Statistix 8, Ninth Edition, Managerial Economics McGraw- 

Hill/Irwin. (ISBN: 0073402818). More information at http://www.statistix.com. 

377

Kassemeyer H H, Seibicke T, Boso S: Resistance versus susceptibility in grapevine - Response of different 

grapevine genotypes to the biotrophic pathogen Plasmopara viticola. In: Feldmann F, Alford D V, Furk C: Crop 



7-1 Resistance versus susceptibility in grapevine - Response of different 

grapevine genotypes to the biotrophic pathogen Plasmopara viticola 

Kassemeyer H H, Seibicke T, Boso S 

Staatliches Weinbauinstitut, Abteilung Biologie, Merzhauser Str. 119, 79100 Freiburg, 

Germany 

E-mail: Hanns-Heinz.Kassemeyer@wbi.bwl.de 

378 

Abstract 

In a series of inoculation experiments we examined the course of colonization of 

leaf mesophyll by the causal agent of grapevine downy mildew, Plasmopara 

viticola, in a susceptible and a resistant grapevine genotype. The aim of our studies 

was to compare the development of the pathogen in the host tissue with the 

activation of defense response. Microscopical studies on the establishment of P. 

viticola and the colonization of the host tissue revealed significant differences 

among the two genotypes. In the susceptible genotype the pathogen colonized the 

intercellular space of the host tissue rapidly and the infection cycle was completed 

within four days after inoculation by the sporulation event. In the resistant genotype 

the invasive growth of P. viticola was delayed, and further development ceased 

before sporangiophores were formed. To study the induction of the defense in both 

genotypes after challenging by P. viticola we analyzed the activation of genes 

involved in defense response such as a grapevine β-1,3-glucanase and a grapevine 

stilbenesynthase by means of quantitative PCR. The analysis revealed an increase of 

the transcript of both the glucanase and the stilbenesynthase in the resistant 

genotype within 12 h after inoculation. In the susceptible genotype no induction of 

the genes was observed up to two days after inoculation. At the same time as a 

prompt and strong activation of defense in the resistant genotype further 

development of the pathogen inhibited, the delayed response in the susceptible 

genotype enables the pathogen to colonize the host tissue and to form propagules.

INTRODUCTION 

Downy mildew, caused by Plasmopara viticola (Peronosporomycetes) is among the most 

important diseases of grapevine, particularly in warm and humid climates. The classical 

cultivars of Vitis vinifera are highly susceptible to P. viticola, resulting in severe epidemics 

under favorable conditions. In comparison, North American Vitis species typically express 

significant resistance to this disease (Staudt & Kassemeyer 1995, Kortekamp et al. 1998, 

Unger et al. 2007, Boso & Kassemeyer 2008). Hybrids between V. vinifera and American 

species, including those resulting from multiple backcrossings with European cultivars, exhibit 

a variable range of intermediate resistance to downy mildew (Spring 2001). In the last two 

decades offspring of these hybrids expressing a high resistance against P. viticola and 

producing a high quality has been introduced in viticulture. 

Downy mildew spreads by asexually formed sporangia, which are released in large quantities 

under favorable conditions. The sporangia are transported by wind, attach to the host surface 

(Kortekamp et al. 1998) and release four to eight zoospores in the presence of free water. The 

pathogen penetrates its host through the stomata, bypassing preformed barriers on the host 

surface such as the cuticle and the epidermal cell wall. For this, the motile zoospores attach 

specifically to the stomata after a swarming phase (Kiefer et al. 2002), encyst by forming a cell 

wall and subsequently develop a penetration peg, which grows through the stomatal pore 

(Kiefer et al. 2002, Riemann et al. 2002). Under optimal conditions at 22 to 24°C, release of 

zoospores and targeting of the stomata occur within 2 h. After an incubation period, which 

depends on temperature the first symptoms arise on infected leaves, inflorescences and berries. 

Sporulation occurs thereafter once the relative humidity exceeds 92% at night (Blaeser & 

Weltzien 1978). Under such conditions, sporangiophores emerge from the stomata within 7 h, 

after which they branch and form sporangia at their tips (Rumbolz et al. 2002). The study of 

growth and development of P. viticola during the incubation period, particularly the 

colonization of the intercellular space and the spatial and temporal development within the 

mesophyll requires specific microscopical methods that have described recently (Unger et al. 

2007). This methods enable the characterization of pathogen development in resistant and 

susceptible grapevine genotypes. 

All plants can defend themselves against attack by microorganisms with different resistance 

mechanisms such as hypersensitive response (HR), cell wall reinforcement, biosynthesis of 

phytoalexins and expression of pathogenesis related proteins (PR-proteins) (Hückelhoven 

2007). PR-proteins induced upon attack by oomycetes, fungi, bacteria and viruses play an 

important role in the defense response (Van Loon et al. 2006). They are expressed through the 

action of the pathogen recognition, a signal cascade and defense gene transcription. Hence the 

transcriptional activity of PR-proteins can be used to study the kinetics of the defense response 

in plants upon challenging by a pathogen. We chose a β-1,3-glucanase, belonging to the PR 2 

family, which we characterized recently to quantify the kinetics of the defense response in 

susceptible and resistant grapevine genotypes. Molecular markers for this PR-protein permit 

the quantitative analysis of the course of the defense gene activation by means of Real-Time 

379

PCR. The combination of the microscopical studies and the molecular analysis of defense gene 

induction provide insight into the nature of resistance and susceptibility in grapevine. 


For the inoculation experiments we used cuttings of Vitis rupestris Michx. representing a 

resistant genotype while V. vinifera L. cv. Müller-Thurgau (Staudt & Kassemeyer 1995) was 

considered highly susceptible. The plants were inoculated by spraying the abaxial surface of 

the leaf with a aqueous suspension containing 2 x 10 4 sporangia ml -1 of Plasmopara viticola 

(Rumbolz et al. 2002). The inoculated plants were incubated in a growth chamber and leaves 

were harvested at distinct intervals from 6 to 96 h post inoculation (hpi). Excised leaf disks 

were cleared and stained with 0.05% aniline blue (Kiefer et al. 2002). Examination was 

performed with a Zeiss Axiophot equipped with an epifluorescence facility (excitation at 395- 

440 nm; FT 460 nm, LP 470 nm) and Plan-Neofluar objectives. The imaging analysis was 

accomplished with a Zeiss AxioCam digital camera and the Zeiss AxioVision software. 

For the quantification of putative defense genes we used a grapevine β-1,3 Glucanase (Seibicke 

et al. in preparation; Accession No. AJ277900) and a pathogen inducible grapevine 

stilbenesynthase (Accession No. S63221). Total RNA was isolated and purified from the 

excised leaf disks, and reverse transcribed using random hexamers and reverse transcriptase 

(Abgene) following the manufacturer’s instructions. The quantification of the transcripts was 

carried out by Real-Time RT-PCR and SYBR ® green method in an ABI 7500 Real-Time PCR 

System (Applied Biosystems). Each Real-Time Assay was also tested in a dissociation protocol 

and sequencing the amplicon to ensure that each PCR amplifies a single specific product. All 

data were normalized on the 18S housekeeping gene. A standard curve was obtained for the 

grapevine β-1,3 glucanase and grapevine stilbenesynthase as well as for the 18S amplicon by 

amplifying known cDNA quantities, and each amplicon was then quantified by comparison 

with the respective standard curve. The n-fold induction of both genes was derived from the 

VGl/18S mean quantity ratio. 

RESULTS 

Course of colonization of the host tissue by Plasmopara viticola in resistant and 

susceptible genotypes 

At 6 h after inoculation (hpi), in both genotypes Plasmopara viticola had penetrated the 

stomata and had reached the substomatal cavity, where substomatal vesicles with a primary 

hypha appeared. The primary hyphae were formed rapidly after penetration, and no 

substomatal vesicles without hyphae occurred 6 hpi. At this time we observed the first 

haustoria on the primary hyphae. Further growth of the hyphae was delayed for some time. In 

the susceptible genotype longitudinal growth of the primary hyphae resumed after a while and 

elongated hyphae were found to invade the intercellular space of the mesophyll by 24 hpi. The 

development of the pathogen advanced rapidly after this point and between 42 and 48 hpi on 

380

the elongating hyphae numerous haustoria were formed. The long hyphae branched and spread 

into the intercellular space of the mesophyll. After 48 hpi, these hyphae formed a mycelium in 

the susceptible genotype which was fully expanded 66 hpi. At 96 hpi hyphae accumulated in 

the substomatal cavities forming secondary vesicles, and sporangiophore initials emerged from 

the stomata. After exposition to favorable humidity conditions overnight, sporangiophores with 

numerous well-developed sporangia at the tips emerged from the stomata. Although initial 

stages of the P. viticola development in the resistant genotype were similar to those in the 

susceptible genotype at 6 hpi, subsequent development was retarded and was only rarely 

completed. The elongation of the primary hyphae proceeded slower than in the susceptible 

genotype and the long hyphae began to branch retarded at 30 hpi only in some lesions. In most 

cases further development of P. viticola ceased and only at a very low frequency the pathogen 

progressed and formed a loose mycelium. We never observed a dense mycelium and only very 

few unbranched, and sterile hyphae emerging from the stomata. The different time course of 

infection and the length of primary hyphae and long hyphae in the two genotypes was evident 

in significant differences between both genotypes. 

Course of defense gene activation in resistant and susceptible genotypes in response to a 

challenge infection by Plasmopara viticola 

Northern blot analysis revealed transcription of the grapevine β-1,3 glucanase and grapevine 

stilbenesynthase after challenging with P. viticola in both genotypes. To discriminate the 

response of susceptible and resistant genotypes following P. viticola attack, the induction 

kinetics of the grapevine β-1,3 glucanase and grapevine stilbenesynthase was quantified by 

Real-Time RT-PCR. For this purpose, inoculation experiments were carried out on susceptible 

V. vinifera cv. Müller-Thurgau and resistant V. riparia. In V. vinifera cv. Müller-Thurgau, the 

specific transcript abundance of the glucanase remained low until 48 hpi and then increased 

between 48 hpi and 60 hpi. In V. riparia the transcript increased 12 hpi and accumulated within 

24 hpi showing a 24-fold induction. A second boost up to 35-fold induction arose between 24 

hpi and 36 hpi followed by a slight decrease until 60 hpi. To confirm the obtained results, the 

induction kinetics of the glucanase was compared with that of a grapevine stilbenesynthase. As 

for the glucanase, the accumulation of the stilbenesynthase as a response to P. viticola 

inoculation was quantified in both genotypes. In this experiment, the course of stilbenesynthase 

induction was nearly identical to that of the glucanase. The transcript abundance in the 

susceptible genotypes remained low until 48 hpi, and subsequently increased between 48 and 

60 hpi. The resistant genotype showed a rapid transcript accumulation up to 17.6 induction 

already at 12 hpi, a considerable drop between 36 hpi and 48 hpi and a second increase after 48 

hpi. 

DISCUSSION 

The transcriptional activity of PR-proteins is a suitable marker for the kinetics of the defense 

response in plants. Among them, hydrolytic enzymes such as β-1,3-glucanases (PR-2) and 

381

chitinases (PR-3, PR-4, PR-8) have been suggested to be involved in plant resistance against 

fungal pathogens and oomycetes (Busam et al. 1997a). Our studies show that a grapevine β- 

1,3-glucanase, belonging to the PR-2 family and a grapevine stilbenesynthase is a suitable 

marker to study the kinetic of defense response in grapevine genotypes. The observed time 

course of beta-1,3-glucanase and stilbenesynthase after challenging by P. viticola in the 

resistant genotype is in accordance to previous reports (Busam et al 1997 a, Busam et al. 

1997b) and the rapid transcript accumulation of both defense related genes corroborate their 

role in the defense against P. viticola. The microscopical studies point out that a rapid 

activation of genes involved in defense response during the initial stages of the plant-pathogen 

interaction is crucial for an effective cease of the invasion by P. viticola and its propagation. 

On the other hand a delayed gene activation in a stage at the beginning of the invasive growth 

and mycelium formation with numerous haustoria causes susceptibility against P. viticola. 

REFERENCES 

Blaeser M; Weltzien H C (1978). Die Bedeutung von Sporangienbildung, -ausbreitung und - 

keimung für die Epidemiebildung von Plasmopara viticola. J. Plant Disease Protection 

85, 155-161. 

Boso S; Kassemeyer H H (2008). Different susceptibility of European grapevine cultivars for 

downy mildew. Vitis 47, 39-49. 

Busam G; Kassemeyer H H; Matern U (1997a). Differential expression of chitinases in Vitis 

vinifera L. responding to systemic acquired resistance activators or fungal challenge. 

Plant Physiol. 115, 1029-1038. 

Busam G; Junghanns K T; Kneusel R E; Kassemeyer H H; Matern U (1997b). Characterization 

and expression of Caffeoyl-coenzyme A 3-O-methyltransferase proposed for the 

induction for the induced resistance response of Vitis vinifera L. Plant Physiol. 115, 

1039-1048. 

Hückelhoven R (2007). Cell wall - associated mechanisms of disease resistance and 

susceptibility. Annu. Rev. Phytopathol. 45, 101-127. 

Kiefer B; Riemann M; Büche C; Kassemeyer H H; Nick P (2002). The host guides 

morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola. 

Planta 215, 387-393. 

Kortekamp A; Wind R; Zyprian E (1998). Investigation of Plasmopara viticola with 

susceptible and resistant grapevine cultivars. J. Plant Disease Protection 105; 475-488. 

Riemann M; Büche C; Kassemeyer H H; Nick P (2002). Cytoskeletal response during early 

development of the downy mildew of grapevine (Plasmopara viticola). Protoplasma 

219, 13-22. 

Rumbolz J; Wirtz S; Kassemeyer H H; Guggenheim R; Schäfer E; Büche C (2002). 

Sporulation of Plasmopara viticola: Differentiation and light regulation. Plant Biol. 4, 

413-422. 

Spring J L (2001). Premieres experiences avec les cepages interspecifiques “Merzling”, 

“Johanniter”, “Bronner” et “Solaris” en Suisse romande. Rev. Suisse Vitic. Arboric. 

Hortic. 33, 57-64. 

382

Staudt G; Kassemeyer H H (1995). Evaluation of downy mildew resistance in various 

accessions of wild Vitis species. Vitis 34, 225-228. 

Unger S; Büche C; Boso S; Kassemeyer H H (2007). The course of colonization of two 

different Vitis genotypes by Plasmopara viticola indicates compatible and incompatible 

host-pathogen interactions. Phytopathology 97, 780-786. 

Van Loon L C; Rep M; Pieterse C M J (2006). Significance of inducible defense-related 

proteins in infected plants. Annu. Rev. Phytopathol. 44, 135-162. 

383

Kochanova M, Prokinova E, Rysanek P: Detection of wheat resistance to bunts by real-time PCR. In: Feldmann F, 



7-2 Detection of wheat resistance to bunts by real-time PCR 

Kochanova M, Prokinova E, Rysanek P 

Czech University of Life Sciences Prague, Kamycka 129, 16521 Prague 6 Suchdol 

Email: kochanova@af.czu.cz 

384 

Abstract 

Bunts belongs to the most dangerous diseases of wheat but can attact other cereals, 

too. Symptoms of common bunt may not be apparent until after heading, although 

infection hyphae attact young seedlings. Hyphae become established initially in 

both resistant and susceptible varieties but in resistant variety there are not created 

sori in spikes, which means the spike is healthy. Using resistant cultivars is included 

in preventative precautions against bunt. In the Czech Republic wheat varieties 

show different tolerance against bunts. This tolerance can be discovered by using 

molecular biological quantification called real-time PCR.

Häffner E, Konietzki S, Socquet-Juglard D, Gerowitt B, Diederichsen E: Genetics of Resistance Against the 

Vascular Pathogen Verticillium Longisporum in Brassica and Arabidopsis Thaliana. In: Feldmann F, Alford D V, 

Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 385-392; ISBN 978-3-941261-05-1; © 


7-3 Genetics of Resistance Against the Vascular Pathogen Verticillium 

Longisporum in Brassica and Arabidopsis Thaliana 

Häffner E 1 , Konietzki S 1 , Socquet-Juglard D 1 , Gerowitt B 2 , Diederichsen E 1 

1 

Freie Universität Berlin, Institut für Biologie - Angewandte Genetik, Albrecht-Thaer-Weg 6, 

D-14195 Berlin, Germany 

² Universität Rostock, Agrar-und Umweltwissenschaftliche Fakultät, Institut für Landnutzung- 

Phytomedizin, Satower Str. 48, D-18059 Rostock, Germany 

Email: elked@zedat.fu-berlin.de 

INTRODUCTION 

Many fungi belonging to the genus Verticillium are major pathogens of crop plants, including 

cotton, tomato, strawberry and, more recently, also crucifers such as oilseed rape or 

cauliflower. Verticillium infections on crucifers are mainly caused by V. longisporum 

(Karapapa et al. 1997) and lead to leaf chlorosis, stem discoloration and – in particular in 

oilseed rape – premature ripening of the seeds. While the effects of the disease on whole crops 

are difficult to determine, Duncker et al. (2008) estimated that severely infected oilseed rape 

plants can show up to 80% reduction of single plant yield. V. longisporum starts its infection 

cycle by colonising the host roots, where fungal hyphae invade the root cortex and establish 

themselves in the vascular system of the root (Eynck et al. 2007). After formation of 

conidiospores the fungus spreads systemically inside the xylem into different parts of the shoot 

(Zhou et al. 2006; Duncker et al. 2008). The quantitative degree of this systemic colonisation 

seems to have an impact on disease symptom severity in Brassica napus (Duncker et al. 2008) 

and should have a significant impact on the pathogen’s reproduction rate. Host genotype, host 

development and environment have an influence on the systemic colonisation. Debode et al. 

(2005) could show in B. oleracea that very susceptible cultivars were colonised in the shoot 

already a few days after inoculation, while resistant cultivars were not systemically colonised 

throughout the whole experiment. A similar difference was found in B. napus between the 

susceptible cultivar cv. Falcon and the moderately susceptible cultivar cv. Talent by Duncker et 

al (2008), where cv. Talent was colonised significantly later and to a lower degree than cv. 

Falcon. However, this difference was observed only in greenhouse experiments. In field 

experiments environmental effects on the degree of shoot colonisation were more significant 

385

(Duncker et al. 2008). Zhou et al. (2006) studied the systemic colonisation of spring oilseed 

rape at different developmental stages and observed that shoot colonisation by V. longisporum 

occurred mainly after the onset of flowering, which is in accordance with the results from 

Duncker et al. (2008) from field trials. 

As host resistance against V. longisporum is the most promising method to control this disease, 

more knowledge about the genetic basis of resistance is needed. Arabidopsis thaliana is also a 

host for V. longisporum, it provides several useful features for studying the genetics of 

Verticillium resistance in crucifers. Previous workers have exploited these advantages and have 

identified several factors involved in resistance. Veronese et al. (2003) have identified a locus, 

Vet1, in the ecotype C24, which confers quantitative resistance and acts at the same time as a 

negative regulator of the transition to flowering. Johansson et al. (2006) identified two loci on 

chromosomes 2 and 3 in the Bay-0 x Shahdara recombinant inbred line (RIL) population which 

increased the resistance of ecotype Shahdara. They further found out that a certain allele of 

rfo1, which has originally been identified to control resistance to Fusarium oxysporum, also 

confers resistance against V. longisporum. The aim of our studies is the identification of genes 

controlling Verticillium resistance in Brassica and in Arabidopsis. Furthermore, genes involved 

in the relation between host development, pathogenesis and different resistance parameters will 

be addressed. Special emphasis is given on the inheritance of resistance against systemic 

colonisation. 


Seeds of B. napus, B. oleracea and B. rapa accessions were given to us by different gene 

banks, the Norddeutsche Pflanzenzucht (NPZ), the cabbage breeding company Rijk Zwaan 

Marne, or were bought as commercial cultivars. The V. longisporum isolate ‘43’ (Zeise & von 

Tiedemann 2002) was used for resistance tests. Ecotypes of Arabidopsis were supplied by 

Nottingham Arabidopsis Stock Centre (NASC; Nottingham, U.K.). Conidiospore suspensions 

for inoculation were produced in liquid Czapek- Dox medium on a shaker at 20°C. Spore 

densities were counted using a haemocytometer (Neubauer improved). 

In all greenhouse assays a root-dip inoculation procedure as described by Zeise & von 

Tiedemann (2002) was applied. For inoculation a conidiospore suspension of ca. 2x 10 6 spores/ 

ml was used and the roots of all plantlets were injured before dipping. Control plants were 

mock-inoculated in Czapek- Dox medium. All statistical analyses were done using the SAS 

9.1.3 statistical package. Resistance tests were carried out in greenhouse conditions with 

regular watering, insect control on demand and supplementary light to have long day 

conditions. 

Some procedures differed for Arabidopsis or Brassica: 

− Brassica seedlings were raised on sterile, moist sand for 12- 14 days before inoculation. 

After 50 min root-dip inoculation the plantlets were transplanted in multipot trays (1 pot 

386

= 4x4 cm) filled with nutrient poor commercial soil (Einheitserde P). From 21 to 49 days 

after inoculation (dai) the plants were scored weekly for disease symptoms on a 9-graded 

scale (1 = no symptoms, 3 = begin of leaf loss, 5 = slight stunting, 7 = severe stunting, 9 

= dead plant). Based on weekly disease scores the area under the disease progression 

curve (AUDPC) was calculated. After the final scoring the shoot fresh weight was 

measured and apical stem segments were sampled, surface sterilized and plated onto half 

concentrated malt agar. One stem segment per plant was cultured to test for fungal 

outgrowth. Plant developmental progress was weekly recorded using a modified BBCH 

scale. The developmental scores were used to calculate the area under the developmental 

progress curve (AUDevPC). 

− ArabidopsisArabidopsis seeds were stratified for 2 days and then transferred into a soilsand-mixture 

(1 l sand per 3 l Einheitserde P). The seedlings were grown for 19 days in a 

climatised greenhouse under long-day conditions at 20°C before inoculation. Inoculated 

plants were transferred to fresh soil (mixture like above) and grown to maturity, i.e. when 

the first siliques turned yellow, under normal greenhouse conditions. At this stage, the 

following parameters were recorded: Fresh weight, days to maturity, plant height, and 

systemic colonisation according to agar plate test (see above). 


Different parameters were recorded to characterize Verticillium resistance. In Arabidopsis 

disease severity based on morphological modifications such as stunting or fresh weight loss 

seems to vary stronger with seasonal influences than in Brassica (data not shown). Resistance 

to systemic colonisation appeared to be a more reliable parameter. Table 1 gives an overlook 

on screening results from selected host genotypes, indicating the degree of variation which can 

be found in the different species. The overall strongest resistance was found in B. oleracea, 

while B. napus was showing much less variation with cv. Oase being the least susceptible and 

cv. Falcon being regularly most susceptible. In B. oleracea and in Arabidopsis the accessions 

varied also for their developmental behaviour. In both species there was a tendency to 

increased colonisation resistance in slowly developing accessions. 

Development of mapping populations and mapping strategy 

To investigate the genetic basis of differences in these traits, we crossed accessions that 

differed strongly for resistance and development. Arabidopsis ecotypes Bur and Ler were 

chosen mainly on the basis of their ability to suppress systemic colonisation of the shoot 

system and their different developmental behaviour. Since all parameters vary as quantitative 

traits, a QTL-mapping approach was chosen. 

387

388 

Table 1. Summary of resistance screening results of selected host genotypes. 

Host Disease 

severity DS 1 

a) Arabidopsis 

Systemic 

colonisation 

Developmental type 

Ws + ++² Fast 

Sha - ++ Fast 

Hodja + ++ Medium 

Ler + ++ Fast 

Col + + Fast 

C24 + + Medium 

Bur variable - Slow 

Cal 

b) B. oleracea cytodeme 

variable - Slow 

B. alboglabra 24 + Medium, no vern.³ 

B. alboglabra 25 + Medium, no vern. 

B. alboglabra 94 ++ ++ Fast, no vern. 

B. alboglabra 99 - - Slow, no vern. 

Cabbage inbred 38 - - vern. 

Cabbage landrace 12 ++ ++ vern. 

Cabbage inbred 104 - - vern. 

cv. Verheul (curly kale) + vern. 

BRA1008 - - vern. 

BRA544 - - vern. 

BRA1398 + vern. 

B. drepanensis 

c) B. napus 

+ Very slow, vern. 

cv. Oase + vern. 

cv. Express + - ++ + vern. 

cv. Falcon ++ ++ vern. 

1 Brassica: DS is based on AUDPC; Arabidopsis: DS is based on stem length reduction 

² ++ = strong, + = medium, - = low 

³ vern. = Vernalisation required

Backcrossing 

followed by selfing 

F5 

(NILs) 

studying effects 

of individual QTL 

P 

(Bur) 

× 

F1 

F2 

F3 

F5 

s 

s 

s 

s 

s 

P 

(Ler) 

Genotyping 

Phenotyping 

s 

F6 

Genotyping 

for fine mapping 

for rough mapping 

s 

F7 

(RILs) 

Phenotyping 

Figure 1. Mapping population and mapping strategy in Arabidopsis. 

For rough mapping, an F2/F3-mapping population was established. A population of F2-plants 

originating from a single F1-plant was used for marker analysis. From each individual F2plant, 

F3-families were generated by selfing and used for phenotyping in resistance tests. A 

linkage map of polymorphic markers is being established with ~ 10 cM average marker 

spacing which should allow detection and rough localisation of major QTL. For fine-mapping a 

RIL population originating from individual F2-plants by selfing and single-seed-descent will 

be studied. The advantages of the RIL population are: 

− High-resolution mapping is possible with a moderate amount of lines to test, since the 

number of recombinations necessary to fine-map a locus is increased by repeated selfing. 

− F3-families are still segregating for many traits such as resistance parameters and 

flowering time, which makes scoring laborious. Recombinant inbred lines are 

homozygous for most loci and are much more homogeneous. 

However, dominance estimations of QTL are only possible in the F2/F3-mapping approach. 

After confining major QTL to relatively small chromosomal regions, identification and cloning 

of the underlying gene(s) is planned. Major QTL shall be studied in greater detail in near 

isogenic lines (NIL) after introgression into the susceptible background. Therefore F5-plants 

389

which are heterozygous for the locus of interest are backcrossed with the susceptible parent. By 

repeated backcrossing a population shall be obtained which segregates only for the locus of 

interest. 

In Brassica, two B. alboglabra accessions were chosen for mapping. B. alboglabra is a close 

relative of B. oleracea, which is fully cross-compatible and belongs to the same cytodeme. 

Opposite to most other B. oleracea forms, B. alboglabra does not require vernalisation. Two 

accessions of this species were identified who showed regularly contrasting reactions to 

Verticillium in terms of DS, colonisation rate and also for development. The resistant parent 99 

flowered much slower than the susceptible parent 94. Therefore, the segregating population can 

be used to study the inheritance of resistance and developmental behaviour. 

Figure 2. Mapping population and mapping strategy in B. alboglabra 

Mapping is done in a F2/ F3 population, comprising 153 F3- families, the major phenotyping 

experiments are made on F3- families. A back cross Population (BC1F1) will be used to verify 

the QTL which are identified in the F2/ F3 mapping. 

Phenotypical results from resistance testing of F3- families 

In B. alboglabra, the F1- generation was showing a similar level of resistance as the resistant 

parent, while the development was inherited in an intermediate mode. The F3- families 

segregated for DS in a more quantitative manner. Significant differences between different 

390 

99 94 

B. alboglabra x B. alboglabra 

(resistant) (susceptible) 

↓ 

F1-generation x B. oleracea 94 

↓ ↓ 

selfing 

↓ BC1F1 → Phenotyping 

Marker and QTL analysis, 

and QTL analysis ← F2-population verification 

↓ 

selfing

families and between controls vs. inoculated variants were proven by ANOVA or t-test, 

respectively. Only half of the families did show some colonisation, and very few families were 

colonised as strong as parent 94. Nearly 50% of the families were significantly delayed in their 

development due to inoculation. Some interesting correlations between different traits could be 

observed, indicating that the more the families were delayed, the more susceptible they were. 

Furthermore, DS was significantly correlated to the colonisation rate. 

The F3-families originating from the Bur x Ler cross in Arabidopsis showed continuous 

variation in the degree of colonisation, indicating polygenic inheritance. The majority of the 

F3-families as well as the F1-generation showed low colonisation rates, suggesting that 

colonisation resistance is inherited in a dominant manner. Development time showed 

transgressive variation in the late direction, suggesting that also the fast-developing ecotype 

Ler harbours genes which delay flowering. Verticillium infection induced an acceleration of the 

development in most F3-families, which was positively correlated with development time, i.e. 

slowly-developing plants were more accelerated. Most Arabidopsis plants showed different 

degrees of stunting due to Verticillium infection. The stunting reaction of the parental lines was 

complex. In summer, Bur was more resistant than Ler whereas in winter, it was more 

susceptible. Among the F3-families there was a significant negative correlation between the 

height of inoculated plants relative to mock-inoculated controls and the time to maturity, i. e. 

slowly-developing plants were more susceptible to stunting. In contrast to expectations, the 

degree of colonisation showed a weak positive correlation with relative height, i. e. severelycolonised 

plants were less stunted. 

OUTLOOK 

The phenotypical observations in segregating populations of both species demonstrate the 

complexity of the interaction between V. longisporum and its hosts. While it has been shown 

by others that V. longisporum follows certain developmental stages in Brassica for major 

events during pathogenesis, i.e. systemic spreading, we can assume that infection by V. 

longisporum itself can also influence the development of the host. Mapping of genes involved 

in this interaction is in good progress and will help to understand the role of plant development 

in this interaction and its interplay with development. 


The project is supported by a grant from Deutsche Forschungsgemeinschaft (DI 1502/1-1) and 

is part of the DFG- Forschergruppe FOR546. Grants given to S. Konietzki or E. Häffner by the 

NaFöG programme or the “Berliner Programm für Chancengleichheit”, respectively, are 

gratefully acknowledged. Excellent resistance testing support by M. Goltermann, Universität 

Rostock, is highly appreciated. Last, but not least, the financial support and encouragement 

given by the Norddeutsche Pflanzenzucht, in particular by Dr. M. Frauen, is gratefully 

acknowledged. 

391

REFERENCES 

Debode J; Declercq B; Höfte M (2005). Identification of cauliflower cultivars that differ in 

susceptibility to Verticillium longisporum using different inoculation methods. J. 


Duncker S; Keunecke, H; Steinbach P; Tiedemann A v (2008). Impact of Verticillium 

longisporum on yield and morphology of winter oilseed rape (Brassica napus) in 

relation to systemic spread in the plant. J. Phytopathology 156, 698-707. 

Eynck C; Koopmann B; Grunewaldt-Stoecker G; Karlovsky P; Tiedemann A v (2007). 

Differencial interactions of Verticillium longisporum and V. dahliae with Brassica 

napus detected with molecular and histological techniques. Eur. J. Plant Pathol. 118, 

259-274. 

Johansson A; Staal J; Dixelius C (2006). Early responses in the Arabidopsis- Verticillium 

longisporum pathosystem are dependent on NDR1, JA- and ET-associated signals via 

cytosolic NPR1 and RFO1. MPMI 19, 958-969. 

Karapapa V K; Bainbridge B W; Heale J B (1997). Morphological and molecular 

characterisation of Verticillium longisporum comb. Nov, pathogenic to oilseed rape. 

Mycol. Res. 101, 1281-1294. 

Veronese P; Narasimhan M; Stevenson R; Zhu J; Weller S; Subbarao K; Bressan R (2003). 

Identification of a locus controlling Verticillium disease symptom response in 

Arabidopsis thaliana. Plant J. 35, 574-587. 

Zeise K; Tiedemann A v (2002). Host specialization among vegetative compatibility groups of 

Verticillium dahliae in relation to Verticillium longisporum. J. Phytopathol. 150, 

112-119. 

Zhou L; Hu Q; Johansson A; Dixelius C (2006). Verticillium longisporum and V. dahliae: 

infection and disease in Brassica napus. Plant Pathol. 55, 137-144. 

392

Herrmann M, Ruge-Wehling B, Hackauf B, Klocke B, Flath K: Genetical Analysis of Resistance to Powdery 

Mildew in Triticale. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

7-4 Genetical Analysis of Resistance to Powdery Mildew in Triticale 

Herrmann M 1 , Ruge-Wehling B 1 , Hackauf B 1 , Klocke B 2 , Flath K 2 

1 

Julius Kühn Institute (JKI) - Institute for Breeding Research on Agricultural Crops, D-18190 

Groß Lüsewitz, Rudolf-Schick-Platz 3a; E-mail: matthias.herrmann@jki.bund.de 

2 

Julius Kühn Institute (JKI) - Institute for Plant Protection in Field Crops and Grassland; 

Stahnsdorfer Damm 81, D-14532 Kleinmachnow 

Abstract 

Epidemic incidence of powdery mildew has become an increasing challenge in 

triticale growing since 2004, demonstrating the need for additional efforts in 

resistance breeding against powdery mildew. We report on the genetic and 

molecular characterization of a novel powdery-mildew resistance gene in triticale 

by means of a detached-leaf test using single-pustule isolates (SPIs) and molecular 

markers for the chromosomal localization of the resistance gene. Detached-leaf 

segment tests with 14 highly virulent powdery mildew SPIs were performed on 15 

triticale cultivars and a JKI breeding line. The tests revealed resistance towards 13 

isolates in line JKI5015. Segregation analyses of BC, F2 and F3 families of crosses 

between JKI5015 and two susceptible triticale strains demonstrated a monogenic 

dominant inheritance of the resistance. The resistance gene was preliminarily 

designated as Pm 5015 and could be mapped to chromosome 6RL using rye-specific 

microsatellite markers. The identified linkage relationships between Pm 5015 and 

EST-derived markers enables the targeted development of additional molecular 

markers using a comparative-genetics approach and the rice genome data as a 

blueprint. 

INTRODUCTION 

The introduction of triticale in Europe was stimulated by its high level of resistance to leaf 

diseases. Particularly in areas with intensive growing a lower input of fungicides enhanced the 

competitive ability of triticale compared with wheat or barley. Since 1998 the growing area of 

triticale in Germany exceeded 300,000 ha, resulting in an adaptation of the powdery-mildew 

population to widespread grown triticale cultivars such as 'Trimaran'. Only sporadic incidence 

of powdery mildew in breeding nurseries and experimental plots of some breeding strains or 

393

cultivars was observed until 2003 (Scholze 1991; Schinkel 2002; Bundessortenamt 2002). In 

France, an incidence of powdery mildew in triticale was found since 1997 (Masson et al. 2003) 

and stronger in some cultivars in 2000 (Bouguennec et al. 2004). First observations of an 

increasing level of powdery-mildew incidence in triticale were reported in Germany and 

Poland in 2004 (Rodemann & Mielke 2007; Tischner et al. 2006; Wakuliński et al. 2005) and 

in Switzerland in 2005 (Mascher et al. 2006). Powdery-mildew isolates from triticale displayed 

virulence for wheat but not for rye, enabling the classification of the triticale mildew as 

Blumeria graminis f. sp. tritici (Scholze 1991; Felsenstein & Jaser 2006; Flath 2005; Arseniuk 

& Strzembicka 2008). 

The epidemic incidence of powdery mildew in triticale since 2004 demonstrated the need for 

additional efforts in resistance breeding against this pathogen. We report on the genetic and 

molecular characterization of a novel powdery-mildew resistance gene in triticale by means of 

detached-leaf tests using single-pustule isolates (SPIs) and molecular markers for the 

chromosomal localization. 


Plant materials and powdery-mildew isolates: A set of 15 cultivars as well as the line 

JKI5015 were tested for resistance to 14 highly virulent single-pustule isolates (SPIs) of 

powdery mildew (Table 2), selected out of a collection of 366 SPIs from all triticale-growing 

areas of Germany and Poland, respectively. The SPIs were obtained from 11 different locations 

and display different levels of virulence. The virulence complexity of the SPIs has been 

estimated on a differential set of 20 triticale lines and varies from 14 to 18. For example, a 

given SPI with a virulence complexity of 18 reacts compatibly with 18 of the 20 triticale lines 

of the differential set. Therefore, the SPIs can be classified as highly virulent. 

For segregation analysis regarding the resistance in JKI5015 the mildew isolate T41 was used, 

an isolate from Poland with complexity 16 selected from the first year of screening of the 366 

isolates. The triticale line JKI5015 is a progeny of the cross 'Aristos'/'Motto'/2/'Vision'/- 

3/'Kimon'/'Hakada'/2'Lasko' and one of the best-yielding lines from a prebreeding programme 

to widen the genetic basis of triticale (Herrmann 2006). For the genetic analyses BC1, F2, and 

F3 progenies derived from a cross between JKI5015 with the two susceptible triticale strains 

V10 and V31 were used (Table 3). A hard red winter wheat line (KS93WGRC2), homozygous 

for T6BS.6RL wheat-rye chromosome translocation, with resistance to powdery mildew, 

conditioned by the gene Pm20 on 6RL was kindly provided by Dr H. Bockelman (National 

Small Grains Collection, U.S. Department of Agriculture Agricultural Research Service). 

Resistance tests: The level of resistance was assessed in detached-leaf tests. For segregation 

tests leaf segments of ca. 2 cm were sampled from seedlings grown in a glasshouse and placed 

on the surface of benzimidazole agar (0.6% agar, 30 ppm benzimidazole) in clear rectangular 

polystyrene boxes. Each box accommodated 192 leaf segments from the seed leaves of 10- to 

16-day-old seedlings. The inoculation with powdery mildew was performed using an infection 

394

tower with an inter-changeable, 5 bar air-pressured plexiglas device. Freshly harvested spores 

of powdery-mildew which had been propagated on leaf segments of a susceptible triticale line 

were dispersed over the leaf segments with 100-600 spores per cm 2 . Following 8-10 days 

incubation in a growth chamber at 18-20°C, 16 h daylength with 4 kLx fluorescent lighting 

(Philips TLD 58W/25), reaction of leaf segments to powdery mildew was scored using a 0-5 

scale (Table 1). For segregation analysis, leaf segments with scores 0-2 were grouped as 

resistant, while scores ranging between 3 and 5 were summarized as susceptible. 

Table 1. Scale for scoring of triticale powdery-mildew resistance in leaf-segment tests 

(modified after Yu & Herrmann 2006). 

Score Symptoms 

0 no symptoms of infection 

1 sparse mycelium; very weak or no sporulation 

2 weak sporulation; mycelium covers up to 10 % of segment surface area 

3 moderate sporulation; mycelium covers 11-30 % of segment surface area 

4 abundant sporulation; mycelium covers 31-80 % of segment surface area 

5 abundant sporulation; mycelium covers more than 80 % of segment surface area 

In virulence tests detached-leaf segments of twelve-day-old primary leaves of every differential 

genotype were maintained in square plastic dishes with 12 compartments containing wateragar 

(5 g/l) with benzimidazole (35 ppm). SPIs were applied to the differential set using a settling 

tower which was placed over the plastic dishes with the leaf segments. Spores were sucked into 

an eyedropper pipette using a rubber teat which was then removed. The wider end if the pipette 

was placed through a hole in the top of the settling tower and spores were blown into the tower 

with a 10ml syringe connected to the narrow end of the pipette. To induce infection the plates 

were incubated at 16°C with continuous light (3000-4000 Lx). Twelve days after inoculation 

leaf-segment reactions were assessed according to the Nover (1972) scale for infection types. 

Infection types 0 to 2 were interpreted as incompatible (resistant/avirulent) and infection types 

3 and 4 as compatible (susceptible/virulent). 

Marker screening and mapping: The resistant and susceptible parents and three susceptible 

and resistant F2 individuals were used to screen molecular markers for polymorphism between 

the parents and the two groups of F2 individuals. A single F2 family (JKI5015/V10) 

encompassing 197 via progeny test defined resistance genotypes (Table 3) was used for linkage 

analysis. 

From the set of wheat-genomic microsatellites (or simple sequence repeats, SSRs) synthesized 

according to the sequences published by Röder et al. (1998), only those allocated to A- and Bgenome 

chromosomes were tested. Genomic and EST-derived rye microsatellite markers were 

analyzed as described previously (Hackauf & Wehling 2002; Hackauf et al. 2009). 

395

396 

Table 2. Resistance patterns of 15 triticale cultivars and JKI5015, tested with 14 

single pustule isolates of powdery mildew in detached-leaf segment test 

(0 to 2 = resistant; 3 and 4 = susceptible) 

Modus 

Logo 

Mildew 

SPI 

D29/17c 3 3 3 3 3 3 3 3 3 3 2-3 3 2-3 3 0 0 

D29/3c 3 3 3 3 3 3 0 3 1 0 2 3 2-3 3 0 0 

D29/9c 4 4 4 3 4 4 3 3 4 4 3 4 3 0 0 0 

D3/13 3 4 4 3 4 4 3 4 4 3 3 4 2 3 0 0 

D3/33 4 4 4 4 3 3 3 3 3 2 4 4 3 4 0 0 

TR10/18 3 3 2-3 3 4 0 3 3 2-3 0 2-3 2 2 2-3 0 0 

TR12/9 3 3 3 3 3 3 2-3 2-3 3 1 2-3 2 0 2-3 0 0 

TR16/7 4 4 4 3 4 3 2-3 3 2-3 2-3 2 3 3 2 0 0 

TR26/7 2-3 4 3 2-3 3 3 3 3 0 2 2-3 3 1-2 2 0 4 

TR35/1 4 4 3 2-3 4 4 2-3 2 2 2 2-3 3 1 2-3 0 0 

TR36/3 3 3 3 4 3 3 2-3 3 4 2-3 2-3 2-3 0 1-2 0 0 

TR38/3 3 3 3 2-3 4 4 2-3 3 2-3 0 3 3 2 2-3 0 0 

TR39/4 3 4 3 4 4 3 4 3 2 2 3 3 3 2-3 0 0 

TR9/10 3 4 3 4 4 4 4 4 2 2-3 4 4 2-3 2 0 0 

Ticino 

Lupus 

Lamberto 

Trimaran 

Vitalis 

Table 3. Reaction of parents; BC1 with male (m) and female (f) parents; F2 and F3 of crosses 

between powdery-mildew resistant line JKI5015 (female) and breeding strains V10 and 

V31 in leaf-segment tests. 

Korpus 

Cross Generation Number of plants Ratio Chi-square 

Resistant Segregating Susceptible tested probability 

V10 P 1 ♂ 0 21 

V31 P 2 ♂ 0 21 

JKI5015 P 3 ♀ 21 0 

JKI5015 1*/ V10 BC1f 19 0 

JKI5015 1*/ V31 BC1f 19 0 

JKI5015 /1* V10 BC1m 160 124 1:1 0.03 

JKI5015 /1* V31 BC1m 118 114 1:1 0.70 

JKI5015 / V10 F2 186 51 3:1 0.22 

JKI5015 / V31 F2 152 66 3:1 0.07 

JKI5015 / V31 F3 60 86 52 1:2:1 0.13 

JKI5015 / V10 F3 59 95 43 1:2:1 0.24 

RESULTS 

Among the set of cultivars tested, five were completely susceptible while 'Grenado' was the 

only one completely resistant to all SPIs tested (Table 1). For JKI5015 only one SPI showed a 

Tremplin 

Agrano 

Madilo 

Benetto 

Massimo 

Cando 

Grenado 

JKI5015

compatible reaction. This result was accomplished after the genetical characterisation of 

JKI5015 resistance. 

The backcrosses (BC1) of F1 progenies JKI5015/V10 and JKI5015/V31with JKI5015 (Table 

3) were invariantly resistant while the backcrosses to both susceptible parents segregated in a 

1:1 ratio. Additionally, the segregations of four F2 families derived from the crosses between 

JKI5015 and V10 and V31, and the lumped segregation data in the F2 populations with 638 

resistant and 212 susceptible plants each fit a 3:1 ratio, supporting the hypothesis that the 

powdery-mildew resistance in JKI5015 is genetically controlled by a single dominant gene. 

This was confirmed by the segregation pattern of 395 F3 families with 119 nonsegregating 

resistant families, 181 segregating families and 95 nonsegregating susceptible families, which 

fit a 1:2:1 F2 genotypic ratio (Table 3). 

All F2 plants with reactions scored 0–2 either gave 

nonsegregating resistant or segregating F3 progeny, while F2 

plants displaying reactions scored 3 or higher invariantly led to 

susceptible F3 progeny. Thus, the grouping of scores reflected 

the underlying resistance genotypes, i.e. PmPm or Pmpm versus 

pmpm. We have designated the analyzed powdery mildew 

resistance gene in line JKI5015 preliminary as Pm 5015 Xssr45 

5.2 

Xtcos1646 

. 

Molecular marker analyses allowed to identify 65 polymorphic 

wheat microsatellites out of 158 (41.1%) screened. A test for 

cross-species amplification of rye SSR revealed that 130 out of 

the 268 primer pairs did not allow to amplify a product from 

10.6 

genomic DNA of the wheat cultivars 'Chinese Spring' and 

'Topper', respectively. Thus, these 130 SSR markers appear to 

be specific for the rye genome. The polymorphism of rye 

microsatellites was lower compared to wheat SSR. Among 268 

primer pairs tested, 73 markers (27.2%) could by identified 

1.0 

Xtcos1659 

Xssr26 

being polymorphic between the parents, 35 of these markers are 

rye-genome specific. 

0.9 

1.0 

2.4 

6RL 

Xssr23 

Xssr40 

Pm5015 Pm5015 Pm5015 Figure 1. Linkage map 

for Pm 5015 . Distances 

are shown in cm at left 

side of the bar. 

be mapped to the target region on chromosome 6RL. 

Based on the chromosomal localization of the used SSR 

markers, linkage analysis allowed to assign Pm 5015 to the long 

arm of rye chromosome 6R (Fig. 1). Within a genetic interval of 

21.1 cM, Pm 5015 maps most distal and closely linked to the 

marker Xssr40. A comparative mapping approach using the 

sequence information of the mapped EST-derived SSR and the 

rice genome data as a blueprint allowed to bridge the Pm 5015 

genomic region on rye chromosome 6RL to rice chromosome 

R2. In an initial attempt, two additional conserved orthologous 

sequence (cos) markers, Xtcos1559 as well as Xtcos1646, could 

397

DISCUSSION 

The majority of triticale cultivars revealed a high level of resistance towards powdery mildew 

up to 2004. This situation has changed within a short period of time due to the adaptation of 

Blumeria graminis f. sp. tritici to the relatively young crop triticale. The durability of 

resistance to powdery mildew in former triticale cultivars was not solely a consequence of the 

limited growing area. For instance, although cv. 'Modus' had a two-year prior release and larger 

growing areas than 'Trimaran', its resistance to powdery mildew remained longer effective 

(Bundessortenamt 2008). The resistance of cv. 'Modus' even outperformed resistant cultivars 

such as 'Lamberto' or 'Versus'. These observations point to the expression of different 

resistances genes in recently released cultivars. 

Results obtained in our study using different SPIs support the assumption of many different 

powdery-mildew resistance genes in triticale. Except for the completely susceptible cultivars, 

most of the analyzed triticales displayed different resistance patterns towards the SPIs used. 

The analysis of the resistance genetics in further cultivars and breeding strains is in progress. 

We were able to map the Pm resistance gene of JKI5015 to the long arm of rye chromosome 

6R. Several rye genes conferring resistance towards powdery mildew in the genetic 

background of wheat have been described (Zeller & Fuchs 1983; Heun & Fischbeck 1987; 

Heun et al. 1990; Huang et al. 2002; Hysing et al. 2007). Heun & Friebe (1990) reported on a 

Pm resistance gene on chromosome 6R, which originated from the rye cv. 'Prolific' as addition 

or substitution in a wheat background. Friebe et al. (1994) developed a hard red winter wheat 

line (KS93WGRC2), homozygous for T6BS.6RL wheat-rye chromosome translocation, with 

resistance to powdery mildew, conditioned by the gene Pm20 on 6RL. We have observed this 

line as susceptible to the triticale powdery mildew isolate T41. Thus, the gene Pm 5015 described 

in this work appears to be not identical with Pm20. 

For marker-aided selection the resistance gene Pm 5015 should be tightly clamped by markers, 

thereby the SSR Xssr40 with 2.4 cM distance is a first important candidate for marker-aided 

selection. 

Actually, most commercial triticale cultivars appear susceptible to powdery mildew in 

detached-leaf tests yet partially resistant in the nursery showing adult plant resistance. The 

rapid adaptation of powdery mildew within the last years shows the need for intensifying 

research of resistance to powdery mildew in triticale to support the breeding of more durable 

resistant cultivars. 


The authors thank Gunda Kölzow, Renate Pfleiderer, Heike Rudolph and Ines Tessenow for 

valuable technical assistance. This work is supported by the Innovation Programme of the 

Federal Ministry of Food, Agriculture and Consumer Protection (BMELV). 

398

REFERENCES 

Arseniuk E; Strzembicka A (2008). Emerging virulences of Blumeria graminis sp. on triticale 

in Poland. http://www.endure-network.eu/international_conference_2008/abstracts- 

_online/posters/p_61_emerging_virulences_of_blumeria_graminis_sp_on_triticale_in_ 

poland (12-15. October 2008) 

Bouguennec A; Bernard M; Jestin L; Trottet M; Lonnet P (2004). Triticale in France. In: 

Triticale improvement and production, eds M Mergoum & H Gomez-Macpherso, FAO, 

pp. 109-114, ftp://ftp.fao.org/docrep/fao/009/y5553e/y5553e02.pdf. 

Bundessortenamt (2002). Beschreibende Sortenliste für Getreide, Mais, Ölfrüchte, 

Leguminosen und Hackfrüchte. Strothe Verlag: Hannover. 

Bundessortenamt (2008). Beschreibende Sortenliste für Getreide, Mais, Ölfrüchte, 

Leguminosen und Hackfrüchte. Strothe Verlag: Hannover. 

Felsenstein F G; Jaser B (2006). Fungizidresistenz bei pilzlichen Getreidepathogenen und 

Wirksamkeit der vertikalen (qualitativen) Mehltauresistenz bei Weizen und Gerste. 

Situationsbericht 2006. http://www.epilogic.de. 

Flath K (2005). Zunehmende Anfälligkeit von Triticale gegenüber Mehltau. 

http://www.jki.bund.de/nn_806762/DE/veroeff/jb/jb2005/A__inst.html (2005). 

Friebe B; Heun M; Tuleen N A; Zellet F L; Gill B S (1994). Cytologically monitored transfer 

of powdery mildew resisance from rye into wheat. Crop Sci. 34, 621-625. 

Hackauf B; Rudd S; van der Voort J R; Miedaner T; Wehling P (2009). Comparative mapping 

of DNA sequences in rye (Secale cereale L.) in relation to the rice genome. Theor Appl 

Genet. DOI 10.1007/s00122-008-0906-0. 

Hackauf B; Wehling P (2002). Identification of microsatellite polymorphisms in an expressed 

portion of the rye genome. Plant Breeding 121, 17-25. 

Herrmann M (2007). Widening the genetic base of triticale via crosses with primary triticale. 

Vortr. Pflanzenzüchtg. 71, 59-61. 

Heun M & Fischbeck G (1987). Identification of wheat powdery mildew resistance genes by 

analysing host—pathogen interactions. Plant Breeding 98, 124-129. 

Heun M; Friebe B (1990). Introgression of powdery mildew resistance from rye into wheat. 


Heun M; Friebe B; Bushuk W (1990). Chromosomal location of the powdery mildew 

resistance gene of Amigo wheat. Phytopathology 80, 1129-1133. 

Huang X, Hsam S L, Zeller F J (2002). Chromosomal location of genes for resistance to 

powdery mildew in Chinese wheat lines Jieyan 94-1-1 and Siyan 94-1-2. Hereditas 

136, 212-218. 

Hysing S H, Hsamb S L K, Sing R P, Huerta-Espinod J, Boyde L A, Koebner R M D, 

Cambron S, Johnsong J W, Bland D E, Liljeroth E, Merker A (2007). Agronomic 

Performance and Multiple Disease Resistance in T2BS.2RL Wheat-Rye Translocation 

Lines. Crop Sci 47, 254-260. 

Mascher F; Reichmann P; Schori A (2006). Einfluss des Mehltaus auf den Triticaleanbau. 

AGRARForschung 13 (11-12), 500-504. 

Masson E; Ruch O; Hébrard J-P (2003). Dossier Triticale. Des atouts consolidés. Perspectives 

Agricoles 288, 26-27. 

Nover I (1972). Untersuchungen mit einer für den Resistenzträger 'Lyallpur 3645' virulenten 

Rasse von Erysiphe graminis DC. f. sp. hordei Marchal. Arch. Pflanzensch. 8, 439-445. 

399

Rodemann B; Mielke H (2007). Zum Anbau und Pflanzenschutz des Triticale. Mitteilungen 

aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem 409, 

Berlin und Braunschweig. 

Röder M S; Korzun V; Wendehake K; Plaschke J; Tixier M H; Leroy P; Ganal M W (1998). A 

microsatellite map of wheat. Genetics 149, 2007-2023. 

Schinkel B (2002). Triticale – still a healthy crop? In: Proceedings of the 5 th International 

Triticale Symposium, Vol. I, June 30-July 5, 2002, ed Arseniuk E, pp. 157-162. 

Radzików: Poland. 

Scholze P (1991). Mehltauspezialisierung und Feldresistenz bei Triticale. Vortr. Pflanzenzüchtg. 

19, 339-340. 

Tischner H; Schenkel B; Eiblmeier P (2006). Monitoring für Getreidekrankheiten in Bayern – 

mehrjährige Auswertungen über die Bedeutung der einzelnen Schaderreger. Gesunde 

Pflanzen 58, 34-44. 

Wakuliński W; Zamorski C; Nowicki B; Schollenberger M; Mirzwa-Mróz E; Mikulski W; 

Konieczny M (2005). Fungus Blumeria graminis (DC) Speer as serious risk for triticale 

in Poland. Prog. Plant Protection/Post. Ochr. Roślin. 45(1), 505-510. 

Yu J; Herrmann M. (2006). Inheritance and mapping of a powdery mildew resistance gene 

introgressed from Avena macrostachya in cultivated oat. Theor Appl Genet (2006) 113, 

429-437. 

Zeller F J; Fuchs N (1983). Cytologie und Krankheitsresistenz einer 1A/1R- und mehrerer 

1B/1R-Weizen-Roggen-Translokationssorten. Z. PflZücht. 90, 285-296. 

400

Kairu G M: Management of Coffee Leaf Rust, Hemileia Vastatrix, in a Changing Climate. In: Feldmann F, Alford 

D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 401-406; ISBN 978-3-941261-05-1; © 


7-5 Management of Coffee Leaf Rust, Hemileia Vastatrix, in a Changing 

Climate 

Kairu G M 

Coffee Research Foundation, P.O. Box 4-00232 Ruiru, Kenya 

E-mail: gmkairu@yahoo.com 

Abstract 

In a fungicide evaluation trial conducted during 2007, copper sprays applied as a 

protective programme failed to control coffee leaf rust effectively. Timing of copper 

sprays was rendered ineffective because of varying climatic conditions. Unexpected 

out-of-season rains fell before protective sprays were applied. Copper sprays 

managed to reduce the incidence of coffee leaf rust by only 15% to 24%, thus 

allowing about 55 to 61% coffee leaf rust to prevail. The resultant tree defoliation in 

sprayed plots was estimated at 80 to 90%. 

INTRODUCTION 

Coffee leaf rust (Hemileia vastatrix) is an important disease of Coffee (Coffea arabica) 

cultivars in Kenya. About 80% of coffee plantations consist of the susceptible cultivars: SL28, 

SL34, K7, and Blue Mountain. The disease was first reported in Kenya in 1913 (Rayner 1960). 

It is currently managed using various recommended fungicides as well as the new resistant 

variety, Ruiru -11. New fungicide active ingredients and formulations are evaluated to 

determine their efficacy against coffee leaf rust as well as any deleterious effects on the tree 

phenology and productivity before they are recommended for use by growers. The data 

generated are also required for pesticide registration purposes. Coffee leaf rust develops in 

polycyclic epidemics after the short rains (October & November) and the long rains season 

(March, April & May). Affected trees are gradually defoliated through out the season. In 

severe epidemics, all leaves may be shed resulting in depletion of carbohydrate reserves in the 

wood and eventual death of trees. This happened around 1880 in Sri Lanka (then known as 

Ceylon) where, Marshall Ward, a British Plant Pathologist who was sent to investigate the 

problem, reported that: “It was not possible to avert the disaster” because “Plant Pathology was 

still too much in its infancy for satisfactory control methods to be found.” The plantations were 

401

abandoned. Consequently, the drinking habits of the British people changed from coffee to tea 

(Jones 1987). This historical reference has created awareness about the devastating effects of 

coffee leaf rust on coffee production if effective control measures are not put in place. 

Uncontrollable coffee leaf rust epidemics therefore have disastrous effects on coffee farming. 

This concern is further compounded by the observation that the incidence of coffee leaf rust is 

increasing in the cooler, higher altitude (>1800m) plantations where the disease was not 

previously prevalent. Against this background, the Coffee Research Foundation has maintained 

a fungicide evaluation research programme in order to keep abreast with modern development 

of active ingredients and formulations against coffee diseases. Seven different active 

ingredients with systemic activity against coffee leaf rust have been evaluated and 

recommended for use by growers since 1979. 

Successful spray timing with protective fungicides against coffee leaf rust is based on 

knowledge about weather conditions, especially predictability of the onset of seasonal rains. 

Protective sprays must be applied before onset of seasonal rains, usually before the 

October/November short rains. These sprays eradicate the spore load on leaf surfaces before 

occurrence of the right weather conditions for germination, formation of appressoria and 

infection. The management of coffee leaf rust using various formulations of copper and the 

implications of changing climate are reported and discussed. 


A fungicide evaluation trial was carried out at the Coffee Research Foundation – Azania Estate 

in 2006/2007 period. The treatments were: Nordox 75 % WP (0.22 %)- cuprous oxide 

formulation of Nordox Industrier, Norway; Kocide 2000 (0.26%); Kocide 2000 (0.35 %)- 

cupric hydroxide formulation of Griffin Corporation; Cobox 50 % WP (0.35 %)- copper 

oxychloride formulation of BASF Corporation and Unsprayed (control). These treatments were 

applied using motorized knapsack sprayers on 25-tree plots in randomized complete block 

design with 4 replications. A spray interval of 2 to 3 weeks was maintained starting before the 

short rains season in October 2006 and the long rains season in 2007. A random sample of 70 

leaves was sampled from each plot and the percentage of leaves bearing one or more H. 

vastatrix pustules determined. Tree defoliation caused by infection of H. vastatrix was 

determined by counting all fallen leaves under the canopy of each tree and expressing the 

number infected as a % of the total. Rainfall measurements were taken on site daily and leaf 

wetness data recorded automatically using a De Wit leaf wetness recorder. 

RESULTS 

Sprays of all copper-based treatments applied on a protective programme failed to manage 

CLR effectively (P≤ 0.05, Table 1). Copper sprays managed to reduce the incidence of CLR by 

only 15% to 24% - thus allowing about 55 to 61% CLR to prevail. 

The incidence of CLR escalated in July and August in 2007 (Fig.1). This was about two 

402

months after the end of the protective spray programme. The CLR peak recorded in August 

was out of season. In normal weather, peak incidence of CLR would have occurred in 

May/June. Comparison of monthly rainfall totals for 2007 with mean monthly rainfall totals for 

a period of 12 years, indicated no major differences to account for the observed upsurge of 

CLR (Fig. 2) The frequency of wet periods lasting over three hours during the night increased 

in June, July, August and September (Fig 3). This period is normally dry. The resultant tree 

defoliation in all sprayed treatments ranged from 80% to 90% (Fig.1). Elsewhere, increasing 

incidence of CLR was recorded in the cooler, higher altitude plantations which were free of the 

disease in the past (Table 2). 

Table1. Effect of copper sprays against Coffee Leaf Rust (CLR) at Azania Estate Trial 

# 8 in 2007 

Treatment* Peak % CLR 

21/8/2007 

Nordox 75 % WP (0.22 % (std) 55.16 A 

Kocide 2000 (0.26%) 61.31 A 

Kocide 2000 (0.35 %) 59.25 A 

Cobox 50 % WP (0.35 % ) (std) 54.84 A 

Unsprayed (control) 72.26 A 

CV (%) 16.01 

* Spray application dates: 11 th , 29 th October, 2006; 11 th March; 8 th , 28 th May, 2007. Means 

sharing the same letter are not significantly different at P≤0.05 

CLR 

% 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

MAR MAY JUN JUL AUG SEP OCT 

Figure 1. Seasonal incidence of coffee leaf rust in unsprayed plots and resultant 

defoliation at Azania VIII - 2007 

403

404 

Table 2. The Incidence of Coffee Leaf Rust at different altitudes during 2007 

SITE ALTITUDE (m) PEAK % CLR 

Azania VIII 1570 72.26 

Rukera II 1600 64.70 

Kamundu I 1830 10.25 

Tinganga I 1875 8.75 

Yara 1950 5.14 

250 

200 

150 

100 

50 

0 

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 

Figure 2. Monthly rainfall pattern (in mm) at Azania Estate for: 2007 compared with 

twelve year means (1979 to 1990) 

DISCUSSION AND CONCLUSIONS 

After the end of copper spray programme at the Azania site in 2007, out-of-season wet weather 

conditions occurred, which favoured the escalation of coffee leaf rust. In the past, two main 

peaks of coffee leaf last occurred each year in the plantations east of the Rift Valley in Kenya, 

one in February/March and the other in May/June (Nutman & Roberts 1972). Successful 

management of these epidemics depended on the use of well timed sprays of protective copper 

fungicides. Spray timing is based on knowledge about weather conditions, especially the onset 

of seasonal rains. Protective sprays must be applied before onset of seasonal rains, usually 

before the October/November short rains. These sprays eradicate the spore load on leaf 

surfaces before occurrence of the right conditions for germination, formation of appressoria 

and infection by H. vastatrix.

Frequency Hrs. wet 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

May June July Aug Sept Oct 

Figure 3. Frequency and duration of wet periods at Azania trial VIII in 2007: 

Frequency of wet periods ≥ 3 hours at night 

Total hours wet at night 

Coffee leaf rust develops under wet, warm weather. Germination of H. vastatrix uredospores 

requires: water film for 1- 3 hrs, usually in darkness, at 20-25°C, optimum 23°C. Heavy, winddriven 

rains play a major role in dispersal of uredospores from leaf to leaf and from tree to tree. 

These critical requirements and especially the frequency and duration of wet periods of 

darkness occurred after the normal disease season from June to September. It is likely that 

these favourable conditions did not prevail during the spray application period, October 2006 

to May 2007 resulting in low incidence of the disease. For instance, the incidence of coffee leaf 

rust remained low in spite of above normal monthly total rainfall in the period April/May, 

2007. In contrast, light out- of-season rains and cloud cover from June to September resulted in 

a higher frequency of wet periods of three or more hours at night. This was conducive to the 

infection of H. vastatrix leading to a massive epidemic in both sprayed and unsprayed plots. 

The timed copper spray programme applied earlier in the season, was therefore rendered 

ineffective because of the contact mode of action of the active ingredient, its limited 

persistence and varying seasonality of the disease instigated by climatic changes. Such 

uncontrollable coffee leaf rust epidemics could have disastrous effects on coffee farming. 

Fortunately, these epidemics can be stopped with sprays of systemic fungicides. The Coffee 

Research Foundation has so far evaluated and recommended seven systemic fungicide 

formulations of various active ingredients for use by growers since 1979. So far these have 

been effective in managing coffee leaf rust epidemics arising from favourable out-of-season 

weather conditions or mistimed sprays of contact fungicides. 

405


This paper is published with the permission of the Director of Research, Coffee Research 

Foundation, Kenya. 

REFERENCES 

Jones D G (1987). Plant Pathology: Principles and Practice. Open University Press: Milton 

Keynes, UK. 

Nutman F J; Roberts F M (1972). Coffee leaf rust. Kenya Coffee 36, 139-159. 

Rayner R W (1960). Rust disease of coffee 2 - Spread of the disease. World Crops 13, 

222-224. 

406

Hammann T, Truberg B, Thieme R: Improving Resistance to Late Blight (Phytophthora Infestans) by Using 

Interspecific Crosses in Potato (Solanum Tuberosum Ssp.). In: Feldmann F, Alford D V, Furk C: Crop Plant 



8-1 Improving Resistance to Late Blight (Phytophthora Infestans) by Using 

Interspecific Crosses in Potato (Solanum Tuberosum Ssp.) 

Hammann T, Truberg B, Thieme R 

Julius Kühn Institute, Institute for Breeding Research on Agricultural Crops, Rudolf-Schick- 

Platz 3a, OT Groß Lüsewitz, D-18190 Sanitz, Germany 

Email: thilo.hammann@jki.bund.de 

ABSTRACT 

Late blight (Phytophthora infestans) (P.i.) is the most serious disease in potato 

production worldwide and causes tremendous losses in yield and high costs in 

chemical plant protection. A worthwhile approach to combat the disease is breeding 

for late-blight resistance. Wild species of potato are potential sources of P.i.resistance 

genes. In the past, though, genes conferring race-specific resistance were 

rapidly overcome due to the extremely high adaptation rate of the pathogen. An 

alternative strategy in breeding for resistance is to use quantitative or non-specific 

resistance allowing the survival of late blight on a low level without genetic 

adaptation. In a long-term breeding programme at Groß Lüsewitz Experimental 

Station, several wild species have been used for many years. In 2007 and 2008 a 

series of advanced breeding clones from a backcross programme involving different 

wild relatives were investigated for their reaction to late blight. A maturitycorrected 

score was used to separate resistance and late-maturity effects. We 

describe 11 breeding clones which display higher degrees of quantitative P.i.resistance 

in the field combined with earlier maturity as compared to standard 

varieties. Besides, the clones showed high resistance against tuber blight, too. 

Notably, besides improved resistance to P.i. these clones possess acceptable 

agronomic traits with regard to starch content, suitability for crisp and chips 

production and acceptable levels of table quality, respectively. 

INTRODUCTION 

Potato (Solanum tuberosum ssp. tuberosum) is one of the most important staple foods. On a 

worldwide scale, potato ranks fourth as a crop for human nutrition. In Germany potatoes were 

grown on 259,800 ha in 2008 (Statistisches Bundesamt 2009). Besides food and processed 

407

potato consumption, potato is also important for industrial purposes like starch production. The 

clonal propagation of potato favours infestation by various diseases and pests. The most 

important disease worldwide is late blight on foliage and tubers caused by the oomycete 

Phytophthora infestans (P.i.). Plant protection against late blight requires high amounts of 

pesticides each year, with up to 14 sprayings per field. Costs caused by this pathogen are due to 

the need of chemical protection as well as to losses in marketable yield and amount to 470 

EUR per ha in Germany (Darsow 2008). Therefore, pre-breeding to improve resistance to 

potato diseases and pests constitutes an important task. In the past, breeding for late-blight 

resistance relied on single dominant genes (R-genes R1 to R11) which were pathotype-specific. 

The simple way of inheritance and an easy procedure in resistance testing are the advantages of 

this approach. The development of DNA markers for selection of resistant plants is 

comparatively easy, too. However, effectiveness of these genes had not been durable due to 

extremely high adaptation rates of the pathogen (Fry & Goodwin 1997). An alternative strategy 

in breeding for resistance is to use quantitative or non-specific resistance which acts 

quantitatively and incompletely, allowing the survival of late blight on a level sufficiently high 

to avoid selection pressure in favour of virulent races and low enough to restrict the impact on 

marketable yield. 

We have focussed, thus, on the quantitative-resistance approach. Quantitative resistance is 

controlled by several to many genes, and maintains to be effective for a long period. Attention 

must be taken, though, that genetically, foliage vs. tuber blight have to be considered as 

different diseases and real quantitative resistance is often mimicked by the strong positive 

correlation between foliage-blight resistance and late maturity. Although breaking this 

correlation seems to be difficult in general, considerable breeding progress may be achieved as 

is demonstrated in the present contribution. Another challenge in potato breeding is put by the 

large number of approximately 70 traits which breeders have to be aware of when introducing 

polygenes for quantitative disease-resistance from non-adapted genetic resources into adapted 

germplasm. 


Sources of blight resistance and breeding strategy 

Wild relatives of cultivated potato are important sources for resistance genes and have been 

used to produce interspecific hybrid plants for many years. Late-blight resistance is taken from 

Solanum wild species which originated in the centres of genetic diversity of potato and its 

pathogens and, thus, have experienced thousands of years of co-evolution with P. infestans. 

For decades late-blight resistance was tried to be introduced from S. demissum, S. stoloniferum, 

S. acaule and S. chacoense in a conventional sexual way. To widen the spectrum of potential 

sources for resistance, bridge crosses and somatic hybridisation with S. circaeifolium, S. 

bulbocastanum, and S. pinnatisectum were used to overcome crossing-barriers between 

tetraploid cultivars and wild diploid Solanum species with high disease resistance (Thieme 

408

1997). Subsequently, interspecific progenies were backcrossed several times to adapt these 

progenies to the cultivated type of potato. 

The breeding clones used in the present study were developed via conventional crosses with 

the wild species of S. demissum, S. stoloniferum and S. okadae as resistance donors or via 

somatic hybridisation with S. circaeifolium, S. bulbocastanum or S. pinnatisectum as resistance 

donors. The wild species used and the number of clones tested are listed in Table 1. 

Additionally, 12 varieties and 19 clones selected from crosses of tetraploid, conventional 

cultivars were tested in the same trial for their leaf-blight and tuber-blight reaction. 

Table 1. Number of breeding clones developed from Solanum wild species by 

interspecific hybridization which were tested for different characters in 2007 

and 2008 

Assessment of late-blight resistance 

Field test 

Solanum wild species used 

for interspecific hybridization 

Number of 

clones 

tested 

Solanum demissum 41 

Solanum okadae 23 

Solanum circaeifolium 8 

Solanum stoloniferum 7 

Solanum bulbocastanum 2 

Solanum pinnatisectum 1 

Clones were cultivated as double-row plots with 12 plants per plot. Inoculation was done at the 

beginning of flowering of cv. 'Adretta'. The inoculum of P. i. consisted of a mixture of 

common races collected in the field in 2006 and 2007. The trial field was bordered by a strip of 

hemp 3 m in width to provide protection against wind and maintain a humid environment. 

Additionally, irrigation was carried out in the evening if necessary. 

The lowest leaves of each first plant in a row were inoculated with 5 ml spore suspension (12 x 

10³ zoosporangia/ml) in the evening. Scoring started 5 dpi as percentage of attacked area of 

potato tops. Scoring was done twice a week until the stage of maturity (80-90% of the leaves 

have turned to yellow). Quantitative resistance of foliage blight was assessed as Area Under 

Disease Progress Curve (AUDPC) (Fry 1978, Colon 1994) and as Relative Area Under Disease 

Progress Curve (rAUDPC) (Hansen et al. 2002, 2003). To settle rAUDPC from the strong 

influence of maturity a transformation into delta (∆) rAUDPC was calculated (Bormann 2003). 

Stage of maturity and other agronomic characters of each clone were recorded in a field trial 

fully treated with fungicides. 

409

Detached-leaf test 

A detached-leaf test was carried out with five leaves per entry. Inoculation was done with one 

drop of a P.i. suspension (apprx. 1 µl; 15 x 10³ zoosporangia/ml) per leaf and incubated for 5 

days at 19 °C and 95% relative humidity (RH) at 150 Lx. Size of necroses and mycelium 

development were estimated after 5 days on a 1 to 9 scale, with score 1 meaning no attack 

visible and score 9 indicating for leaf area completely necrotic and covered with mycelium. 

Tuber Test 

The tuber test was carried out with 30 washed tubers of each clone. The tubers were dipped in 

a spore suspension (12 x 10³ zoosporangia/ml) and stored in the dark at 100 % RH and 19 °C 

for 1 day. After inoculation tubers were incubated for 7 days at 19 °C and 85 % RH in the dark. 

Tuber resistance was scored individually for each tuber on a 1 to 9 scale and a mean score was 

calculated, with score 1 meaning no attack visible and score 9 indicating for 100% decay of 

tuber tissue. 

Tubers showing no symptoms of late-blight infestation were scored a second time 12 days later 

and their indices summarised with the ones from the first assessment to yield a combined 

infestation index (Darsow 2008). 


In Table 2 the Phytophthora reaction and other key traits of 21 advanced breeding clones, the 

two breeding clones 99.8084.01 and 03.5143.07 used as highly susceptible standards and the 

standard varieties 'Adretta' and 'Sarpo Mira' are summarised. The breeding clones showed 

significantly higher degrees of quantitative P.i.-resistance in the field and varied between 0% 

and 26.8% for foliage-blight attack on the average of 2007 and 2008. The year 2007 generally 

showed very high degrees of natural infection all over Germany. In both years the clones 

04.5170.02 and 04.5197.01 did not show any leaf attack in the field. Scores between 1.1 for 

resistant clones and 5.6 for the highly susceptible clone 03.5143.07 were determined in the test 

of detached leaves. The older variety 'Adretta' had a score of 4.9, which indicated for a higher 

susceptibility to the pathogen. Most of the clones which were resistant in the field test 

displayed good resistance in the detached-leaf test, too. However, there were a few clones 

displaying higher susceptibility in the detached-leaf test, e.g., clones 04.5214.03 and 

03.5131.01. 

Most of the clones with a high level of resistance against foliage blight in the field trial 

demonstrated a moderate susceptibility in the tuber test. The standard varieties and the highly 

susceptible clones explained higher degrees of susceptibility against tuber attack. In contrast, 

clone 04.5170.02 which was completely resistant to leaf blight in the field, showed a higher 

degree of attack on tubers. Leaf- and tuber-blight resistance indices were not correlated in these 

trials and confirmed that foliage vs. tuber blight have to be considered as different diseases. 

410

Breeding 

Clone 

Table 2. Reaction to Phytophthora and key traits of selected clones in comparison to 

standard varieties (in bold) in the years 2007 and 2008 

Field 

test 

Phytophthora attack 

(%) rAUDPC Test score 

Field 

test 

Field 

test 

Leaf 

test a 

Tuber 

test b 

2007 2008 Mean Score Index 

Matu 

rity c 

Starch 

content 

(%) 

Suitability 

for 

Crisps d Chips d 

Table quality 

99.8084.01 70.9 65.8 68.4 3.9 5.1 4.1 18.6 2 6 medium/good 

03.5066.02 7.5 0.3 3.9 1.1 3.2 6.5 21.2 2 5 low/medium 

03.5066.03 4.9 0.1 2.5 1.2 3.2 6.5 19.3 3 5 low/medium 

03.5067.04 7.4 5.1 6.3 1.3 2.0 3.3 16.9 1 4 good 

03.5131.01 32.5 21.1 26.8 5.2 4.1 4.3 23.5 3 5 medium 

03.5143.07 73.7 62.8 68.3 5.6 5.1 5.1 21.8 2 5 medium 

04.1465.03 7.7 12.3 10.0 1.1 2.2 5.4 14.6 1 4 medium/good 

04.5170.02 0.0 0.0 0.0 3.8 5.3 7.1 19.2 2 5 medium 

04.5175.02 11.0 14.0 12.5 1.2 2.4 3.7 16.0 1 5 medium/good 

04.5182.09 4.3 6.6 5.5 4.1 3.5 5.0 19.0 2 4 medium/good 

04.5191.01 1.9 4.4 3.2 1.5 1.9 3.4 18.2 2 5 medium 

04.5197.01 0.1 0.0 0.1 1.2 3.4 5.1 21.0 3 6 medium 

04.5214.03 7.0 6.4 6.7 4.5 4.2 5.1 24.1 3 6 medium/good 

04.5224.01 18.6 13.9 16.3 1.4 1.2 2.7 17.7 3 4 medium/good 

04.5228.07 3.6 1.7 2.7 1.2 2.4 4.0 22.8 3 5 medium 

04.5228.08 3.7 3.9 3.8 1.4 2.3 2.9 21.6 2 7 medium 

04.5229.01 0.1 8.2 4.2 1.1 3.8 3.1 16.4 2 6 medium 

04.5229.02 2.8 3.7 3.3 1.2 3.1 4.4 17.1 1 5 good 

04.5229.04 11.3 5.8 8.6 1.2 3.1 3.3 20.2 3 6 medium 

04.5230.04 29.3 12.4 20.9 1.3 2.0 3.7 20.2 3 7 medium 

04.5230.07 14.1 4.1 9.1 1.2 2.3 5.4 16.9 4 7 good 

04.5230.14 27.9 10.6 19.3 1.9 2.1 3.7 18.2 5 7 medium 

04.5230.16 16.0 13.6 14.8 1.2 2.1 3.2 17.6 4 8 medium 

Sarpo Mira 2.1 3.4 2.8 4.4 6.6 7.3 17.4 4 6 medium 

Adretta 

Mean 

86.0 68.8 77.4 4.9 5.4 3.6 17.3 2 4 medium 

(n=113) 46.0 39.0 42.0 2.4 3.3 4.5 19.1 2.5 5.5 

LSD (5%) 19.0 

a b 

Score 1= no attack, 9 = leaf area completely necroticised and covered with mycelium, Index 1 = no attack, 9 = 

100 % decay of tuber tissue, c Maturity 1 = very early, 9 = very late, d Suitability for Crisps and Chips 1 = low, 9 = 

high 

411

Phytophthora attack (%) rAUDPC 

Phytophthora attack (∆ rAUDPC ) 

412 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Eersteling 

04.5224.01 


Maturity 

LSD 5% for 


04.5228.08 

04.5229.01 

03.5067.04 

04.5191.01 

Adretta 

04.5229.02 

04.5182.09 

Esprit 

04.5214.03 

04.5230.07 

Bintje 

03.5066.03 

04.5170.02 

Kuras 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

Maturity (1 = early; 9 = late) 

Sarpo Mira 

Figure 1. Phytophthora attack (rAUDPC) vs maturity scores of selected breeding clones 

and varieties in the mean of the years 2007 and 2008 

0,4 

0,3 

0,2 

0,1 

0 

-0,1 

-0,2 

-0,3 

-0,4 

-0,5 

-0,6 

Eersteling 

04.5224.01 

04.5228.08 

04.5229.01 

03.5067.04 

04.5191.01 

Adretta 

04.5229.02 

04.5182.09 

Esprit 

04.5214.03 

04.5230.07 

Bintje 

2007 2008 

03.5066.03 

Mean (113) 

Sarpo Mira 

Kuras 

04.5170.02 

Figure 2. Maturity-corrected (∆ rAUDPC) Phytophthora reaction of selected breeding 

clones in comparison to check varieties in the years 2007 and 2008

Notably, besides an improved resistance to P.i., most of these clones possessed acceptable 

levels with regard to starch content, suitability for chips production, or table quality, 

respectively. In contrast, it was difficult to identify late-blight resistant clones with good 

suitability for crisp production after being kept at a stock temperature of 4°C. The other quality 

characters were also tested at stock temperatures of 4°C (Table 2). 

During the last two years breeding clones were identified which displayed a significantly 

higher resistance level explained as rAUDPC against leaf blight as compared to the check cvs. 

'Eersteling', 'Adretta', 'Esprit', 'Bintje', 'Kuras' and 'Sarpo Mira' and showed an earlier or similar 

maturity time (Figure 1). Generally there is a high correlation between late maturity and 

decreasing vulnerability of the plant to late blight. This was visible in 'Sarpo Mira' and in clone 

04.5170.02. 

The maturity-corrected value of ∆ rAUDPC was used to separate late-blight resistance from 

late-maturity effects. Low values of ∆ rAUDPC describe low levels of susceptibility. In Figure 

2, all the clones showed negative ∆ rAUDPC values and can, thus, be considered as little 

susceptible. For instance, clone 04.5228.08 is less susceptible than clone 04.5224.01. 

Finally, field trials and tuber tests will have to be continued for a third year, because the 

environmental effect on quantitative P.i.-resistance is high (Darsow 2008). Furthermore, the 

resistant genotypes have to be selected for acceptable agronomic characters and early to 

medium-early maturing type. 

The clones 04.5170.02 and 04.5197.01 did not show any symptoms of foliage-blight attack in 

the field and the type of resistance, i.e. monogenic or polygenic, of these clones is not clear at 

present. 

REFERENCES 

Bormann C A (2003). Genetic and molecular analysis of quantitative and qualitative late 

blight resistance in tetraploid potato. PhD-Thesis University Hohenheim: Hohenheim. 

Colon L (1994). Resistance to Phytophthora infestans in Solanum tuberosum and wild 

Solanum species. Thesis Agricultural University Wageningen: Wageningen. 

Darsow U (2008). Vorlaufzüchtung der Kartoffel auf quantitative Phytophthora-Resistenz im 

ILK Groß Lüsewitz in der Ressortforschung des BMELV. Mitt. Julius Kühn-Institut 415. 

Quedlinburg. 

Fry W E (1978). Quantification of general resistance of potato cultivars and fungizide effects 

for integrated control of late blight. Phytopathology 68, 1650-1655. 

Fry W E; Goodwin S B (1997). Re-emergence of potato and tomato late blight in the United 

States. Plant Disease 81, 1349-1357. 

Hansen J; Bodger L; Nielsen B J (2002). Implementation of variety resistance control strategies 

of potato late blight. In: Proceedings of the 6 th Workshop of an European Network for 

Development of an Integrated Control Strategy of Potato Late Blight. Special Report 

No. 8, PPO 304, 111-123. 

413

Hansen J G; Lassen P; Koppel M; Valskyte A; Turka I; Kapsa J (2003). Web-blight – regional 

late blight monitoring and variety resistance information on Internet. Journal of Plant 

Protection Research 43 (3), 263-273. 

Statistisches Bundesamt (2009). Bodennutzung 2008. In: Statistischer Monatsbericht 01-2009, 

ed Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz, 

www.bmelv-statistik.de. 

Thieme R; Darsow U; Gavrilenko T; Dorokhov D; Tiemann H (1997). Production of somatic 

hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species. 

Euphytica 97, 189–200. 

414

Truberg B, Thieme R, Hammann T, Darsow U: A QTL-Study on Quantitative Maturity-Corrected Resistance to 

late Blight (Phytophthora infestans) in Tetraploid Potato (Solanum tuberosum). In: Feldmann F, Alford D V, Furk 

C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 415-418; ISBN 978-3-941261-05-1; © Deutsche 


8-2 A QTL-Study on Quantitative Maturity-Corrected Resistance to late 

Blight (Phytophthora infestans) in Tetraploid Potato (Solanum 

tuberosum) 

Truberg B, Thieme R, Hammann T, Darsow U 

Julius Kühn Institute; Federal Research Centre for Cultivated Plants, Institute for Breeding 

Research on Agricultural Crops, Rudolf-Schick-Platz 3a, D-18190 Sanitz 

ABSTRACT 

Late blight (Phytophthora infestans) is the most serious disease in potato production 

worldwide and causes tremendous losses in yield and high costs in plant protection. 

In order to facilitate breeding for quantitative resistance against late blight, a QTLstudy 

was initiated. A full-sib population of 302 tetraploid clones derived from a 

cross between the tetraploid clone GL-93.7015.04 and the commercial variety 

‘Delikat’ was phenotyped and genotyped. Phenotyping was done as scoring of 

percent leaf area infested by P. infestans in a field trial untreated with fungicides 

over a period of three years with two replications at one location in each year. In 

parallel an experiment treated with fungicides was run to collect maturity data for 

each clone. To eliminate the influence of maturity a maturity-corrected resistance 

parameter was calculated and used as phenotypic trait in the QTL-study. 

Genotyping was done by AFLP markers. A single-allele test was performed using 

the Kruskal-Wallis test. Four AFLP markers tagging three locations in the genome 

were found to be associated with maturity-corrected resistance to late blight. 

INTRODUCTION 

Late blight caused by the oomycete Phytophthora infestans in potato leads to tremendous yield 

losses in all parts of the world. Dominant resistance genes (R-genes) from wild species have 

been used to introduce qualitative resistance to late blight into the cultivated potato. However, 

these R-genes are usually overcome by newly appearing races of P. infestans. Selection for 

horizontal resistance appears to be the most promising breeding approach to combat this 

disease (Darsow 2000). A positive genetic correlation between horizontal resistance to late 

415

light and late maturity is a major obstacle in breeding programmes, since late maturity is 

considered an undesired trait. A maturity-corrected resistance parameter is needed to select for 

horizontal resistance to late blight without unintended indirect selection for late maturity. 


Plant Material 

A full-sib population of 302 tetraploid clones derived from a cross between the tetraploid clone 

GL-93.7015.04 (female parent) and the commercial variety ‘Delikat’ (male parent) was used in 

this experiment. ‘Delikat’shows little to moderate resistance to late blight and early maturity, 

whereas GL-93.7015.04 shows moderate resistance to late blight and medium maturity. 

Late blight field evaluation 

Data were collected over a period of three years with two replications at one location in each 

year. In parallel an experiment treated with fungicides was run to collect maturity data for each 

clone. Artificial inoculation with subsequent sprinkler irrigation of the test plots was used to 

ensure infection each year. A solution containing about 15,000 zoosporangia/ml was used for 

the inoculation. The solution was applied to the first plants in the row during the evening. Data 

were collected as percent of foliage diseased at 16 dates during the vegetation period. 

Molecular markers 

Leaf samples were collected from plants grown in the greenhouse. These samples were stored 

at -80°C. Genomic DNA was isolated from the frozen samples. Genotyping using amplified 

fragment length polymorphism (AFLP) was done as described by Vos et al. (1995). Digestion 

was done with EcoRI and MseI. Eight primer combinations yielded 58 scorable markers. 

Data analysis 

To eliminate the influence of maturity a maturity-corrected resistance parameter was calculated 

and used as trait in the QTL study. For the correction a regression of AUDPC-values on 

maturity scores was done. The difference between observed AUDPC and predicted AUDPC 

was defined as maturity-corrected resistance (Bormann 2003). The influence of each AFLP 

marker on the trait maturity-corrected resistance was tested using the Kruskal-Wallis test in 

SAS. A cluster analysis for the AFLP markers was done in R. (1-r) was used as distance 

measure with r being the correlation between two markers. 


Four of the tested AFLP markers showed a p-value of less than 0.01 for the Kruskal-Wallis test 

(Tab. 1). 

416

The cluster analysis showed three independent loci with an influence on maturity-corrected 

resistance to be marked by the AFLP markers. So far, these loci have to be considered 

anonymous in respect to their location in the genome. For a localization of these loci it is 

planned to continue the genotyping of the population with microsatellites that have been 

mapped (Feingold et al. 2005). This way it will be possible to assign linkage groups to the 

AFLP markers. The mapping of AFLP markers and microsatellites is intended to be done using 

TetraploidMap (Hackett et al. 2006). 

Table 1. AFLP markers showing a p-value of less than 0.01 for the Kruskal-Wallis test 

Marker p-Value Primer Combination Fragment Size 

AFLP_6 0,0046 E32/M51 322 

AFLP_46 0,0021 E35/M58 160 

AFLP_48 0,0026 E32/M51 110 

AFLP_52 0,0095 E45/M60 470 

A cluster analysis revealed that three independent loci are marked by these four AFLP markers 

(Figure 1). 

Figure 1. Cluster dendrogram of the AFLP markers. Marked with arrows are the 

markers showing a p-value of less than 0.01 for the Kruskal-Wallis test. 

417


This study is funded by the German Federal Ministry of Economics and Technology (BMWi) 

by resolution of the German Bundestag.” 

REFERENCES 


blight resistance in tetraploid potato. PhD-Thesis University Hohenheim: Hohenheim 

Darsow U ( 2000). Sources of and breeding for relative late blight resistance of potato. In: 

Potato, global research and development. Proceedings of the Global Conference on 

Potato, New Delhi, India, 6-11 December, 1999: Volume 1, eds S M P Khurana et al., 

pp. 561 – 570. 

Feingold S; Lloyd J; Norero N; Bonierbale M; Lorenzen J (2005). Mapping and 

characterization of new EST-derived microsatellites for potato (Solanum tuberosum L.). 

Theor Appl Genet 111, 456 – 466. 

Hackett C A; Milne I; Bradshaw J E; Luo Z W (2006). TetraploidMap for Windows. SAS 

Institute Inc.: Cary, NC, USA. 

R Development Core Team (2008). R: A language and environment for statistical computing. 

R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL 

http://www.R-project.org. 

Vos P; Hogers R; Bleeker M; Reijans M; van de Lee T; Hornes M; Frijters A; Pot J; Peleman 

J; Kuiper M; Zabeau M (1995). AFLP: a new technique for DNA fingerprinting. 

Nucleic Acids Research, 23 (21), 4407 – 4414. 

418

Askarianzadeh A, Birch A N E, McKenzie G, Ramsay G, Minaeimoghadam M: Study of Wild Solanum Species 

to Identify Sources of Resistance Against the Green Potato Aphid, Myzus Persicae. In: Feldmann F, Alford D V, 



8-3 Study of Wild Solanum Species to Identify Sources of Resistance 

Against the Green Potato Aphid, Myzus Persicae 

Askarianzadeh 1 A, Birch 2 A NE, McKenzie 2 G, Ramsay 2 G, Minaeimoghadam 3 M 

1 

Plant Protection Department, College of Agricultural Science, Shahed University, Tehran, 

Iran 

2 

Scottish Crop Research Institute, Dundee, UK 

3 Plant Protection Department, Agricultural College, Chamran University, Ahwaz, Iran 

Emai: askarianzadeh@shahed.ac.ir 

Abstract 

The green peach-potato aphid, Myzus persicae, damages potato worldwide, both 

directly by their feeding and by spreading important viruses such as potato leaf roll 

virus (PLRV), potato virus Y (PVY) and potato virus X (PVX). In this study, we 

investigated 21 Commonwealth Potato Collection (CPC) accessions from seven 

wild Solanum species (S. jamesii, S. ehrenbergii, S. chomatophilum, S. sanctaerosae, 

S. palustre, S. trifidum and S. infundibuliforme) in order to identify sources 

of resistance against the green potato aphid. Experimental cultures of aphids (clone 

Mp1) were reared on Solanum tuberosum L. Test plants of wild potato species were 

grown from seeds sown in 15 cm diameter pots. After 4 weeks, 5 wingless adult 

aphids were put onto each test plant and maintained in a glasshouse. The effects of 

plant resistance on aphids were calculated as a resistance index. The number of 

aphids (adults and nymphs surviving) was counted after 24 hour, 7 and 10 days. At 

the second screening stage, aphids were removed from the test potatoes using 

nicotine fumigation, left for 3-4 weeks and then re-tested by releasing new aphids 

onto each test plant and counting them at intervals as in the first stage screen. The 

status of glandular hairs at the each Solanum species was also investigated, using a 

stereomicroscope at x50 magnification). Results showed that most of young wild 

Solanum plants tested from the CPC collection were susceptible to aphids. The most 

resistant Solanum species to aphid belonged to S. trifidum and S. palustre and the 

most susceptible species tested was S. sanctae-rosae. Stability of the detected aphid 

resistance during plant development as measured by the correlation of repeat tests 

for S. jamesii (CPC 7166, r 2 = 0.78) and S. trifidum (CPC 7123, r 2 = 0.47) were more 

than other accessions tested. The number of glandular hairs on these two resistant 

419

420 

species was low and medium respectively. Therefore, resistance of these CPC 

accessions does not appear to be related to the presence of glandular hairs. 

Therefore, these two CPC accessions are advantageous to identify molecular 

markers for novel aphid resistance traits in potato to Myzus persicae. 

INTRODUCTION 

The history and economic importance of potato 

The potato (Solanum tuberosum L.) ranks as the fourth most important food crop in the world 

after wheat, rice and maize. It is grown from 55° N to 50° S at altitudes between sea level up to 

5000 m and under a wide range of temperature and humidity regimes (Mendoza 1994; Pandey 

& Kaushik 2003). Today potato is grown on about 19.2 million ha in 153 countries. Its world 

total annual production in 2003 was about 311 x 10 6 tons (FAO STAT). The potato (Solanum 

tuberosum) is an annual herbaceous dicotyledonous plant that reproduces asexually by tubers, 

the only edible part of the plant. Tubers are formed at the end of underground stems, called 

stolons. The plant also flowers and forms small green or purplish-green berries in which the 

true potato seeds (TPS) are produced. True potato seeds are used for breeding and in recent 

years also for propagation. The genus Solanum belongs to the plant family Solanaceae 

(Rabinowitch & Levy 2001). Over 2000 species have been described in the genus Solanum. 

The basic chromosome number (x) of S. tuberosum is 12. The ploidy level for three quarters of 

potato species has been determined and varies from 2x to 6x. Most of the potato species (73%) 

are diploid but 4% triploid, 15% tetraploid, 2% pentaploid and 6% hexaploid occur. The main 

cultivated potato species, S. tuberosum, is tetraploid (2n=4x=48) in its most common form, 

Group Tuberosum, is adapted to long days and cultivated worldwide. The other tetraploid 

form is the Andean Group Andigena which is adapted to short days (Gopal et al. 2003). The 

diploid cultivated forms have had specific and subspecific rank but are now placed in S. 

tuberosum as Groups Phureja and Stenotomum. There are many wild species of potato, of 

which a subsample was tested in this experiment (Table 1). 

The most important pests and diseases of potato 

The potato is vulnerable to attack by a large number of pests and pathogens that individually 

and in combination can cause severe reductions in the yield and quality of potato crops. In each 

region and production system, however, there are certain key pests that need to be monitored 

for control. In seed tuber production systems, for instance, the most important of pests are 

usually aphid vectors of potato viruses, particularly the green peach-potato aphid, Myzus 

persicae, whereas in ware production in more temperate zones, the key pests may be insects 

which attach tubers, such as potato tuber moth (Phthorimaea operculella), Andean potato 

weevil (Premnotrypes spp.) or wireworms (Agrotes spp.). In other situations, foliage feeders 

such as Colorado potato beetle (Leptinotarsa decemlineata) may be considered as the key pests 

(Raman & Radcliffe 1992). Plant viruses are the other important group of potato infecting

Table 1. Accessions of wild Solanum species used in this study. 

Species Accession Shared origin with USDA accessions 

S. jamesii CPC 3850 From a cross with CPC 1439 (PI 195190) 1. 

CPC 7166 PI 275169 1. 

CPC 5845 

S. ehrenbergii CPC 5908 

CPC 7507 

S. chomatophilum CPC 3558 

CPC 5855 

CPC 5861 

S. sanctae-rosae CPC 3269 

CPC 7204 

CPC 7325 

S. palustre CPC 1576 

CPC 2452 

CPC 7034 

CPC 7134 

S. trifidum CPC 7123 PI 255536 1. 

CPC 7125 PI 283104 1. 

S. infundibuliforme CPC 2479 

CPC 7051 

CPC 7249 

1. 

These PI accessions have been recorded in the USDA database as possessing possible resistance to 

Myzus persicae. 

pathogens of which some of them, such as potato leaf roll virus (PLRV), potato virus Y (PVY) 

and potato virus X (PVX) are among the most significant biotic yield constraints of potato 

crops (Mendoza & Sawyer 1985). The aphid’s method of feeding has made them one of the 

most successful and important pests in agriculture. Their mouthparts consist of two pairs of 

flexible stylets, the outer (mandibular) and the inner pair (maxillary) are held within a groove 

of the labium. They are extended from the labium during feeding. The mouthparts have been 

perfectly adapted for piercing plant tissues and extracting sap for food and they are also the 

direct means of acquisition and transmission of plant viruses (Forbes 1977). The aphid family 

Aphididae contains 10 subfamilies, one of which, the Aphidinae, contains more than half of the 

most important pest species of aphids and most of the economically important virus vectors 

such as Myzus persicae (Eastop 1977). 

421

Resistance to aphid 

Aphids damage crops both directly by their feeding and by spreading viruses. In the case of M. 

persicae, the damage it does directly is minor compared to the damage due to the viruses it 

transmits. Aphicides have proved very effective at protecting crops against aphids and 

preventing the spread of some aphid-borne viruses. However, the constant and increasing use 

of pesticides has caused new problems such as ecological side-effects, the danger of selection 

for insecticide-resistant aphids and destruction of predators. Moreover, the success of aphicides 

in controlling aphids has overshadowed the value of plant resistance against aphids. Therefore, 

the use of plant genotypes resistant against either viruses or vectors or against both viruses and 

vectors can reduce their effects on crops without having the problems resulting form the 

repeated application of pesticides. When there are only one or few vector species and several 

viruses, breeding for resistance to the vector is simpler and more advantageous than to viruses 

(Gibson & Plumb 1977). 

In this study, several wild Solanum species were investigated to identify sources of resistance 

against the green potato aphid, Myzus persicae. 


Rearing Myzus persicae: 

In order to rear aphids, several pots of the susceptible potato cv Desiree were put in two cages 

and adult aphids placed on leaves. The cages were maintained in a constant environment room 

at 20ºC and 16:8 hour photoperiod respectively for the population of aphids increased. During 

the screening program, adult aphids were taken from these stock plants for inoculation onto test 

plants. 

List of Commonwealth Potato Collection (CPC) accessions: 

A set of accessions were assembled from the CPC from species which have had putative 

resistance recorded previously (Table 1). In some cases CPC accessions could be linked to 

accessions in the USDA collected at the same locality and time from a comparison of collector 

numbers of accessions in both collections. 

Rearing material plant 

For the first screen, about 25 seeds from each CPC accession were sown in 10 cm pots. Control 

seeds from self-pollinated cv Desiree were also sown. After two weeks, seedlings were 

transferred individually to single pots. At the second screen, accessions that showed 

segregation for resistance were re-sown in batches of 100 seeds. 

422

The first aphid resistance screen 

Screening was delayed until plants were about 4 weeks old. Ten healthy plants were selected 

and transferred into another glasshouse for screening. The distance between pots was 20 cm. 

Five wingless adult aphids were put on each plant at day 0. After 24 hours, the number of 

settled aphids was counted aphids on each plant. Plants that had less than 5 aphids were reinoculated 

so that the total of aphids on each plant became 5. After 24 hours the plants were 

carefully watered by hand daily to avoid disturbing feeding aphids. After 7 days and 10 days, 

the following measurements were recorded: Number of winged adults, number of wingless 

adults, number of winged nymphs, number of wingless nymphs, and plant development stage 

(number of leaves each plant). 

The second aphid resistance screen 

Selected CPC lines from the first screen were re-screened using the same procedure (e.g. 5 

aphids put on each plant and after 24 hour counted then after 7 and 10 days measured the same 

variables above). 

Status of leaf hairs in selected CPC accessions 

For the assessment of this plant trait, a fully expanded, healthy leaf from each CPC accession 

was cut and was investigated with a binocular microscope at x50 magnification. A four-stage 

scale was used to score hair density of short, long and glandular hairs. 


The correlation between the results of the two screens was performed using Excel spreadsheets. 


Screening experiments 

The mean number of aphids on 10 plants from each CPC accession at the first and second 

screening tests is shown in Table 2. The most resistant CPC accessions belonged to the species 

S. trifidum and S. palustre. The most susceptible CPC accessions screened belonged to the 

species S. sanctae-rosae. For investigating the stability of genetic resistance in the tested CPC 

accessions, correlations were calculated between the first and second screening for each CPC 

accession (Table 3). Based on the criteria published by Davis (1971), the correlation coefficient 

analysis showed that only CPC accessions, S. jamesii (CPC 7166) and S. trifidum (CPC 7123), 

gave strong (r 2 =0.78) and medium correlations (r 2 =0.47) respectively. Correlation coefficients 

for some CPC accessions were too low to be acceptable. On this basis, these two CPC 

accessions were selected for molecular marker experiments. 

423

424 

Table 2. Mean (±SDV) number of aphids on CPC lines after 24 hour, 7 days and 10 

days. 

Species CPC 24 

Hour 

Mean of No. Aphid in Screening 1 Mean of No. Aphid in Screening 2 

7 

Day 

10 

Day 

24 

Hour 

S. demissum 3850 3.60±0.97 13.80±8.75 18.30±15.47 4.20±1.03 35.10±19.60 56.50±22.30 

S. jamesii 7166 2.70±0.82 17.20±9.43 24.00±16.72 3.10±1.79 15.50±21.95 28.10±28.27 

S. jamesii 5845 4.10±0.74 23.60±12.29 49.10±27.37 - - 

S. palustre 5908 1.80±1.32 13.20±6.25 18.10±12.65 4.20±1.55 40.30±35.588 51.30±41.85 

S. ehrenbergii 7507 3.10±1.37 16.40±6.59 19.10±7.28 0.80±1.23 0.20±0.42 0.30±0.67 

S. chomatophilum 3558 3.70±1.34 18.10±7.62 31.00±19.53 4.40±0.84 31.80±16.36 63.10±44.54 

7 

Day 

S. chomatophilum 5855 3.50±0.97 16.60±5.83 38.90±13.02 - - - 



S. sanctae-rosae 3269 4.30±0.67 24.00±6.78 41.20±19.15 - - - 

S. sanctae-rosae 7204 4.20±079 15.80±6.94 28.90±19.48 - - - 

S. sanctae-rosae 7325 3.50±0.97 10.40±6.04 20.80±11.41 4.80±0.63 68.50±33.08 112.20±56.81 

S. palustre 1576 4.80±0.42 15.90±10.08 11.50±11.82 3.80±1.32 11.80±14.34 5.90±4.61 

S. palustre 2451 4.60±0.70 16.30±8.22 17.00±14.08 3.50±1.08 4.30±3.27 5.60±5.30 

S. palustre 7034 4.60±3.70 9.70±4.22 14.80±12.53 4.30±0.82 16.70±18.52 21.00±22.90 

S. palustre 7134 3.40±1.26 14.5±8.91 21.20±16.20 3.10±1.10 14.70±9.75 10.20±5.65 

S. trifidum 7123 3.80±1.23 16.10±8.39 26.00±15.56 3.10±1.45 4.10±6.72 2.60±5.04 

S. trifidum 7125 2.90±1.29 13.00±4.64 19.80±8.05 0.40±0.70 1.20±1.55 0.80±1.23 

S. infundibuliforme 2479 2.30±1.42 19.10±8.56 21.60±11.48 3.90±1.37 11.30±10.30 18.90±21.37 

S. infundibuliforme 7051 3.10±1.60 16.00±10.45 47.40±25.61 - - - 

S. infundibuliforme 7249 3.70±1.34 20.00±8.27 25.90±10.75 - - - 

S. tuberosum 4.10±1.10 28.60±13.87 34.90±21.40 4.10±1.29 21.30±16.53 42.40±29.46 

10 

Day

Table 3. R- squared value between the first and the second screening of selected CPC’s 

No. Species CPC No. R- squared 

1 S. demissum 3850 0.27 

2 S. jamesii 7166 0.78 

4 S. palustre 5908 0.04 

5 S. ehrenbergii 7507 0.20 

6 S. chomatophilum 3558 0.001 

12 S. sanctae-rosae 7325 0.13 

13 S. palustre 1576 0.07 

14 S. palustre 2451 0.35 

15 S. palustre 7034 0.08 

16 S. palustre 7134 0.00 

17 S. trifidum 7123 0.47 

18 S. trifidum 7125 0.03 

19 S. infundibuliforme 2479 0.20 

Desiree S. tuberosum 0.002 

Status of hairs in CPC lines 

Glandular hairs are one of resistance factors in potato to aphid. However, the genetic basis of 

glandular hairs is often complex and difficult to select for in breeding programmes. Our results 

indicated that species S. palustre had the highest number of glandular hairs and species S. 

ehrenbergii and S. chomatophilum lacked glandular hairs. The number of glandular hairs for 

the two most resistant CPC accessions, S. jamesii (CPC 7166) and S. trifidum (CPC 7123, were 

low and medium respectively (Table 4). 

Based on resistance to aphids, S. palustre was also more resistant than S. ehrenbergii and S. 

chomatophilum. This suggests that in S. palustre, glandular hairs may be a resistance factors to 

M. persicae. The stability of resistance during plant growth (young and older plants) for S. 

jamesii (CPC 7166) and S. trifidum (CPC 7123) was greater than for the other wild Solanum 

species assessed. The number of glandular hairs for these two recent CPC accessions was low 

and medium respectively. This result indicates that the detected aphid resistance of these CPC 

accessions is not related to the presence of dense glandular hairs and may be due to a resistance 

mechanism based on an R-gene of use in future plant breeding. Therefore, these two wild 

Solanum species are potentially useful in studies to identify molecular markers of potato for 

novel mechanisms of resistance to Myzus persicae in potato. In tomato for example, the Mi-1 

gene is a nucleotide-binding LRR-type R gene conferring resistance to both the nematode 

Meloidogyne incognita and the potato aphid Macrosiphum euphorbiae (Vos et al. 1998). 

Future work should focus on the possibility that similar genes exist in wild potato species and 

that they could confer resistance to the main virus-transmitting aphid pest Myzus persicae. 

425

426 

Table 4. Status of hairs in CPC lines 

No. Species CPC.No. Short hairs Long hairs Glandular 

hairs 

1 S. demissum 3850 Low Nil Moderate 

2 S. jamesii 7166 Nil Low Low 

3 S. jamesii 5845 

4 S. ehrenbergii 

(=S. cardiophyllum) 

5908 Medium Nil Nil 

5 S. ehrenbergii 7507 Low Low Nil 

6 S. chomatophilum 3558 Nil Nil Nil 

7 S. chomatophilum 5855 - - - 



10 S. sanctae-rosae 3269 - - - 

11 S. sanctae-rosae 7204 - - - 

12 S. sanctae-rosae 7325 Nil Medium Medium 

13 S. etuberosum 

(=S. brevidens) 

1576 High High High 



2451 Medium Medium High 



7034 Medium Medium Medium 



7134 High High High 

17 S. trifidum 7123 Nil Medium Medium 

18 S. trifidum 7125 High Low Nil 

19 S. infundibuliforme 2479 Medium Medium Medium 

20 S. infundibuliforme 7051 

21 S. infundibuliforme 7249 

Desiree S. tuberosum Low Low Low 


This work was conducted at the Scottish Crop Research Institute/Dundee, UK. The authors 

thank the Scottish Government’s RERAD and the Iranian Ministry of Agriculture (Sugarcane 

Development & Industrial By-Product Co.) for funding.

REFERENCES 

Davis J A (1971). Elementary Survey Analyses. Prentice Hall: Englewood Cliffs, NJ, USA. 

Eastop V F (1977). Worldwide importance of aphids as virus vectors. In: Aphids as Virus 

Vectors, eds K F Harris & K Maramorosch, pp. 3-61. Academic Press: London, UK. 

FAO STAT (2003). Annual Report. 

Forbes A R 1977. The mouthparts and feeding mechanism of aphids In: Aphids as Virus 

Vectors, eds K F Harris & K Maramorosch, pp. 83-103. Academic Press: London, UK. 

Gibson R W; Plumb R T (1977). Breeding plants for resistance to aphid infestation. In: Aphids 

as Virus Vectors, eds K F Harris & K Maramorosch, pp. 473-500. Academic Press: 

London, UK. 

Gopal J; Kumar V; Sandhu S K (2003). Biosystematics and genetic resources of potato. In: The 

Potato: Production and Utilization in Sub-tropics, eds S M P Khurana, P S Naik, J S 

Minhas & S K Pandey, pp. 31-47. Mehta Publishers: New Delhi, India. 

Mendoza H A; Sawyer R L (1985). The breeding program at the International Potato Center. 

In: Progress in Plant Breeding, ed G E Russell, pp. 117-137. Butterworths, UK. 

Mendoza H M (1994). Development of potato with multiple resistance to biotic and abiotic 

stresses: The international potato centre approach. In: Advances in Potato Pests Biology 

and Management, eds G W Zehnder, R K Powelson & K V Raman, pp. 627-642. APS 

Press: St. Paul, Minnesota, USA. 

Pandey S K; Kaushik S K (2003). Origin, evolution, history and spread of potato. In: The 

Potato: Production and Utilization in Sub-tropics, eds S M P Khurana, P S Naik, J S 

Minhas & S K Pandey, pp. 15-24. Mehta Publishers: New Delhi, India. 

Rabinowitch H D; Levy D (2001). Biology and physiology of potato. In: Virus and Virus-like 

Diseases of Potatoes and Production of Seed-potatoes, eds G Loebenstein, P H Berger, 

A A Brunt & R H Lawson, pp. 19-37. Kluwer Academic Publishers: Amsterdam. 

Raman K V; Radcliffe E B (1992). Pest Aspects of Potato Production, Part 2, Insect Pests. In: 

The Potato Crop, the Scientific Basis for Improvement, ed P Harris, pp. 477-506. 

Chapman & Hall: London, UK. 

Vos P, Simons G, Jesse T, Wijbrandi J, Heinen L, Hogers R, Frijters A, Groenendijk J, 

Diergaarde P, Reijans M, Fierens-Onstenk J, de Both M, Peleman J, Liharska T, 

Hontelez J; Zabeau M (1998). The tomato Mi-1 gene confers resistance to both rootknot 

nematodes and potato aphids. Nature Biotechnology 16, 1365 – 1369 

doi: 10.1038/4350. 

427

Thieme R, Schubert J, Nachtigall M, Hammann T, Truberg B, Heimbach U, Thieme T: Wild Potato Species of the 

Series Pinnatisecta - Progress in their Utilisation in Potato Breeding. In: Feldmann F, Alford D V, Furk C: Crop 

Plant Resistance to Biotic and Abiotic Factors (2009), 428-437. ISBN 978-3-941261-05-1; © Deutsche 


8-4 Wild Potato Species of the Series Pinnatisecta - Progress in their 

Utilisation in Potato Breeding 

Thieme R 1 , Schubert J 2 , Nachtigall M 1 , Hammann T 1 , Truberg B 1 , Heimbach U 3 , Thieme T 4 

1 1 

Julius Kühn Institute, Institute for Breeding Research on Agricultural Crops, Rudolf-Schick- 

Platz 3a, OT Groß Lüsewitz, D-18190 Sanitz, Germany 

2 

Institute for Biosafety of Genetically Modified Plants, Erwin-Baur-Str. 27, D-06484 

Quedlinburg, Germany 

3 

Institute for Plant Protection in Field Crops and Grassland, Messeweg 11-12, D-38104 


4 

BTL Bio-Test Lab GmbH Sagerheide, Birkenallee 19, D-18184 Sagerheide, Germany 

Email: ramona.thieme@jki.bund.de 

428 

Abstract 

Somatic hybrids between genebank accessions of three species of the series 

Pinnatisecta: Solanum cardiophyllum, S. pinnatisectum and S. tarnii, and 

commercial cultivars were produced and characterised for resistance to potato virus 

Y (PVY), foliage blight (Phytophthora infestans) and agronomic traits. The somatic 

hybrids were backcrossed with cultivated potato to produce BC progenies. 

Resistance to PVY was assessed by mechanical inoculation of green-house grown 

plants using isolates of five virus strains (N, O, NTN, NW, C) and by exposure to 

viruliferous aphid vectors in the field. The tuber quality and tuber yield of selected 

hybrids and BC clones were evaluated in the field. 

Parental clones, somatic hybrids and BC progenies were assessed for resistance to 

foliage blight by the detached-leaflet assay and artificial inoculation in the field 

with zoospores of P. infestans. Results suggest that both resistance traits were 

transferred to somatic hybrids by protoplast fusion and that they persist in BC 

clones with some segregation for resistance to foliage blight and PVY occurring in 

the BC1 and BC2 population.

INTRODUCTION 

To-date, introgression breeding for resistances to late blight (P. infestans) and viruses has 

exploited a limited number of wild Solanum species, mainly S. demissum, S. stoloniferum, S. 

chacoense and S. acaule. This is mainly due to the presence of crossing barriers of the majority 

of wild potato species. Somatic hybridization via protoplast fusion is one way of using more of 

the wild species as resources of genetic variation in the potato breeding process. The known 

crossing barriers between cultivars of potato and wild Mexican potato species are easily 

overcome by this technology. This material should be also of interest for molecular analyses 

and identification and characterization of the genetic background of resistance to both of these 

pathogens. 


Material 

The following species: S. cardiophyllum (cph) accession GLKS 108, S. pinnatisectum (pnt) 

accession GLKS 1607, S. tarnii (trn) accession GLKS 2870, (IPK Genebank, External Branch 

‘North‘, Groß Lüsewitz) and Solanum tuberosum ssp. tuberosum, cvs. Delikat, Agave, Quarta 

and Rasant were used. 

Methods 

Production and identification of somatic hybrids 

Interspecific somatic hybrids were produced by electrofusion of protoplasts (Thieme et al. 

2008). Plants were regenerated from callus and their ploidy determined by flow cytometry 

(Thieme et al. 1997). DNA samples were prepared from leaf tissue of in vitro potato plants. 

Standard procedures such as CTAB-based DNA isolation were carried out (Saghai-Maroof et 

al. 1984). A mini-preparation method (Dorokhov et al. 1997) was applied. The hybrid nature 

was proved by SSR analyses (Dinu & Thieme 2001). The PCR reactions were performed in a 

total volume of 20 µl containing 25 ng template DNA, 1 x PCR reaction buffer, 1.5 mM 

MgCl2, 0.25 µM of forward and reverse primers, 200 µM dNTP mix and 0.5 unit Taq 

polymerase. PCR profiles and primer sequences are described by Provan et al. (1996) and 

Feingold et al. (2005). The amplification products were separated on a 6% polyacrylamide 

denaturing gel in a Sequi-Gen GT sequencing cell (Bio-Rad Laboratories, Inc.). The DNA 

fragments were detected using the silver-staining. 

Production of backcross progeny 

The somatic hybrids were crossed in a greenhouse by pollination with cvs. Delikat, Sonate and 

Romanze. 

429

Assessment of resistance to Potato virus Y (PVY) and agronomic traits 

From the parental lines and somatic hybrids five plants were assayed for the presence of PVY 

in their leaves using an enzyme-linked immunosorbent assay (ELISA) after mechanical 

inoculation in a greenhouse with virus strains (of isolates): N - PVY N (CH605, P. Gugerli, 

RAC, Nyon, Switzerland), O - PVY O (205, JKI, Germany), C - PVY C (Q3, I. Browning, 

SASA, Edinburgh, Scotland), NTN - PVY NTN (Linda, JKI, Germany ), W - PVY N W (Wilga O, 

M. Chrzanowska, IHAR, Mlochow, Poland), N* - PVY N (Amigo-N150/1, JKI, Germany) and 

testing sprouts of field-grown tubers. For field trials tubers of the parental lines and standard 

varieties and tubers or in vitro plants of each of the selected somatic hybrids and BC clones 

were transferred from a greenhouse to the field. Five plants per genotype were planted in each 

of four replicates, total area: 12 m x 63 m. Three additional rows with PVY infected tubers of 

the cv. Linda were planted among these clones. The occurrence of aphids was scored in the 

field by counting the number of alatae, apterae and nymphs of the potato- colonizing species of 

the genera Myzus, Aphis, Aulacorthum and Macrosiphum, which are known vectors of PVY. 

After three months storage the harvested tubers (n = 2-87, ~40 tubers per clone) were planted 

in a greenhouse. The excised-bud-assay using ELISA was applied to determine the presence of 

PVY. 

Tuber number and tuber weight of the field grown plants in each of the 4 replicates (for some 

clones 2 replicates) were determined separately. 

Assessment of resistance to late blight 

Greenhouse-grown plants were assessed for resistance to foliage blight using the detached 

leaflet assay method (Darsow et al. 1988; Thieme et al. 2008). Five days after inoculation the 

intensities of necrosis and sporulation were scored and expressed on a scale of 1 (resistant) to 9 

(susceptible). In the field tests inoculation was done at the beginning of flowering of cv. 

Adretta. The inoculum of P. infestans consisted of a mixture of common races collected in the 

field in 2006 and 2007. The trial field was bordered by a strip of hemp 3 m in width, which 

provided protection against wind and maintained a humid environment. Additionally, the 

plants were irrigated in the evening if necessary. The lowest leaves of each first plant in a row 

were inoculated with 5 ml spore suspension (12 x 10³ zoosporangia/ml) in the evening. Scoring 

the percentage of the area of potato tops attacked was started 5 dpi. Scoring was done twice a 

week until the stage of maturity (80-90% of the leaves are yellow). Quantitative resistance to 

foliage blight was assessed as Area Under Disease Progress Curve (AUDPC) (Fry, 1978) and 

Relative Area Under Disease Progress Curve (rAUDPC) (Hansen et al. 2003). 


A total of 240 somatic hybrids between three wild species of the series Pinnatisecta and 

commercial cultivars of S. tuberosum were generated by protoplast fusion. The interspecific 

somatic hybrids were identified using SSR markers (Table 1, Fig. 1). Depending on the fusion 

430

combination somatic hybrids were successfully used as females for the production of BC 

progenies (Table 1). 

Table 1. Number of somatic hybrids between wild species of the series Pinnatisecta 

and potato cultivars obtained by cell fusion and production of BC progenies. 

Combination Somatic hybrids (n) BC generation 

Solanum cardiophyllum + cv. Agave 68 BC1 

Solanum cardiophyllum + cv. Delikat 70 - 

Solanum tarnii+ cv. Delikat 63 BC1, BC2, BC3 

Solanum pinnatisectum + cv. Quarta 14 BC1 

Solanum pinnatisectum + cv. Rasant 25 BC1, BC2 

Figure 1. SSR analysis of regenerated plants that used the ST13ST marker to identify 

somatic hybrids (H) produced by protoplast fusion of A: S. pinnatisectum (pnt) 

+ cv. Quarta, B: S. tarnii (trn) + cv. Delikat, C: S. pinnatisectum (pnt) + cv. 

Rasant, D: S. cardiophyllum (cph) + cv. Delikat. 

431

Genotype 

432 

Table 2. Results of the assessment of the resistance to PVY and foliage blight of 

selected somatic hybrids with different levels of ploidy in the fusion 

combinations Solanum cardiophyllum (cph) + cvs. Agave, Delikat, S. 

pinnatisectum (pnt) + cvs. Quarta, Rasant and S. tarnii (trn) + cv. Delikat and 

progeny compared to that of the parental genotypes (selection of hybrids in 

bold letters). 

Ploidy 

PVY 1 

Number of tested plants/Number of plants infected 

Greenhouse Field 

Foliage blight 2 

Score ± SD 

Parents N O C NTN W N* 

cph 2x 5/0 5/0 5/0 5/0 5/0 20/0 14/0 + 1.1 ± 0.3 

pnt 2x 5/0 5/0 5/0 5/0 5/0 20/0 77/0 + 1.1 ± 0.3 

trn 2x 5/0 5/0 5/0 5/0 5/0 20/0 nt 1.3 ± 0.5 

cv. Agave 4x 5/5 5/5 5/1 5/5 5/5 20/5 87/72 + 5.1 ± 0.3 

cv. Delikat 4x 5/5 5/5 5/5 5/5 5/4 20/20 37/37 4.7 ± 1.0 

cv. Quarta 4x 5/5 5/5 5/5 5/5 5/5 nt 40/40 + 4.7 ± 0.5 

cv. Rasant 4x 5/4 5/5 5/5 5/5 5/5 nt 26/36 + 5.0 ± 0.5 

Somatic hybrids cph + cv. Agave 

1187/6 6x 5/0 5/0 5/0 5/0 5/0 19/0 22/0 6.0 ± 0.7 

1183/1 8x 5/0 5/0 5/0 5/0 5/0 20/0 32/0 + 5.0 ± 2.6 

1154/2 6x 5/0 5/0 5/0 5/0 5/0 20/0 12/0 + 4.8 ± 1.4 

1186/3 6x 5/0 5/0 5/0 5/0 5/0 20/0 27/1 + 1.8 ± 1.1 

Somatic hybrids cph + cv. Delikat 

1255/2 8x 5/0 5/0 5/0 5/0 5/0 20/0 36/0 2.7 ± 2.1 

1263/3 6x 5/0 5/0 5/0 5/0 5/0 20/0 32/0 3.8 ± 0.7 

1263/12 6x 5/0 5/0 5/0 5/0 5/0 20/0 20/0 2.8 ± 2.3 

1265/2 6x 5/0 5/0 5/0 5/0 5/0 20/0 34/0 4.1 ± 0.7 

1265/5 6x nt nt nt nt nt 20/0 22/0 1.7 ± 1.4 

1266/4 6x nt nt nt nt nt 20/0 24/0 2.0 ± 1.5 

1263/2 m 5/0 5/0 5/0 5/0 5/0 20/0 28/0 + 3.0 ± 1.5 

1255/2 8x 5/0 5/0 5/0 5/0 5/0 20/0 36/0 2.7 ± 2.1 

1262/15 6x 5/0 5/0 5/0 5/0 5/0 20/0 2/0 1.7 ± 0.5 

Somatic hybrids pnt + cv. Delikat 

2195/2 6x 5/0 5/0 5/0 5/0 5/0 nt nt 4.1 ± 0.6 

2226/1 6x 5/0 5/0 5/0 5/0 5/0 nt nt 4.0 ± 0.0 

Somatic hybrids pnt + cv. Quarta 

1798/1 6x 5/0 5/0 5/0 5/0 5/0 nt nt 3.2 ± 0.4 

1802/4 6x 5/0 5/0 5/0 5/0 5/0 nt nt 4.0 ± 0.0 

Somatic hybrids pnt + cv. Rasant 

2044/1 6x 5/0 5/0 5/0 5/0 5/0 nt nt 4.1 ± 0.3 

2045/2 6x 5/0 5/0 5/0 5/0 5/0 nt nt 3.6 ± 0.5

Table 2. (continued) Results of the assessment of the resistance to PVY and foliage 

blight of selected somatic hybrids with different levels of ploidy in the fusion 

combinations Solanum cardiophyllum (cph) + cvs. Agave, Delikat, S. 

pinnatisectum (pnt) + cvs. Quarta, Rasant and S. tarnii (trn) + cv. Delikat and 

progeny compared to that of the parental genotypes (selection of hybrids in 

bold letters). 

Somatic hybrids trn + cv. Delikat, BC clones 

H 2 6x 5/0 5/0 5/0 5/0 5/0 20/0 47/0 1.0 ± 0.0 

BC1 2/80 ~5x 5/0 5/0 5/0 5/0 5/0 nt 47/0 1.7 ± 0.8 

BC2 2/80/1 ~4x 5/5 5/0 5/4 5/5 5/3 nt 48/47 3.5 ± 0.5 

BC2 2/80/4 ~4x 5/0 5/0 5/0 5/0 5/0 nt 48/0 4.1 ± 0.6 

BC2 2/80/5 ~4x 5/0 5/0 5/0 5/0 5/0 nt 32/0 3.2 ± 0.4 

BC2 2/80/8 ~4x 5/0 5/0 5/0 5/0 5/0 nt 43/0 4.2 ± 0.4 

BC2 2/80/9 ~4x 5/0 5/0 5/0 5/0 5/0 nt 46/0 2.8 ± 0.4 

H 7 6x 5/0 5/0 5/0 5/0 5/0 20/0 48/0 2.9 ± 0.9 

BC1 7/27 ~5x 5/0 5/0 5/0 5/0 5/0 nt 47/0 4.1 ± 0.3 

BC2 7/27/8 ~4x 5/0 5/0 5/0 5/0 5/0 nt 42/0 4.5 ± 0.7 

1 N O C NTN N 

After mechanical inoculation with virus strains: N - PVY , O - PVY , C - PVY , NTN - PVY , W - PVY W, 

N* - PVY N in the greenhouse and excised-bud-assay of field-grown tubers (Field/ 2006 + , 2007). 

2 

Using the detached leaflet assay with scores from 1 (resistant) - 9 (susceptible), m = mixoploid; nt = not tested; 

SD = standard deviation. 

The hybrids differed in ploidy, vigour, leaf and flower morphology and level of resistance to 

PVY and late blight. The majority of the hybrids show resistance to PVY after mechanical 

inoculation and field trials in 2006 and 2007 (Table 2). This resistance was confirmed by the 

excised-bud-assay (March 2009) from field-grown tubers (2008) for the hybrids and most of 

the clones of the BC progeny of combination with trn and pnt (Table 3). 

The detached leaflet assay indicated that the resistance to foliage blight of these hybrids varied 

from susceptible to resistant (Table 2). Only those somatic hybrids with morphological traits 

similar to the cultivar, with resistance to foliage blight (score < 3.0) and/ or PVY resistance 

were selected for backcrosses (Table 2, bold letters). 

The trn + Delikat somatic hybrids and some BC clones cultivated in the field produced a 

number and weight of tubers comparable to the standard cultivars (clone 2/80/4, Table 3). 

Although in vitro plants were used for field trials, clones of the first backcross of the 

combination pnt + cv. Quarta and cv. Rasant showed a 2-3fold higher number of tubers of 

comparable tuber weight (clone 1798/1/8, 1798/1/15, 1802/4/8, 2044/1/8, 2045/2/9) in 

comparison to the standard cvs. Agave, Delikat and Sonate (Table 3). In general there was 

great variability in yield and tuber characters. The field trials should be repeated for several 

years. Further backcrosses are needed to improve the tuber characters, such as shape and eye 

depth. 

Among the first and second BC of the fusion combination trn + cv. Delikat there were clones 

with slightly lower rAUDPC values (clone 2/17, 2/57, 2/87, 7/18, 7/27 and 7/27/8). The six 

somatic hybrids cph + cv. Delikat had significantly lower rAUDPC values than cvs. Delikat, 

Sonate and Agave (Fig. 2), which indicates a higher resistance to foliage blight in the field. 

433

434 

rAUDPC 

0,9 

0,8 

0,7 

0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0 

cv. Agave 

HA 1187/9 

HA 1187/6 

HD 1266/4 

HD 1265/5 

HD 1265/2 

HD 1263/12 

HD 1263/3 

HD 1255/2 

cv. Delikat 

BC2_7/27/8 

BC1_7/27 

BC1_7/23 

BC1_7/18 

BC1_7/13 

BC1_7/6 

BC1_7/1 

H 7 

BC2_2/80/9 

BC2_2/80/8 

BC2_2/80/5 

BC2_2/80/4 

BC2_2/80/2 

BC1_2/80 

BC1_2/87 

BC1_2/57 

BC1_2/38 

BC1_2/36 

BC1_2/17 

BC1_2/14 

BC1_2/6 

H 2 

cv. Sonate 

cv. Delikat 

Figure 2. Assessment quantitative resistance to late blight using Relative Area Under 

Disease Progress Curve (rAUDPC) of the somatic hybrids (H) S. tarnii + cv. 

Delikat and BC progeny, the somatic hybrids S. cardiophyllum + cvs. Agave 

(HA) and Delikat (HD) and cvs. Delikat, Sonate and Agave grown and 

inoculated with P. infestance in the field. 

These clones were selected for determining their relative resistance in future field experiments. 

Besides the artificial inoculation with P. infestans it is important to determine the maturity of 

separately field-grown plants in order to transfer rAUDPC data into delta (∆) rAUDPC, which 

is an important criterion for characterizing and comparing the resistance to late blight of 

breeding clones and cultivars, and determining standards of resistance (Bormann 2003).

It was shown that potato breeding clones can be produced by somatic hybridization and 

backcrossing exploiting wild species that are sexually incompatible with S. tuberosum, which 

offers the possibility of increasing genetic diversity in respect to resistance to PVY and late 

blight for potato breeding. 

Table 3. Assessment of resistance to PVY using excised-bud-assay on field-grown 

tubers 2008 in the greenhouse 2009, number and weight of tubers produced by 

somatic hybrids (H) Solanum tarnii (trn) + cv. Delikat, S. pinnatisectum (pnt) 

+ cvs. Quarta, Rasant, BC progenies and cultivars grown in the field (4 

replicates x 5 plants = 20 plants), + 2 replicates = 10 plants, *instead of tubers 

in vitro plants transferred to the field, nt – not tested yet. 

Tubers 

Combination Status cv./ Clone 

PVY Number Total weight 

tested/ infected harvested (± SD) (mean ± SD) 

Standard cv. Delikat 39/35 173 ± 15.0 14.55 ± 1.78 

Standard cv. Agave 39/26 249 ± 8.1 18.21 ± 0.62 

Standard cv. Sonate 40/37 156 ± 7.4 17.91 ± 0.29 

Standard cv. Quarta + 40/40 + 68 + ± 2.8 5.80 + ± 0.07 

Standard cv. Rasant + 38/26 + 57 + ± 2.1 7.10 + ± 1.13 

trn + Delikat H 2 34/0 184 ± 7.3 17.96 ± 0.60 

trn + Delikat H 7 56/0 159 ± 7.4 12.50 ± 1.48 

trn + Delikat BC1 2/80 52/0 124 ± 10.0 12.26 ± 0.72 

trn + Delikat BC1 7/27 40/0 205 ± 12.6 16.36 ± 1.38 

trn + Delikat BC2 2/80/2 39/0 189 ± 9.4 12.53 ± 0.80 

trn + Delikat BC2 2/80/4 40/0 326 ± 3.3 21.75 ± 1.00 

trn + Delikat BC2 2/80/5 27/0 148 ± 9.0 7.68 ± 0.50 

trn + Delikat BC2 2/80/8 39/0 185 ± 8.5 7.19 ± 0.15 

trn + Delikat BC2 2/80/9 37/0 175 ± 6.0 14.13 ± 0.48 

trn + Delikat BC2 7/27/7 39/11 149 ± 7.9 10.86 ± 0.16 

pnt + Quarta H 1798/1 + 6/0 57 + ± 0.7 2.90 + ± 0.42 

pnt + Quarta H 1802/4 + nt 48 + ± 8.5 4.35 + ± 1.17 

pnt + Quarta BC1 1798/1/8* 33/0* 894* ± 51.4 21.07* ± 0.97 

pnt + Quarta BC1 1798/1/11* 35/0* 355* ± 10.1 13.58* ± 2.18 

pnt + Quarta BC1 1798/1/15* 39/0* 759* ± 47.6 16.98* ± 1.33 

pnt + Quarta 

pnt + Rasant 

BC1 

H 

1802/4/8* 

2044/1 

34/0* 472* ± 25.6 21.27* ± 0.53 

+ nt 42 + ± 4.2 5.10 + ± 0.00 

pnt + Rasant H 2045/2 + 34/0 + 61 + ± 9.2 4.55 + ± 0.53 

pnt + Rasant BC1 2044/1/2* 38/0* 289* ± 12.5 22.60* ± 1.77 

pnt + Rasant BC1 2044/1/8* 40/0* 465* ± 19.9 15.75* ± 0.89 

pnt + Rasant BC1 2045/2/7* 39/0* 295* ± 15.5 16.20* ± 1.33 

pnt + Rasant BC1 2045/2/9* 38/0* 506* ± 20.4 16.45* ± 0.23 

435


The authors thank H. Baumann, B. Deumlich, P. Hertling, K. Böhm, U. Dominik, U. Busch 

and R. Ionasku for technical assistance. 

REFERENCES 


blight resistance in tetraploid potato. PhD-Thesis University Hohenheim: Hohenheim. 

Darsow U; Junges W; Oertel H (1988). Die Bedeutung der Prädisposition für die Laborprüfung 

von Kartoffelblättern auf relative Resistenz gegenüber Phytophthora infestans (Mont.) 

de Bary. Archiv für Phytopathologie und Pflanzenschutz 24, 109-119. 

Dinu I; Thieme R (2001). Utilization of genetic resources in Solanum for potato breeding 

through biotechnological methods. Schriften zu Genetischen Ressourcen 16,120-127. 

Dorokhov DB; Klocke E (1997). A rapid and economic technique for RAPD analysis of plant 

genomes. Russian Journal Genetics 33, 358-365. 

Fry W E (1978). Quantification of general resistance of potato cultivars and fungizide effects 

for integrated control of late blight. Phytopathology 68, 1650-1655. 

Feingold S; Lloyd J; Norero N; Bonierbale M; Lorenzen J (2005). Mapping and 

characterization of new EST-derived microsatellites for potato (Solanum tuberosum L.). 

Theoretical and Applied Genetics 111, 456-466. 

Hansen J G; Lassen P; Koppel M; Valskyte A; Turka I; Kapsa J (2003). Web-blight – regional 

late blight monitoring and variety resistance information on Internet. Journal of Plant 

Protection Research 43 (3), 263-273. 

Provan J; Powell W; Waugh R (1996). Micosatellite analysis of relationships within cultivated 

potato (Solanum tuberosum). Theoretical and Applied Genetics 92, 1078-1084. 

Saghai-Maroof M A; Soliman K M; Jorgensen R A; Allard R W (1984). Ribosomal DNA 

spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location 

and population dynamics. Proceedings of the National Academy of Sciences USA 81, 

8014-8018. 

Thieme R; Darsow U; Gavrilenko T; Dorokhov D; Tiemann H (1997). Production of somatic 

hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species. 

Euphytica 97, 189-200. 

Thieme R; Rakosy-Tican E; Gavrilenko T; Antonova O; Schubert J; Nachtigall M; Heimbach 

U; Thieme T (2008). Novel somatic hybrids and their fertile BC1 progenies of 

potato (Solanum tuberosum L.) + S. tarnii, extremely resistant to potato virus Y 

and resistant to late blight. Theoretical and Applied Genetics 116, 691-700. 

436

Varrelmann M: Can natural resistance help to control nematode transmissible tobacco rattle virus in potato? In: 

Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), …-…… ISBN 978- 


8-5 Can natural resistance help to control nematode transmissible tobacco 

rattle virus in potato? 

Varrelmann M 

Institute of Sugar Beet Research, Holtenser Landstr. 77, 37079 Göttingen 

Email: mvarrel@gwdg.de 

Abstract 

Tobacco rattle virus (TRV), genus Tobravirus infects a broad range of plant species 

worldwide and is mainly transmitted by plant-parasitic nematodes. TRV is a singlestranded 

bipartite plus-sense RNA virus possessing an RNA1 which can replicate 

autonomously without forming particles (NM-isolates). RNA2 encodes the viral 

coat protein and in some isolates proteins responsible for vector transmission. In 

potato TRV can induce a disease called “spraing”, characterized by arcs or flecks of 

brown, corky tissue present in the tuber flesh or on the surface, which has 

significant economic implications. In contrast to aphid transmissible potato 

infecting viruses like potato virus Y, chemical vector control does not allow for 

specific targeting and is mostly prohibited. Production of virus free propagation 

material is not reliable as immunological virus detection does not capture NMisolates. 

In addition and in contrast to the other potato infecting viruses with 

economic importance, natural resistance, although existent, has not been 

investigated in detail. Nevertheless, resistance assessment in naturally infected soil 

inhabiting viruliferous trichodorid nematodes by scoring of tuber symptoms is 

unreliable because of uneven vector distribution and unpredictable weather 

conditions in field trials. Recently, identification of hypersensitive resistance 

reaction in different potato genotypes has been described and is supposed to be 

based on monogenic dominant genes. As matching viral avirulence gene, the RNA1 

encoded 29K movement protein was isolated. A fast and reliable resistance test 

based on transient 29K expression in potato leaf tissue was developed allowing for 

the first time the development of genetic markers for selection. Finally a method for 

fast and reliable TRV isolation from soil samples was established, which enables 

the characterisation of 29K variability and the estimation of spatial resistance 

usability and sustainability. This review describes the current knowledge of TRV 

resistance and summarizes the prospects for future TRV control based on natural 

resistance. 

437

Giesemann, A, Balko, C: delta13C values - an indicator for drought tolerance of different potato genotypes. In: 



9-1 delta13C values - an indicator for drought tolerance of different potato 

genotypes 

Giesemann, A 1 , Balko, C 2 

1 

Institut für Agrarrelevante Klimaforschung, Johann Heinrich von Thünen-Institut (vTI), 

Bundesforschungsinstitut für Ländliche Räume, Wald und Fischerei, Bundesallee 50, 38116 

Braunschweig 

2 

Institut für Resistenzforschung und Stresstoleranz, Julius Kühn-Institut 

(JKI),Bundesforschungsinstitut für Kulturpflanzen, OT Groß Lüsewitz, Rudolf-Schick-Platz 3, 

18190 Sanitz 

Email: anette.giesemann@vti.bund.de 

438 

Abstract 

d13C values - an indicator for drought tolerance of different potato genotypes The 

potato belongs to the crops responding relatively sensitive to drought stress. With 

regard to the climate change, the occurrence of drought periods will increase even 

in Middle Europe. Breeding is one approach to create new cultivars which are 

adapted to such drought stress conditions. Hence it is desirable to find indirect 

selection criteria to easily characterise breeding relevant genotypes d13C is an 

integrative measure for water use efficiency (WUE), which is an important 

physiological character of crop plants with regard to drought stress. The C isotopic 

composition of plants is different under different water regimes. Following drought 

stress conditions, stomata are (more or less) closed and the discrimination against 

13C is reduced. Contrary, under sufficient water supply, stomata are wide open, gas 

exchange is ensured and 13C discrimination is high. In our investigations, the C 

isotopic composition of various parts of the potato plant (leaf, stem, stolons, tuber, 

root) was determined to evaluate their drought stress response. Furthermore, 

different potato genotypes of early to intermediate maturity were cultivated under 

drought stress conditions from the beginning of tuber formation. Water 

consumption, WUE as well as 13C discrimination in the leaf were monitored and 

related to yield. A significant correlation between d13C values and water 

consumption, WUE and yield was found.

Hackauf B, Truberg B, Wortmann H, Fromme F J, Wilde P, Menzel J, Korzun V, Stojałowski S: Minimizing 

Ergot Infection in Hybrid Rye by a SMART Breeding Approach. In: Feldmann F, Alford D V, Furk C: Crop Plant 



9-2 Minimizing Ergot Infection in Hybrid Rye by a SMART Breeding 

Approach 

Hackauf B 1 , Truberg B 1 , Wortmann H 2 , Fromme F J 2 , Wilde P 3 , Menzel J 3 , Korzun V 3 , 

Stojałowski S 4 

1 

Julius Kühn Institute, Institute for Breeding Research on Agricultural Crops, D-06484 

Quedlinburg 

2 

HYBRO GmbH & Co KG, D-17291 Schenkenberg 

3 

KWS LOCHOW GmbH, D-29303 Bergen 

4 

Agricultural University Szczecin, Dep. of Genetics and Plant Breeding, Szczecin Poland 

Email: bernd.hackauf@jki.bund.de 

Abstract 

Restoration of male fertility is currently the most favourable approach to minimize 

ergot infection in hybrid rye varieties. Novel gene-based STS markers proved to be 

efficient tools for marker assisted introgression of the restorer gene Rfp1 in hybrid 

rye varieties. In the present study, we were able to detect linkage of these markers 

to a second major restorer gene of the P cytoplasm, Rfp2, as well. Our analysis of 

Rfp2 revealed, that the expression of this major restorer gene is largely influenced 

by modifier genes. As an additional result, we identified microsatellite markers 

linked to minor restorer genes located on chromosomes 1R, 3R, 6R and 7R. 

Although the the SSR markers provide a significant progress in addressing these 

minor restorer genes, the complex genetics of male-fertility restoration based on 

Rfp2 increases the efforts of marker assisted introgression of this restorer gene in 

elite breeding lines. The observed linkage of the STS markers to Rfp2 as well as to 

Rfp1 and Rfc1 in a previous study supports the assumption, that the restorer genes 

identified on chromosome 4RL are either alleles of a single restorer gene or 

represent different linked genes located in this sub-genomic region. The gene-based 

markers on chromosome 4RL, together with a set of microsatellite markers 

dispersed throughout the entire rye genome, proved to be efficient tools to elucidate 

the genetics and to examine the value of restorer genes for practical hybrid rye 

breeding. 

439

INTRODUCTION 

Ergot (Claviceps purpurea) infection counts among the economically most important diseases 

in rye (Secale cereale). Susceptibility to ergot upon artificial inoculation is a trait published for 

registered rye varieties in the descriptive variety lists of the Bundessortenamt since 2008. The 

compliance of defined thresholds for ergot contamination of the harvest (0.05% for human 

consumption, 0.1% for feeding purposes) is critical for a reliable marketing. Breeding of 

improved varieties is the only way in the combat of ergot, as the plants can not be chemically 

protected against the fungus. 

Highly productive hybrid rye varieties, which are cultivated on more than 65% of the German 

rye production area, are notably susceptible to ergot. This susceptibility is caused by the 

utilization of a cytoplasmic male sterility system, which is needed as a genetic fertilization 

control mechanism for hybrid seed production. Although different sterility inducing 

cytoplasms have been described in rye, basically the Pampa (P) cytoplasm (Geiger & Schnell 

1970) has been implemented in hybrid rye breeding. While recent results indicate, that a 

genetically controlled resistance towards ergot has been evolved in rye (Mirdita et al. 2008), 

the restoration of male fertility is currently the most favourable approach to minimize ergot 

infection in hybrid rye varieties. 

Restorer genes identified in European rye germplasms result in an incomplete restoration of the 

male fertility in the P cytoplasm (Miedaner et al. 2000). Incomplete restoration, in particular 

under unfavourable weather conditions during flowering time, increases the venture of an ergot 

infection. Effective restorer genes like Rfp1 and Rfp2, which have been identified in unadapted 

genetic resources of rye (Miedaner et al. 2000; Stracke et al. 2003), result in an almost 

complete restoration of male fertility in hybrid rye varieties and, thus, contribute to minimize 

harvest contamination with ergot. In the proximity of these restorer genes, however, other 

gene(s) are located that have a negative influence on yield. To remove this linkage drag, 

molecular markers are needed to identify and select individuals with recombinant haplotypes. 

In an initial attempt, sequence-tagged site (STS) markers for the restorer genes Rfp1 and Rfp2 

have been developed by Stracke et al. (2003). Using a comparative genetic approach and the 

rice genome data as a blueprint, we were able to develop novel STS markers tightly linked to 

the restorer gene Rfp1 (Hackauf et al. 2006, 2007). These gene-derived markers are codominantly 

inherited and provide a cost effective strategy to identify recombinants with a 

precision not feasible before. The novel markers, which allow a clear visualization of 

genotypes connected to the locus (locus haplotyping), have been validated in elite breeding 

lines of rye and proved to be efficient selection tools for Rfp1 in practical rye breeding 

(Hackauf et al. 2009a). Here, we report on the linkage analysis of these markers relative to 

Rfp2, a second restorer gene of the P cytoplasm in rye. 

440


Three F1 genotypes were generated by crossing individual plants of the male-sterile singlecross-tester 

L2039-P x L145-N from the non-restorer genepool with the BC2 breeding lines 

L3362, L3360 and L401, which all originate from the restorer genepool of Hybro GmbH & Co 

KG. Each BC2 line carries a 4RL chromosome-segment with the restorer gene Rfp2, which has 

its source in the self-incompatible population ‘Pico Gentario’ (Miedaner et al. 2000). 

Introgression of this donor-chromosome segment was achieved by marker-assisted 

backcrossing using the dominant STS marker SCY03 (Stracke et al. 2003). Each of the three F1 

genotypes was selfed to produce the F2 families JKI-1309, JKI-1310 and JKI-1311, 

respectively. From each of these populations 106, 90 and 92 plants were cloned with two 

clones per individual plant. Male fertility was visually assessed according to Geiger & 

Morgenstern (1975), with one clonal part of each family being independently scored at the 

Hybro station in Kleptow and the other at JKI in Groß Lüsewitz. 

The genetic mapping in the three F2 families was performed in a two step approach. Upon 

genotyping with 4RL markers, a bulked segregant analysis (Michelmore et al. 1991) was 

performed to identify SSR markers for restorer genes distinct from Rfp2. For this purpose, two 

DNA bulks were compiled within each population encompassing 10 individual plants each, 

which were phenotypically scored as male sterile (male-fertility score 1-3) or male fertile 

(male-fertility score 7-9) in both repetitions. In addition, the male sterile and male fertile bulks 

were balanced with respect to their genetic constitution on chromosome 4RL, i.e. in both bulks 

established for populations JKI-1309 and JKI-1310 each plant was heterozygous at all 

genotyped 4RL marker loci, while in population JKI-1311, next to the 10 samples representing 

the male fertile bulk, almost 4 samples in the male sterile bulk were scored as heterozygous for 

the Rfp2 genomic region on chromosome 4RL. Application and mapping of SSR and STS 

markers were done as described before (Hackauf & Wehling 2002; Hackauf et al. 2006, 2007). 

Analyses of the male fertility data were computed for each locus using standard procedures 

(Snedecor & Cochran 1989). Mendelian segregation ratios were tested by the standard χ 2 

method using a gene model with male-fertility scores 1-3 being considered as male sterile and 

4-9 being considered as male-fertile (Miedaner et al. 2000). As the male-fertility data 

significantly deviated from a normal distribution, all statistical tests are only approximate for 

this trait. For each marker, a one-way analysis of variance (ANOVA) was performed using the 

GLM procedure (SAS 2003) to test whether the phenotypic trait was significantly (P= 0.01) 

different between marker classes. The Scheffé test (Snedecor & Cochran 1989) with an error 

probability of P= 0.01 was applied to perfom comparisons among the means. Epistatic 

interaction was tested using the MIXED procedure in SAS (SAS 2003) in a two-marker model. 

All possible marker pairs were tested. 

441

RESULTS 

In all F1 genotypes obtained from the described crosses complete restoration of male fertility 

(male-fertility score 8) could be observed. The assessment of male-fertility in the segregating 

F2 populations revealed in each case a bimodal distribution of the means across both locations 

with peaks in the phenotypic classes ‘male-sterile’ and ‘fully male-fertile’ (Table 1). In 

populations JKI-1309 and JKI-1311, the observed segregation ratios fitted the hypothesis of a 

monogenic dominant inheritance of male-fertility restoration, while the segregation ratio 

observed in population JKI-1310 can be explained by the complementary action of two 

dominant restorer genes. 

442 

Table 1. Frequency distribution of mean fertility scores (1-9) in three F2 populations 

and χ 2 tests for two Mendelian segregation ratios. Classes with full male 

fertility are printed in bold. 

Population Male-fertility score Chi-Square 

value 

1 2 3 4 5 6 7 8 9 N Mean 1:3 7:9 

JKI-1309 10 12 11 1 1 1 10 52 8 106 5,7 2,13 

JKI-1310 13 23 5 1 0 2 14 28 4 90 4,8 0,11 

JKI-1311 12 10 5 1 0 1 11 47 5 92 5,8 0,93 

Figure 1. A consensus map of 

rye chromosome 4RL integrating 

the data of the F2 populations 

JKI-1309, JKI-1310 

and JKI-1311. 

The gene-derived STS marker allowed to address a genomic 

region on chromosome 4RL covering 56.6 cM with seven of the 

10 marker clustering to a short genetic interval of 4.7 cM 

(Figure 1). One-way analysis of variance revealed significantly 

different male-fertility means between the three marker classes 

of the 4RL markers in each mapping population (Table 2). 

However, the 4RL marker genotypes did not completely explain 

the observed male-fertility phenotypes. In the analyzed 

populations 12 (JKI-1309), 20 (JKI-1310) and 4 (JKI-1311) 

plants were phenotypically scored as male-sterile and, as 

deduced by the marker genotypes, concurrently carried the Rfp2 

donor-chromosome segment at least in the heterozygous status. 

Using SSR markers covering the entire rye genome, bulked 

segregant analysis allowed us to identify polymorphisms 

between male-sterile and male-fertile bulks for markers located 

on chromosomes 1R, 2R, 3R, 6R and 7R. Four additional 

restorer loci on chromosomes 1R, 3R, 6R and 7R were detected 

by one-way analysis of variance (Table 2). Based on the malefertility 

scores for the three marker classes of the most closely 

linked SSR markers in populations JKI-1309 and JKI-1311, 

only the restorer gene Rfp2 on chromosome 4RL can be 

classified as major gene and the remaining four as minor genes.

Table 2. Chromosomal localization of sequence-specific PCR markers significantly (P= 

0.01) associated with male-fertility restoration in three F2 populations. Mean 

fertility scores (1-9) for the marker classes are estimates obtained by one-way 

ANOVA. Markers Xpsr167 as well as Xiac69 are dominantly inherited. 

Mapping 

population 

Chromosome 

Marker Markerclass a 

A H B C 

Male-fertility score b 

JKI-1309 1R Xscm177 3,38 a 

6,44 b 

6,00 b 

2R Xscm357 5,76 a 

6,08 a 

5,21 a 

3R Xscm294 5,64 a 

5,53 a 

6,03 a 

3R Xscm117 5,10 a 

5,96 a 

5,88 a 

4R Xiac105 4,20 a 

5,84 b 

6,46 b 

4R Xiac81 3,69 a 

6,14 b 

7,14 b 

4R Xiac70 2,80 a 

6,58 b 

7,19 b 

4R Xiac69 2,63 a 

6,64 b 

7,04 b 

4R Xiac74 2,63 a 

6,55 b 

7,22 b 

4R Xiac67 2,63 a 

6,55 b 

7,22 b 

4R Xiac86 2,44 a 

6,57 b 

7,22 b 

4R Xiac76 2,69 a 

6,52 b 

7,22 b 

4R Xiac66 2,84 a 

6,30 b 

7,28 b 

4R Xpsr167 4,29 a 

6R Xscm280 4,89 a 

5,37 a 

6,90 b 

6R Xscm176 3,82 a 

5,40 b 

7,37 c 

6R Xrms1090 4,00 a 

5,29 b 

7,24 c 

6R Xscm68 4,71 a 

a, b 

5,61 6,43 b 

7R Xscm19 4,89 a 

6,16 b 

a, b 

5,75 

JKI-1310 3R Xscm117 2,38 a 

4R Xiac70 2,22 a 

4R Xiac69 1,87 a 

4R Xiac74 1,84 a 

4R Xiac76 1,57 a 

6R Xscm176 2,91 a 

6R Xscm294 3,68 a 

JKI-1311 2R Xscm357 5,56 a 

4R Xscm155 6,04 a 

4R Xiac105 5,86 a 

4R Xiac70 2,81 a 

4R Xiac74 2,72 a 

4R Xiac67 2,75 a 

4R Xiac86 2,75 a 

4R Xiac76 2,75 a 

4R Xiac66 3,03 a 

4R Xpsr167 4,23 a 

7R Xscm19 5,69 a 

5,10 b 

5,63 b 

5,60 b 

5,74 b 

4,92 b 

4,68 

a, b 

5,64 a 

6,14 a 

5,91 a 

6,94 b 

7,03 b 

7,12 b 

7,12 b 

7,12 b 

7,24 b 

6,08 a 

5,70 b 

5,79 b 

5,95 b 

5,85 b 

5,71 b 

5,89 b 

6,64 a 

5,50 a 

5,72 a 

7,86 c 

7,83 b 

7,83 b 

7,83 b 

7,83 b 

7,79 b 

5,15 a 

a Different indices within a row designate significantly (P ≥0.05) different means (Scheffé-Test). 

b 1 = highly degenerated, non-dehiscent, empty anthers; 9 = full-sized, abundantly pollen-shedding anthers. 

6,26 b 

5,74 b 

7,21 b 

443

Interactions between the restorer loci was further analyzed by a two-locus model. In population 

JKI-1311 no interaction between the Rfp2 locus on chromosome 4RL and other restorer loci 

could be detected. In contrast, the marker class means revealed epistatic interactions between 

restorer genes on chromosomes 1R and 7R, 3R and 6R, 3R and 7R, 4R and 1R, 4R and 6R, 4R 

and 7R in population JKI-1309 (Table 3) and in population JKI-1310 between restorer loci on 

chromosomes 4R and 3R, 6R and 3R as well as 4R and 6R (Table 4). The strongest epistatic 

interaction could be observed between the restorer genes located on chromosomes 1R and 4R 

as well as 4R and 6 R and 4R and 7R in population JKI-1309. In this mapping population, 

complete male-fertility (mean male-fertility score ≥ 7 ) was reached in the presence of at least 

one restorer allele at each of two loci analyzed. If the Rfp2 locus on 4RL was homozygous for 

the non-restorer allele (mama), full male-fertility could not be induced by the restorer allele of 

the second locus. If the second restorer locus was homozygous for the non-restorer allele, the 

restorer allele (Ma .) at the Rfp2 locus resulted only in partial restored male-fertility in all but 

one cases. A mean male-fertility score ≥ 7 could be observed for the interaction between Rfp2 

and the non-restorer allele of the minor restorer locus linked to Xscm177 on rye chromosome 

1R, if Rfp2 was homozygous for the restorer allele (MaMa). A complex, albeit statistically 

significant pattern of epistatic interactions could be observed in this population between the 

minor restorer genes on chromosomes 1R and 7R, 3R and 7R as well as between 3R and 6R. 

Complete male-fertility could be observed for individual allele-combinations in different gene 

combinations. 

Remarkably, in population JKI-1310 the Rfp2 donor-chromosome segment was associated with 

complete male-fertility in both investigated restorer gene combinations only, if the second 

restorer locus was homozygous for the restorer allele (Table 4). Based on a two-gene model 

with male-fertility scores 4-9 being considered as male-fertile, only the interaction between the 

restorer gene Rfp2 on chromosome 4RL and the restorer locus linked to Xscm176 on rye 

chromosome 3R fitted (χ 2 = 1,85) the 9:7 segregation ratio expected for two complementary, 

dominant acting restorer genes. Thus, it appears likely, that the phenotypic 9:7 segregation 

ratio observed for male-fertility restoration in this population is governed by the 

complementary action of Rfp2 and the restorer gene linked to the SSR marker Xscm176 on 

chromosome 3R. 

DISCUSSION 

The improvement of modern hybrid rye varieties with respect to their ability to produce a 

sufficient amount of pollen is still the most favourable strategy to minimize harvest 

contamination with ergot. Genes have been identified in unadapted germplasms of rye, which 

can efficiently restore male fertility in the P plasma (Miedaner et al. 2000). However, visual 

assessment of these restorer genes at flowering is a time and labour consuming process. Thus, 

marker assisted selection of restorer genes significantly contributes to speed up the breeding 

process, as information on the presence of a restorer gene can be generated in early stages of 

plant development and without conducting test crosses and visual assessment of male-fertility. 

444

Table 3. Two-way table of marker class means for male-fertility scores in the F2 

population JKI-1309 at individual STS marker loci combinations. Plant 

numbers are given in parentheses, classes representing complete male-fertility 

are highlighted. 

Locus Genotype Xscm177-1R Mean 

Xscm19-7R 

MaMa Mama mama 

MbMb 6,4 (12) 6,3 (13) 2,7 (5) 5,1 

Mbmb 6,8 (17) 6,8 (21) 3,1 (9) 6,0 

mbmb 3,6 (8) 5,9 (13) 4,3 (6) 4,6 

mean 5,6 6,4 3,4 


Xscm176-6R 


MbMb 7,7 (8) 7,3 (16) 7,2 (11) 7,4 

Mbmb 5,5 (21) 6,0 (18) 4,1 (10) 5,2 

mbmb 5,2 (9) 3,1 (8) 2,6 (5) 3,6 

mean 6,1 5,5 4,6 


Xscm19-7R 


MbMb 6,9 (9) 4,4 (10) 6,0 (11) 5,8 

Mbmb 6,5 (16) 6,8 (20) 4,8 (13) 6,0 

mbmb 4,4 (13) 6,0 (12) 1,5 (2) 4,0 

mean 5,9 5,7 4,1 

Locus Genotype Xiac86-4RL Mean 

Xscm177-1R 


MbMb 7,7 (12) 7,4 (15) 1,8 (10) 5,6 

Mbmb 6,7 (13) 6,9 (27) 4,0 (7) 5,9 

mbmb 7,8 (2) 3,8 (9) 1,9 (9) 4,5 

mean 7,4 6,4 2,6 


Xscm176-6R 


MbMb 7,9 (10) 7,4 (22) 5,5 (3) 6,9 

Mbmb 7,2 (13) 6,9 (19) 2,4 (17) 5,5 

mbmb 5,6 (4) 4,5 (12) 1,2 (6) 3,8 

mean 6,9 6,3 3,0 

445

446 

Table 3. (continued) Two-way table of marker class means for male-fertility scores in 

the F2 population JKI-1309 at individual STS marker loci combinations. Plant 


are highlighted. 


Xscm19-7R 


MbMb 7,7 (8) 6,8 (14) 1,9 (8) 5,5 

Mbmb 7,3 (14) 6,6 (28) 2,3 (7) 5,4 

mbmb 6,4 (5) 6,2 (11) 2,9 (11) 5,2 

mean 7,1 6,5 2,4 

The common evolutionary origin of rye and rice (Oryza sativa) and the conserved gene order 

in sub-genomic regions of both species facilitates the use of informations on the rice genome 

data for the development of gene-derived markers in the large genome of rye (Hackauf et al. 

2009b). This strategy allowed us to establish a molecular technique based on gene-derived 

markers, which is diagnostic for the restorer gene Rfp1 on chromosome 4RL in rye (Hackauf et 

al. 2006, 2007, 2009a). Rfp1 could be mapped within a genetic interval of 1.3 cM, which is 

defined by the marker loci Xiac76 and Xiac86 (Hackauf et al. 2006). As demonstrated here, 

these markers are linked to the restorer gene Rfp2 as well and, thus, allow for an effective 

genotyping with respect to this second major restorer gene in the P plasma. The estimated 

genetic interval of 4.7 cM defined by the markers Xiac70 and Xiac66, respectively, compares 

well to the genetic distance estimated between marker locus SCY03 and Rfp2 in rye (Stracke et 

al. 2003). 

Traits governed by one or a few major genes like the restorer genes Rfp1 or Rfp2 in rye can 

efficiently transferred from unadapted into elite germplasm by backcross breeding. However, 

linkage drag (Brinkman & Frey 1977; Tanksley et al. 1989), i.e. genomic segments carrying 

undesirable genes linked to the target gene, often hamper backcross projects. Separation of 

such gene complexes by naturally occurring recombination is a seldom event and asks for a 

fast and precise method to identify favourable recombinants. The gene-derived STS markers 

used in this study represent powerful tools to identify individual plants carrying recombinantly 

reduced donor-chromosome segments on chromosome 4RL. Further research on recombinant 

4RL haplotypes will clarify, if an observed correlation between male-fertility restoration and 

undesired plant height (Miedaner et al. 2000) could be forced open. Together with additional 

markers covering the entire rye genome (Saal & Wricke 1999; Hackauf & Wehling 2003; 

Klestkina et al. 2004, 2005; Hackauf et al. 2009) the gene-derived markers linked to Rfp1 and 

Rfp2 allow for an efficient marker-assited selection approach (for review see Collard & 

Mackill 2008) to transfer the desired donor-chromosome segment into elite germplasm. A 

SMART (Selection with Markers and Advanced Reproductive Technologies, Davis et al.

1997) breeding approach like this has been applied for instance to improve submergence 

tolerance in rice (Xu et al. 2006). 

Table 4. Two-way table of marker class means for male-fertility scores in the F2 

population JKI-1310 at individual STS marker loci combinations. Plant 


are highlighted 


Xscm117-3R 


MbMb 7,7 (6) 6,8 (9) 1,4 (5) 5,3 

Mbmb 5,7 (18) 6,3 (24) 1,8 (12) 4,6 

mbmb 1,5 (2) 3,2 (9) 1,2 (5) 2 

mean 5 5,4 1,5 


Xscm176-6R 


MbMb 6,8 (8) 5,8 (13) 2,7 (3) 5,1 

Mbmb 6,2 (14) 6,4 (21) 1,4 (14) 4,7 

mbmb 2,9 (4) 3,9 (8) 1,3 (5) 2,7 

mean 5,3 5,4 1,8 


Xscm117-3R 


MbMb 6,3 (5) 6,0 (12) 3,3 (3) 5,2 

Mbmb 5,8 (16) 5,1 (30) 3,8 (8) 4,9 

mbmb 4,0 (3) 2,4 (7) 1,5 (6) 2,6 

mean 5,4 4,5 2,9 

Our study on the restorer gene Rfp2 revealed, that the expression of this gene is largely 

influenced by modifier genes. These observation confirms previous results decribed for Rfp2 

and minor restorer genes located on chromosomes 1RS, 3RL, 5R and 6RL (Miedaner et al. 

2000). In their study, Miedaner et al. (2000) observed a strong epistatic interaction of the 

complementary type between Rfp2 and a minor restorer gene on chromosome 6RL. Although 

447

epistatic interaction between Rfp2 and a restorer gene on chromosome 6R could be detected in 

population JKI-1310, the observed phenotypic segregation ratio perfectly corresponds to the 

observed complementary interaction between Rfp2 and a restorer gene on chromosome 3R. 

The minor restorer gene located on chromosome 7R in rye has not been described before. As a 

consequence of the observed epistatic interactions, the male-fertility level of a hybrid may be 

unsatisfactory, if the modifying genes are lacking. This phenomenon is particularly illustrated 

in the mapping population JKI-1310, where complete male-fertility means could only scarcly 

be observed and 22% of the population was scored as male-sterile despite of the presence of 

the donor-chromosome segment carrying Rfp2. Although the identified microsatellite markers 

linked to the minor restorer genes provide a significant progress in addressing these genes, the 

complex genetics of male-fertility restoration using Rfp2 increases the efforts of its marker 

assisted introduction in elite breeding lines. In their study Miedaner et al. (2000) were able to 

identify the minor restorer gene on chromosome 6RL originating from the non-restorer parent 

of the mapping population. We are currently not able to assign the origin of the marker alleles 

to either of the parents used. However, it should be noted that in population JKI-1311 no 

epistatic interaction between Rfp2 and any other restorer gene could be detected although the 

male-sterile non-restorer genotype was identical in each of the crosses analyzed in our 

experiments. The characterization of the parental genotypes is in progress and should clarify 

the origin of the minor restorer genes described here. 

In summary, we were able to detect linkage between the gene-derived markers on chromosome 

4RL an the dominant restorer gene Rfp2 in rye. The observed linkage of the STS markers to 

Rfp2 as well as to Rfp1 and Rfc1 in a previous study (Hackauf et al. 2009a) supports the 

assumption that the restorer genes identified on chromosome 4RL are either alleles of a single 

restorer gene or represent different linked genes located in this sub-genomic region. These 

markers, together with the set of microsatellite markers dispersed throughout the entire rye 

genome, proved to be efficient tools to elucidate the genetics and to examine the value of 

restorer genes located on chromosome 4RL for practical hybrid rye breeding. 


We gratefully acknowledge technical assistance of Daniela Kempke, Marion Hos, Regina Voss 

and Kirsten Jantzen. 

REFERENCES 

Brinkman M A; Frey K J (1977). Yield component analysis of oat isolines that produce 

different grain yield. Crop Sci. 17, 165-168. 

Collard B C Y; Mackill D J (2008). Marker-assisted selection: an approach for precision plant 

breeding in the twenty-first century. Phil Trans R Soc B 363, 557 - 572. 

Davis G P; D’Occhio M J; Hetzel D J S (1997). SMART Breeding: Selection with markers and 

advanced reproductive technologies. 12 th Conf. Assoc. Adv. Anim. Breed Genet. 12, 

429-432. 

448

Geiger H H; Schnell F W (1970). Cytoplasmic male sterility in rye (Secale cereale L.). Crop 

Sci 10, 590–593. 

Geiger H H; Morgenstern K K (1975). Angewandt-genetische Studien zur cytoplasmatischen 

Pollensterilität bei Winterroggen. Theor Appl Genet 46, 269–276. 

Hackauf B; Wehling P (2002). Identification of microsatellite polymorphisms in an expressed 

portion of the rye genome. Plant Breed. 121, 17-25. 

Hackauf B; Wehling P (2003). Development of microsatellite markers in rye: map 

construction. Plant Breed. Seed Sci. 48, 143-151. 

Hackauf B; Wortmann H; Wehling P (2006). Nutzung von genomischen Ressourcen aus Reis 

und Gerste zur gezielten Markierung von Genen der Befruchtungskontrolle bei Roggen. 

Ber. 57. Tag. Vereinig. Pflanzenzüchter und Saatgutkaufleute Österreichs, 33-36. 

Hackauf B; Wortmann H; Wehling P (2007). Unravelling genomic regions involved in 

fertilization control in rye: advances and prospects. Vortr. Pflanzenzüchtung 71, 

210-216. 

Hackauf B; Stojałowski S; Wortmann H; Wilde P; Fromme F J; Menzel J; Korzun V; Wehling 

P (2009a). Minimierung des Mutterkornbefalls im Hybridroggen durch Ansätze der 

Präzisions-züchtung. J. Kulturpfl. 1, 15-20. 

Hackauf B; Rudd S; van der Voort J R; Miedaner T; Wehling P (2009b). Comparative mapping 

of DNA sequences in rye (Secale cereale L.) in relation to the rice genome. Theor. 

Appl. Genet. 118, 371-384. 

Khlestkina E K; Than M H M; Pestsova E G; Roder M S; Malyshev S V; Korzun V; Borner A 

(2004). Mapping of 99 new microsatellite derived loci in rye (Secale cereale L.) 

including 39 expressed sequence tags. Theor. Appl. Genet. 109, 725-732. 

Khlestkina E K; Than M H M; Pestsova E G; Roder M S; Malyshev S V; Korzun V; Borner A 

(2005). Erratum: Mapping of 99 new microsatellite derived loci in rye (Secale cereale 

L.) including 39 expressed sequence tags. Theor. Appl. Genet. 110, 990-991. 

Michelmore R W; Paran I; Kesseli R V (1991). Identification of markers linked to diseaseresistance 

genes by bulked segregant analysis: a rapid method to detect markers in 

specific genomic regions by using segregating populations. Proc Natl Acad Sci U S A 

88, 9828-9832. 

Miedaner T; Glass C; Dreyer F; Wilde P; Wortmann H; Geiger H H (2000). Mapping of genes 

for male-fertility restoration in ‘Pampa’ CMS winter rye (Secale cereale L.). Theor 

Appl Genet 101, 1226–1233. 

Mirdita V; Dhillon B S; Geiger H H; Miedaner T (2008). Genetic variation for resistance to 

ergot (Claviceps purpurea [Fr.] Tul.) among full-sib families of five populations of 

winter rye (Secale cereale L.). Theor Appl Genet. 118, 85-90. 

Saal B; Wricke G (1999). Development of simple sequence repeat markers in rye (Secale 

cereale L.). Genome 42, 964-972. 

SAS Institute Inc. (2003). SAS/STAT User’s Guide. Release 9.1. Cary, North Carolina, USA 

Snedecor G W; Cochran W G (1989). Statistical methods. 8 th Ed. Iowa State University: Ames. 

Stracke S; Schilling A G; Förster J; Weiss C; Glass C; Miedaner T; Geiger H H (2003). 

Development of PCR-based markers linked to dominant genes for male-fertility 

restoration in Pampa CMS of rye (Secale cereale L.). Theor Appl Genet 106, 

1184-1190. 

449

Tanksley S D; Young N D; Paterson A H; Bonierbale M W (1989). RFLP mapping in plant 

breeding: new tools for an old science. Bio/Technology 7, 257-264. 

Xu K; Xu X; Fukao T; Canlas P; Maghirang-Rodriguez R; Heuer S; Ismail A M; Bailey-Serres 

J; Ronald P C; Mackill D J (2006). Sub1A is an ethylene-response-factor-like gene that 

confers sub-mergence tolerance to rice. Nature 442, 705-708. 

450

Hannah M, Metzlaff M: Maintenance of NAD homeostasis - a promising route towards multiple stress tolerance 

in plants. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 451; 


9-3 Maintenance of NAD homeostasis - a promising route towards multiple 

stress tolerance in plants 

Hannah M, Metzlaff M 

Bayer BioScience N.V., Technologiepark 38, 9052 Gent, Belgium 

Email: Michael.Metzlaff@bayercropscience.com 

Abstract 

Maintenance of NAD+ homeostasis - a promising route towards multiple stress 

tolerance in plants Matthew Hannah & Michael Metzlaff Bayer BioScience N.V., 

Gent, Belgium Biotic and abiotic stresses can result in harvest losses in all major 

crops of up to 80 %. Worldwide modern solutions exist for fighting biotic stresses 

caused by insects, fungi, bacteria, viruses and weeds. In contrast, efficient 

technologies for reducing the impact of abiotic stresses like drought, heat, cold, 

salinity and ozone still have to be developed. Because of the expected global 

climate changes the introduction of the trait “abiotic stress tolerance” into crops has 

become a major challenge in modern agriculture. NAD+ is a key co-factor for many 

enzymes activated during plant stress response. The re-synthesis of NAD+ 

molecules is an energy-consuming process requiring 8 molecules ATP/1 molecule 

NAD+. We modulated NAD+-consuming pathways either by silencing of stressresponding 

enzymes, i.e. PARP or by over-expressing genes of the NAD+ salvage 

pathway. Both strategies resulted in pronounced resistance of Arabidopsis and 

oilseed rape plants to various abiotic stresses. First field trials proved stress 

tolerance and yield maintenance of transgenic oilseed rape lines cultivated in 

drought conditions. Genome-wide expression profiling revealed the resetting of 

expression of a number of stress-related and ABA-controlled genes suggesting 

novel links of signaling pathways underlying plant stress response. 

451

Molodchenkova O O, Adamovskaya V G: The Formation of Biochemical Resistance to Biotic and Abiotic Stress 

in Cereals. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 452- 


9-4 The Formation of Biochemical Resistance to Biotic and Abiotic Stress 

in Cereals 

Molodchenkova O O, Adamovskaya V G 

Plant Breeding Genetic Institute, Ovidiopolskaya doroga 3, Odessa, 65036, Ukraine 

Email: adam@paco.net 

452 

ABSTRACT 

Changes in the activity of lipoxygenase, H + -ATPase, NADPH-oxidase, signal 

molecule, oxidative and anti-oxidative processes during defense reactions of cereals 

infected by Fusarium were studied, as were the influences of drought and salicylic 

acid. Differentiated changes.depended on the genotype resistance to Fusarium. It is 

supposed that SA, hydrogen peroxide and nitric oxide are the components of the 

system that regulates the activation of mechanisms of plant resistance by 

pathogenesis. 

INTRODUCTION 

Problem of increasing the plant resistance by the growth of anthropogenic load to ecosystem 

and obtaining the stable harvests at the zones of risky farming cause the special actuality of 

studying the mechanisms of plants adaptation to the adverse effects of the environment. 

Adaptation of plants to the impact of biotic and abiotic factors is associated with the activation 

of defense reactions that are responsible for the retention of vital functions and reorganization 

of the plant’s metabolism in response to stress. Common as well as highly specific mechanisms 

of adaptation were found for each kind of stress. Formation of stress protein, the activation of 

ferments, changes in oxidative and anti-oxidative processes, and changes in phenol and lipid 

metabolism of cell are reactions that relate to biochemical processes connected with the 

resistance to stress (Ilyinskaya 1991, Apel 2004, Taran 2004). One of inductors of plant 

resistance to disease is salicylic acid (SA) (Raskin 1992). The aim of this work was to study 

changes in the activity of lipoxygenase (LOX), H + -ATPase, NADPH-oxidase, signal molecule, 

oxidative and anti-oxidative processes during defense reactions of cereals infected by 

Fusarium, as well as in the influence of drought and SA.

METHODS 

Activity of a LOX was measured by absorption at 440 nm using linoleic acid as a substrate, 

following the method of Budnitskaya (Budnitskaya 1955). SA concentration was quantified by 

HPLC (Raskin et al. 1989). Lipid peroxidation was measured by MDA accumulation (Uchida 

1999). Glutathione content was determined using Ellman reagent (Grishko & Sushikov 2002). 

Activity of glutathionereductase was measured following Iovata & Tanaka (1977). Activity of 

glutathioneperoxidase was determined following Sushikov& Grishko (2004). A catalase 

activity was determined by its reaction with H2O2 and DAP (Koroljuk et al. 1988). H2O2 was 

measured by the fluorometric method (Ebermann & Couperus 1987). NO content was 

measured by levels of nitric oxide stabile metabolites: NO2 – and NO3 – (Komarevtseva et al. 

2002). Activity of H + -ATPase was determined by method (Rudashevskaya et al. 2005). 

Activity of NAD(P)H-oxidase was determined for oxidation of NAD(P)H (Pinton et al. 1994). 

This research was done on cultivars of winter wheat, spring barley and maize that differed in 

their resistance to Fusarium infection and drought and on four methods of germination (in pure 

water, in presence of 2 mM SA, in presence of Fusarium, Bipolaris and in drought conditions). 

RESULTS 

The dynamics of activity of cereal seedlings LOX with the infection by Fusarium and SA 

influence had different directions depending on the level of resistance and kind of crop, which 

indicated the participation of the ferment in the response to defense reactions of cereal plants 

by the given influences. The function and role of SA, hydrogen peroxide and nitric oxide in the 

tissues of cereal seedlings by pathogenesis were researched. Differentiated changes of the 

given indexes depended on the genotype resistance to Fusarium. It is supposed that SA, 

hydrogen peroxide and nitric oxide are the components of the system that regulates the 

activation of mechanisms of plant resistance by pathogenesis. Dynamics of changes in the 

intensity of oxidative and anti-oxidative processes as well as of activity of H + -ATPase, 

NADPH-oxidase membrane ferments showed different character response reaction of the 

resistant and susceptible cereal genotypes to the influence of pathogen, drought, high 

temperature and SA. Retention of antioxidants level (renovation of glutathione, activity of 

glutathione independent ferments, catalase) with the lowering of intensity level of lipid 

peroxidation processes in cereal seedlings in the conditions of water shortage can be attributed 

to the qualitative adaptation reaction of plants by drought and as a result to the keeping the 

physiological processes in norm. Increase of lipids peroxidation with the following 

mobilization of antioxidants can serve as one of the defense reactions of cereals by being 

infected by Fusarium. It is supposed that there is a connection between H + -ATPase activity and 

lipids peroxidation while studying the stress factors. The connection was ascertained between 

NADPH-oxidase activity and the content of hydrogen peroxide and endogenous SA in the 

tissues of cereals seedlings by infection with Fusarium and SA action. 

453

DISCUSSION AND CONCLUSIONS 

The researched biochemical indexes take part in the formation of response defense reactions of 

cereals with infection by Fusarium, influence of water shortage, high temperature and SA. The 

character of response reactions varied depending on the genotype resistance to the Fusarium 

and drought, genus of crop and the nature of the influencing stress factor. The uneven 

orientation of changes of researched biochemical indexes in the tissues of different genus of 

cereal seedlings that differ by resistance to Fusarium, drought, with biotic and abiotic 

influences is connected with the different contribution of those substances to the formation to 

the plants defense reactions and most likely has a genus specific character. 

REFERENCES 

Budnichkaya E V (1955). Research of lipoxygenase activity of grasses by a method of 

oxidation of carotin. Biochemistry 20, 615-621. 

Grishko V N; Sushikov D V(2002). Method of estimation of restorative form of glutathione in 

the vegetative organs pf plants.Ukr. Biochemistry Journal 74, 123-124. 

Sushikov D V; Grishko V N (2004). Function of glutathione-dependent antioxidant system in 

peas, soya-bean and corn at the action of substance of cadmium. Physiology and 

biochemistry of cultural plants V.36. N 6, 503-509. 

Ebermann R; Couperus A A (1987). A nonenzymatic method for determination of hydrogen 

peroxide and organic peroxides. Analytical biochemistry 165, 414-419. 

Iljinskaja L I; Vasjukova NI; Ozerechkovskaya O L (1991). Biochemical aspects of induced 

resistance and a susceptibility of plants. Results of a science and technics(technical 

equipment). A series: Protection of plants. M.: VINITI: 4-71. 

Komarevtseva L A; Orlova E A; Blagodarenko E A (2002). Level of nitric oxide of apoptosis 

in kidney. Ukr. Biochemical Journal 74, 116-119. 

Koroljuk M A; Tokarev V M; Маyorova I G (1988).Definition of catalase activity. Lab. 

Delo. 1, 16-18. 

Raskin I (1992) Salicylate a new plant hormone. Plant Physiol 99, 799-803. 

Rudashevskaya E L; Kirpichnikova A A; Shishova M P (2005). Activity of H + ATP-ase of 

plasmalemma of coleoptyle’ cell in the process of seedling of corn development. Plant 

Physiol.(Russian) 52, 566-572. 

Pinton R; Cakmak I; Marschner H (1994). Zinc deficiency enhanced NADP(H)-dependent 

superoxide radical production in plasma memrane vesicles isolated from roots of bean 

plants.J. Exp. Bot 45, 45-50. 

Uchida K; Shiraishi M; Naito Y; Torii Y; Nakamura Y; Osaka T (1999). Activation of stress 

signaling pathways by the end product of lipid peroxidation. JBC Online 274, 

2234-2242. 

454

Brahm L, Gladwin R, Semar M, Gomes-de-Oliveira C: Contribution of chemical treatments to Crop Stress 

Tolerance. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 455; 


9-5 Contribution of chemical treatments to Crop Stress Tolerance 

Brahm L, Gladwin R, Semar M, Gomes-de-Oliveira C 

BASF SE, Carl-Bosch-Str. 62, 67117 Limburgerhof 

Email: lutz.brahm@basf.com 

Abstract 

Major challenge of the up-coming years will be to meet the demands of a growing 

world population and prospering societies in Asia and Latin-America for food, feed 

and energy. The potential to take more land into agricultural production is limited 

and is partly counteracted by loss of arable land for infrastructure and building. 

Therefore, the higher production has to come mainly from a higher productivity in 

agriculture. Productivity is limited by several factors. As we know from history, the 

increase in cropping intensity can be a key in parts of the world, where still basic 

inputs like herbicides, fungicides, insecticides and fertilizer inputs can be increased 

Major threats to production are biotic and abiotic stresses that crops are exposed to. 

Biotic stresses like weeds, diseases and pests can be controlled by crop protection 

products and crop resistance, a practice well established in the major production 

areas. Abiotic stresses are known to be as well a major limitation to yield. With the 

climatic change we are facing, abiotic stresses, especially drought and heat stress, 

are coming more and more into focus. We discuss the potential of agro-chemicals to 

contribute to crop tolerance to abiotic stresses. The control of weeds, diseases and 

pests keeps the crop strong/healthy enabling it to withstand a-biotic stresses better. 

Furthermore certain crop protection compounds (like strobilurins or PGR´s) interact 

with the crop physiology and may provide anti-stress properties. 

455

Perovic D, Welz G, Förster J, Kopahnke D, Lein V, Löschenberger F, Buerstmayr H, Ordon F: Breeding 

Strategies for Wheat Improvement: Creating Semi-Dwarf Phenotypes with Superior Fusarium Head Blight 

Resistance. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 456- 


10-1 Breeding Strategies for Wheat Improvement: Creating Semi-Dwarf 

Phenotypes with Superior Fusarium Head Blight Resistance 

Perovic D 1 , Welz G 2 , Förster J 3 , Kopahnke D 1 , Lein V 4 , Löschenberger F 5 , Buerstmayr H 6 , 

Ordon F 1 

1 

Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Resistance 

Research and Stress Tolerance, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 

2 

Fr. Strube Saatzucht GmbH Co. KG, Hauptstraße 1, 38387 Söllingen, Germany 

3 

Saaten-Union Resistenzlabor GmbH, Hovedisser Str. 92, 33818 Leopoldshöhe, Germany 

4 Saaten Union Recherche SARL, 163 avenue de Flandre, 60190 Estrées St Denis, France 

5 Saatzucht Donau,GesmbH & CoKG, Saatzuchtstraße 11, 2301 Probstdorf, Austria 

6 

University of Natural Resources and Applied Life Sciences, Department for 

Agrobiotechnology (IFA-Tulln), Institute for Plant Production Biotechnology, Konrad-Lorenz- 

Strasse 20; 3430 Tulln, Austria 

Email: dragan.perovic@jki.bund.de 

456 

ABSTRACT 

In order to study the association of “reduced height” (Rht) genes and Fusarium head 

blight (FHB) resistance, Rht-B1b, Rht-D1b and Rht8 genes were introduced in tall 

high-yielding wheat cultivars expressing a good level of FHB resistance via markerassisted 

selection (MAS). The F1 plants of five crosses were backcrossed and in 

successive generations 189 BC1, 395 BC1S1 and 420 BC2 plants were genotyped. 

MAS was performed with Rht-B1b and Rht-D1b specific markers, with SSR 

Xgwm261 being diagnostic for Rht8, and with a Ppd-D1 specific marker that is 

tightly linked to Rht8. During the first phase of the BC1 selection, chromosomes 

harbouring Rht genes were screened with 48 SSRs for polymorphisms followed by 

the use of 3 to 5 polymorphic SSRs for the detection of recombination around these 

loci. Based on this procedure a total of 42 lines were selected for the second 

selection phase of the BC1. For this purpose 152 genome-wide SSRs were screened 

for polymorphisms between parental lines and 48 to 68 polymorphic SSRs per cross 

combination were used for background fingerprinting of the 42 lines. Finally eight

BC1 plants were selected to be backcrossed again. Among the analyzed BC1 plants 

the percentage of the recurrent parent genome varied between 65% and 78%. To 

further test the efficiency of this selection procedure, the BC1S1 and BC2 

generations were produced in parallel and genotyped. During the BC1S1 selection a 

set of 395 plants derived by embryo rescue from the eight BC1 plants were 

genotyped in three steps. Rht and Ppd gene marker selection resulted in reduction to 

78 BC1S1 plants, while the target chromosome genotyping reduced the number of 

plants to 18 followed by the selection of 8 plants based on recurrent parent genome 

recovery. These plants are now used for doubled-haploid line development. 

Genotyping of the BC2 generation is ongoing. The emerging BC1S1- and BC2S1derived 

DH lines will be tested in multi-location field trials to analyse the 

association of the three Rht genes and FHB susceptibility. Results up to now have 

shown that MAS in combination with embryo rescue saved time and reduced the 

number of plants to be backcrossed. We expect that Rht alleles will be quickly 

integrated into tall and high-yielding cultivars with excellent quantitative FHB 

resistance carrying only small chromosomal fragments of the Rht donor lines. 

Factors influencing the efficiency of applied marker-assisted selection procedures 

are discussed in this paper. 

INTRODUCTION 

Semi-dwarf wheat varieties were the back-bone of the so-called “Green Revolution” in the 

1960s and 1970s. These short-strawed varieties, introduced through the work of Norman 

Borlaug at CIMMYT (Borlaug 1968), were rapidly adopted on the Indian subcontinent as they 

were less prone to lodging, especially when fertilized with nitrogen, producing more grain 

yield at the expense of straw. The “reduced height” (Rht) genes utilized in the process were 

Rht-B1b and Rht-D1b originating from the Japanese variety Norin10. Today, the majority of 

wheat cultivars from the UK, France and Germany carry Norin10-derived semi-dwarf genes. In 

Europe, the Italian wheat breeder Strampelli pioneered the development of short-strawed wheat 

varieties in 1913, when he crossed the Japanese variety Akakomugi, the source of the Rht8 

gene, with western wheat cultivars and released many varieties that became very successful 

throughout southern and south-eastern Europe (Borojevic & Borojevic 2005). The dwarfing 

gene Rht8 is tightly linked with a gene for photoperiod insensitivity, Ppd-D1 (Korzun et al. 

1998), which improves adaptation to southern environments with shorter days. Rht8 and Ppd- 

D1 together reduce plant height by 10 cm, increase spikelet fertility and shorten the time to 

flowering by 8 days (Gale & Youssefian 1985). 

Fusarium head blight (FHB) is considered the most serious wheat disease in Europe. Therefore, 

FHB-resistant, semi-dwarf varieties would help to increase yield, to lower production costs 

(fungicide use) and to improve food and feed security. Among wheat breeders and pathologists 

it is common knowledge that shorter varieties tend to become more severely infected by FHB 

than taller varieties (reviews in Miedaner 1997; Mesterházy 2003; Srinivasachary et al. 2008). 

457

Also, very recently it was shown that Rht-B1b and Rht-D1b loci differ significantly in their 

influence on FHB resistance (Srinivasachary et al. 2009; Miedaner & Voss 2009). However, it 

is not yet clear whether tight linkage between Rht and susceptibility genes, or pleiotropic 

effects of Rht mutants are responsible. 

Rht genes vary in their physiological mechanism – gibberellic acid (GA) insensitivity – and, 

hypothetically, their effect on FHB susceptibility since the pathogenesis-related signal 

transduction pathway may be affected by GA. Therefore, our aim is to analyse the association 

of various Rht alleles with FHB susceptibility and use a combination of marker-assisted 

selection (MAS) and doubled-haploid (DH) technology for accelerated crop improvement. 


Two high-yielding FHB-resistant wheat cultivars, i.e. Midas and Phönix, were crossed with 

various Rht lines (Table 1). Donors of Rht-B1b and Rht-D1b were cvs. ‘Striker’ and ‘Toras’ 

respectively, while donors of Rht8 were cvs. ‘Delabrad’, ‘Pobeda’ and ‘Bezostaya Dwarf’. 

Developing karyopses of F1, BC1, BC1S1 and BC2 (ongoing) plants were subjected to embryo 

rescue in order to abridge the backcrossing procedure. Specific markers for Rht-B1b, Rht-D1b 

and Ppd-D1 were amplified according to Ellis et al. (2002) and Beales et al. (2007), 

respectively. A total of 200 genomic and EST-derived SSR markers located on 21 wheat 

chromosomes (48 from 2D, 4B and 4D chromosomes and 152 from the other 18 chromosomes) 

for the Rht and background selection were amplified according to Röder et al. (1998) and 

Somers et al. (2004). PCRs were carried out in an AB9700 Thermal Cycler (Applied 

Biosystems) and products were resolved on an AB3130xl DNA analyzer according to the 

manufacturer’s (Applied Biosystems) instruction. Data analysis was carried out using 

GeneMapper software v.4 (Applied Biosystems). The proportion of recurrent parent recovery 

(PRPR) was calculated according to the formula: 

458 

Table 1. Winter wheat crosses used for introgression of Rht genes into FHB-resistant 

cultivars 

BC1 population Selected genotype class 

Target 

chr. 

Total No. 

BC1 plants 

Rht/Ppd 

sel. BC1 's 

No. BC1s 

recomb. on 

target chr. 

Midas/Delabrad//Midas Rht8, Ppd-D1 or ppd-D1 2D 41 8 4 2 

Midas/Pobeda//Midas Rht8, Ppd-D1 or ppd-D1 2D 19 9 9 2 

Phönix/Bezostaya Dwarf//Phönix Rht8, Ppd-D1 or ppd-D1 2D 69 41 21 2 

Midas/Striker//Midas Rht-B1b 4B 12 5 4 1 

Midas/Toras//Midas Rht-D1b 4D 48 18 4 1 

No. BC1 

plants 

for BC2 

PRPR= [(RP + H/2)/ n] x 100, where RP is the sum of recurrent parents alleles, H is the 

number of heterozygous loci and n is the number of investigated SSRs.

Production of BC1S1- and BC2S1-derived DH lines is ongoing. These lines will be 

investigated for their association of different Rht alleles with FHB susceptibility. 


MAS of desirable gene/marker combinations was an efficient way to reduce the number of 

plants to be used in further crossing schemes. Out of the total number of BC1 plants available 

those carrying the respective Rht genes and, subsequently, showing recombination at the Rht 

chromosomal region were selected. The number of genotypes selected due to this criterion in 

the BC1 generation varied between 4 and 21 plants (Table 1). The next selection criterion was 

the degree of restoration of the recurrent parent genome estimated by scanning the non-target 

chromosomes. Out of 152 SSRs that were used for parent screening, between 48 and 68 

markers per cross combination were polymorphic. Finally, after background fingerprinting as 

the third step of MAS, eight BC1 plants were selected for further propagation. Recovery of the 

recurrent parent genome in the BC1 generation was rather low and varied between 65% and 

78% (Table 3). 

Table 2. Results of marker assisted selection procedure for five BC1S1 cross 

combinations 

BC1 population 

Total No. 

BC1S1 

plants 

Rht/Ppd 

selected 

BC1S1 's 

No. BC1S1's 

recomb. on 

target chr. 

No. BC1S1's 

plants for DHs 

production 

Midas/Delabrad//Midas 114 19 4 2 

Midas/Pobeda//Midas 86 22 4 2 

Phönix/Bezostaya Dwarf//Phönix 100 21 7 2 

Midas/Striker//Midas 42 10 2 1 

Midas/Toras//Midas 56 10 3 1 

Table 3. Proportions of recurrent parent recovery at BC1 and BC1S1 generations 

BC1 population 

BC1 % RP 

min. 

BC1 % RP 

max. 

BC1S1 % 

RP min. 

BC1S1 % 

RP max. 

Midas/Delabrad//Midas 67.2 75.4 79.2 94.0 

Midas/Pobeda//Midas 65.5 75.4 80.0 88.6 

Phönix/Bezostaya Dwarf//Phönix 65.6 73.5 86.2 93.3 

Midas/Striker//Midas 68.9 72.8 82.0 84.8 

Midas/Toras//Midas 73.5 78.7 83.3 85.4 

459

In the second cycle of selection, a set of 395 BC1S1 plants derived by embryo rescue from the 

eight BC1 plants were genotyped in three steps (Table 2). Rht-Ppd gene marker selection 

resulted in a reduction to 82 BC1S1 plants. Out of these, 20 plants, i.e. between 2 and 7 per 

combination, were selected based on target chromosome genotyping. After background (i.e. 

non-target chromosome) genotyping, eight out of 20 plants were selected for doubled-haploid 

line production which is ongoing. In the BC1S1 generation, MAS-based recovery of the 

recurrent parent genome varied between 79.2% and 93.3% (Table 3). 

MAS in combination with embryo rescue actually saved time and reduced the number of plants 

to be backcrossed. Another goal was to quickly recover the recurrent parent genome, as it has 

been shown in other crops, e.g. Sorghum bicolor , that already in BC1 individuals with a very 

high proportion of the recurrent parent genome (> 75%) can be identified (Uptmoor et al. 

2006). Since in the BC1 generation the observed recovery of the recurrent parent genome did 

not differ significantly from the expected proportion of 75%, the superiority of MAS vs. 

phenotypic selection remains to be confirmed for dominantly inherited traits. 

Plants showing the shortest donor segment around the respective Rht loci and the highest 

proportion of recurrent donor genome are used to produce BC1S1 and BC2 derived DH-lines. 

These will be tested in multi-location field trials to analyse the association of the three Rht 

genes and FHB susceptibility. The DH lines developed from BC1S1 and BC2 plants will on 

the one hand be suited for direct use in wheat breeding and on the other hand may help to 

answer the question whether genotypes carrying Rht8 are less negatively affected by FHB than 

lines carrying Rht-B1b or Rht-D1b, or, more generally, whether the gibberellic acid status of a 

wheat plant would be relevant to FHB. Due to their physiologically different mode of action, 

the Rht genes may well have different effects on the interaction of wheat with its pathogens, 

such as Fusarium graminearum or F. culmorum. In this case, Rht-B1b or Rht-D1b had truly 

pleiotropic effects on FHB resistance. If genes closely linked to Rht-B1b or Rht-D1b were 

causing the elevated FHB susceptibility, the effect should have been removed by MAS in the 

BC2S1DH lines. 


Project SHORTWHEAT is funded within the ERANet EUROTRANS-BIO-1 programme 

(0315009). We thank Dr. David Laurie, John Innes Centre, UK, for providing an unpublished 

protocol for Ppd-D1 genotyping and B. Knüpfer for excellent technical assistance. 

REFERENCES 

Beales J; Turner A; Griffiths S; Snape J W; Laurie D A (2007). A Pseudo-Response Regulator 

is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat (Triticum 

aestivum L.). Theoretical & Applied Genetics 115, 721-733. 

Borlaug N (1968). Wheat breeding and its impact on world food supply. In: Proceedings of 3 rd 

International Wheat Genetics Symposyum, pp. 1-36. Australian Academy Of Science: 

Canberra. 

460

Borojevic Ka; Borojevic Ks (2005). The transfer and history of "reduced height genes" (Rht) in 

wheat from Japan to Europe. Journal of Heredity 96, 455-459. 

Ellis M H; Spielmeyer W; Gale K R; Rebetzke G J; Richards R A (2002). "Perfect" markers 

for the Rht-B1b and Rht-D1b dwarfing genes in wheat. Theoretical & Applied Genetics 

105, 1038-1042. 

Gale M D; Youssefian S (1985). Dwarfing genes in wheat. In: Progress in Plant Breeding, eds 

G E Russel,. pp. 1-35. Butterworth: London. 

Korzun V; Röder M S; Ganal M W; Worland A J; Law C N (1998). Genetic analysis of the 

dwarfing gene (Rht8) in wheat. Part I. Molecular mapping of Rht8 on the short arm of 

chromosome 2D of bread wheat (Triticum aestivum L.). Theoretical & Applied 

Genetics 96, 1104-1109. 

Mesterházy Á (2003). Breeding wheat for Fusrium head blight resistance in Europe. In: 

Fusarium Head Blight of Wheat and Barley, eds K J Leonard & W R Bushnell, pp. 211- 

240. American Phytopathological Society Press: St. Paul, Minnesota, USA. 

Miedaner T (1997). Breeding wheat and rye for resistance to Fusarium diseases. Plant 

Breeding 116, 201-220. 

Miedaner T; Voss H H (2009). Effect of Dwarfing Rht Genes on Fusarium Head Blight 

Resistance in Two Sets of Near-Isogenic Lines of Wheat and Check Cultivars. Crop 

Science 48, 2115-2122. 

Röder M S; Korzun V; Wendehake K; Plaschke J; Tixier M H; Leroy P; Ganal M W (1998). A 

microsatellite map of wheat. Genetics 149, 2007-2023. 

Somers D J; Peter I; Edwards K (2004). A high-density microsatellite consensus map for bread 

wheat (Triticum aestivum L.). Theoretical & Applied Genetics 109, 1105-1114. 

Srinivasachary N; Gosman A; Steed J; Simmonds M; Leverington-Waite M; Wang Y; Snape J; 

Nicholson P (2008). Susceptibility to Fusarium head blight is associated with the Rht- 

D1b semi-dwarfing allele in wheat. Theoretical & Applied Genetics 116, 1145–1153. 

Srinivasachary N; Gosman A; Steed T W; Hollins R; Bayles; Jennings P; Nicholson P (2009). 

Semi-dwarfing Rht-B1 and Rht-D1 loci of wheat differ significantly in their influence 

on resistance to Fusarium head blight. Theoretical & Applied Genetics 118, 695–702. 

Uptmoor R; Wenzel W; Ayisi K; Donaldson G; Gehringer A; Friedt W; Ordon F (2006). 

Variation of the genomic portion of the recurrent parent in BC1 and ist relation to yield 

performance in sorghum (Sorghum bicolor) breeding for low-input conditions. Plant 

Breeding 125, 532-534. 

461

Beckuzhina S, Kochieva, E: Wheat double haploid lines with improved salt tolerance: in vitro selection and 

RAPD analysis. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 


10-2 Wheat double haploid lines with improved salt tolerance: in vitro 

selection and RAPD analysis 

Beckuzhina S 1 , Kochieva E 2 

1 

Astana Agricultural University, Prospect Pobedy 62, 010011, Astana, Kazakhstan 

2 Moscow Agricultural Academy, Timiryazevskaya 49, 127550, Moscow, Russia, sara- 

bek@yandex.ru; ekochieva@yandex.ru 

In the major grain crops anther culture is the commonly used method to develop haploids and 

double haploids (DH). Double haploid plants have been increasingly used by breeders to 

develop and release new cultivars with improved agronomic traits. Combination of microspore 

embryogenesis with in vitro selection can provide an efficient screen for desired gametoclonal 

variants. In this method the selection agent is introduced into culture medium. Surviving 

embryos/plants are doubled and grown in the greenhouse. Verification takes place in the in the 

next generation. Several double haploid lines resistant to herbicides have been developed in 

rapeseed by this process (Swanson et al. 1988). Similar systems of in vitro mutagenesis and 

selection were developed for generating DH lines with improved tolerance to Sclerotinia 

sclerotiorum in B. napus (Liu et al. 2005) and Erwinia carotovora in B. campestris (Zhang & 

Takahata 1999). In addition to herbicide and disease resistance, mutants for seed quality traits 

in rapeseed (Kott 1998) and for salt tolerance in rice (Rahman et al.. 1995) have been selected. 

An essential component of this system is the molecular characteristic of selected genotypes. 

Several techniques of molecular biology are available for detection of genetic polymorphism at 

the DNA level. The randomly amplified polymorphism (RAPD) method has been widely used 

to estimate genetic diversity (Araujo et al. 2001; Bocianowski et al. 2003). The objectives of 

this study were (I) to screen salt tolerant digaploid wheat lines via anther culture and (II) to 

investigate the genetic diversity of anther-derived plants by RAPD analysis. 

The spring wheat cv. Tselinnaya-Jubileinaya was used in the experiments. To screen salt 

tolerant embryoids wheat anthers were cultivated on the selective media containing 0.01, 0.05 

and 0.1% NaCI (Table 1). The selection was performed in the population of 4,380 anthers. The 

anther response varied from 0.52% to 1.1%. The spontaneous digaploid line U-580 was 

selected and grown in the greenhouse to maturity. The F1 generation of this line was subjected 

to the second cycle of in vitro anther culture. We were able to screen three gametoclonal lines 

LGV-1, LGV-3 and LGV-20 under selective conditions (NaCI). The response of selected lines 

to salt salinity was investigated at the field site in the Agricultural Research Centre, 

462

Kazakhstan (Table 2). There was a significant difference between the control wheat cultivar 

and double haploids. The gametoclonal line LGV-3 demonstrated the highest yield in saline 

conditions. The field test has revealed that stress tolerance was manifested at the level of whole 

plant and inherited. 

Table 1. Screening of wheat gametoclonal variants in selective conditions. 

Genotype NaCI No anthers No Embryogenesis 

concentration (%) 

First cycle 

embryoids efficiency (%) 

Tselinnaya-Jubileinaya 0.01 2000 16 1.1 

0.05 1180 13 0.52 

0.1 1200 9 1.0 

U-580 0.01 

Second cycle 

500 17 3.4 

0.05 500 12 2.4 

0.1 580 13 2.2 

Table 2. 

Genotype 

Field test in saline conditions. 

Crop yield, 

centner/ha 

2004 2005 2006 Mean 

control 20.2 21.7 26.0 22.6 

U-580 24.1 22.3 26.2 24.2 

LGV-1 24.6 21.9 26.1 24.2 

LGV-3 28.1 20.8 29.6 26.2 

LGV-20 24.7 19.6 25.6 23.3 

After observing the inheritance of salt tolerance in field trails RAPD analysis was performed to 

investigate the genetic basis of this variation. The 9 decamber primers amplified 24 

polymorphic fragments. The RAPD profiles of three gametoclonal lines LGV-1, LGV-3, LGV- 

20 differentiated this group from parental U-580 line: 13 polymorphic lines were scored. The 

dendrogram generated by cluster analysis of RAPD polymorphism using coefficient of 

similarity of Jaccard for investigated genotypes can be divided into two groups (Fig. 1). The 

first one includes LGV-1 and LGV-20 gametoclones. The original cv. Tselinnaya-Jubileinaya, 

DH line U-580 and gametoclone LGV-3 belong to the second subgroup. 

The data presented here provide further evidence that the anther culture technique has the 

potential to increase wheat stress tolerance. The phenotypic variation for salt tolerance was 

related to genetic variability between the parental cultivar (U-580) and gametoclonal variants 

(LGV-1, LGV-3 and LGV-20), as was shown by RAPD analysis. Double haploid lines 

designed in this study can be used in breeding programmes to design salt-tolerant genotypes 

and in basic research to study the mechanisms of salt tolerance. 

463

464 

Figure 1. Dendrogram generated by cluster analysis of RAPD polymorphism showing 

genetic divergence between wheat cultivars Akmola-2 (1), Tselinnaya- 

Jubileinaya (3) and gametoclonal lines U-580 (2), LGV-1 (4), LGV-3 (6) and 

LGV-20 (5). 

REFERENCES 

Araujo L G; Prabhu A S; Filippi M C; Chaves L J (2001). RAPD analysis of blast resistant 

somaclones from upland rice cultivar IAC 47 for genetic divergence. Plant Cell Tissue 

and organ Culture 67, 16-172. 

Bocianowski J; Chelkowski J; Kuczynska A; Wisniewska H; Surma M; Adamski T (2003). 

Assessment of RAPD markers for barley doubled haploid lines resistant and susceptible 

to Fusarium culmorum at seedling and adult plant growth stages. Applied Genetic 44, 

355-360. 

Kott L (1988). Application of doubled haploid technology in breeding of oilseed Brassica 

napus. AgBiotech News Inform 10, 69N-74N. 

Liu S; Wang H; Zhang J; Fitt B D L; Xu Z; Evans N; Liu Y; Yang W, Gao X (2005). In vitro 

mutation and selection of doubled haploids Brassica napus lines with improved 

resistance to Sclerotinia sclerotorium. Plant Cell Reports 24, 133-144. 

Rahman M H; Krishinaraj S; Thorpe T A (1995). Selection for salt tolerance in vitro using 

microspore-derived embryos of Brassica napus CV Topas, and the characterization of 

putative tolerant plants. In vitro cellular and developmental biology. Plant 31, 116-121. 

Swanson E B; Coumans M P; Brown G L; Patel J D; Beversdorf W D (1988). The 

characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection 

of microspores and protoplasts. Plant Cell Reports 7, 83-87. 

Zhang F-L; Takahata Y (1999). Microspore mutagenesis and in vitro selection for resistance to 

soft rot disease in Chinese cabbage (Brassica campestris L. ssp. Pekinesis). Breeding 

Science 49, 161-166.

Körösi K, Virányi F: Induction of defense-related genes in downy-mildew infected sunflower plants treated with 

resistance inducer. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

10-3 Induction of defense-related genes in downy-mildew infected sunflower 

plants treated with resistance inducer 

Körösi K, Virányi F 

Institute of Plant Protection, Szent István University, Páter K. u. 1., 2103 Gödöllő, Hungary, 

Korosi.Katalin@mkk.szie.hu 

Abstract 

In our previous study it was shown that three plant activators, benzothiadiazole 

(BTH), β-aminobutyric acid (BABA) and dichloroisonicotinic acid (INA) could 

reduce downy mildew infection in sunflower. In the present work, we examined the 

gene expression of glutation S-tranferase (GST), defensin (PDF) and catalase 

(CAT) in different sunflower lines following BTH treatment and downy mildew 

inoculation. These genes are considered to play a crucial role in the plant defense 

responses against pathogen attack, based on their antixenobiotic, antimicrobial and 

antioxidant activities, respectively. 

Among the untreated sunflowers, resistant plants had higher GST and PDF 

transcript levels compared with the susceptible ones. It should be noted that all three 

genes were induced significantly in the activator-treated susceptible genotype. 

Furthermore, BTH treatment induced GST and PDF in the resistant sunflower, as 

well. 

Induced expression of three genes (GST, PDF and CAT) may play important role in 

the beneficial effect of this plant activator on defense response of sunflower against 

P. halstedii. 

INTRODUCTION 

Although downy mildew of sunflower (Plasmopara halstedii) can be effectively controlled by 

using genetic resistant plants and seed treatment with fungicides, protection can be hindered by 

the genetic variability of the fungus (Albourie et al. 1998; Gulya 2007). Thus, beside the 

traditional control strategies there was a need of looking for alternative methods to provide 

effective disease control. One solution can be the use of systemic induced resistance, i.e. the 

activation of the defense system of plants. 

465

Chemicals, such as BTH (benzo (1, 2, 3) thiadiazole-7-carbothioic acid S-methyl ester), BABA 

(DL-β-amino butyric acid) or INA (2, 6-dichloroisonicotinic acid) have already been shown to 

activate the plant’s defense system with no detectable antifungal activity in vitro or in planta. 

Thus, BTH is documented to induce systemic acquired resistance (SAR) in a number of hostparasite 

interactions (Bán et al. 2004; Sauerborn et al. 2002; Schweizer et al. 1999; Stadnik & 

Buchenauer 1999; Tosi et al. 1999). BABA has been reported to activate disease resistance in 

various crops when used at relatively high rates (Cohen et al. 1994; Pajot et al. 2001; Tosi et 

al. 1998). In addition, another SAR inducer, INA has also been shown to induce resistance to 

various fungal and bacterial diseases (Dann et al. 1998; Kogel et al. 1994). BTH appears to be 

able to restrict downy mildew symptoms in sunflower under greenhouse conditions (Bán et al. 

2004). Microscopic observations showed that BTH treatment significantly decreased the 

development of fungal structures associated with cell necrosis and H2O2 accumulation in the 

BTH treated susceptible sunflower hypocotyls. 

Glutation S-transferase (GST) has a well defined role in plant detoxification reactions. It is 

capable of catalyzing the binding of various xenobiotics, like pathogens. Various abiotic 

stressors are the inducers of GST activity in plants (Dean et al. 1990). GST is also considered 

one of the antioxidative enzymes, because it plays an important role in the protection against 

oxidative membrane damage and necrotic disease symptoms. Enhanced GST activity has been 

found in plants after pathogen infection, for example in barley plants infected by powdery 

mildew (El-Zahaby et al. 1995), and tobacco plants infected by TMV (Fodor et al. 1997). 

To protect themselves against pathogenic attack, plants evolve diverse strategies, for example 

the synthesis of antimicrobial peptides, like defensin. Defensin is a small, cysteine-rich 

antimicrobial peptide, existing in a wide range of plants and animals. Urdangarin et al. (2000) 

described full length sunflower cDNA from Helianthus annuus flowers encoding for defensin, 

and the authors supposed there was a relationship between enhanced expression of a defensin 

gene and decreased susceptibility to Sclerotinia sclerotiorum. Solis et al. (2006) isolated a 

defensin gene from Lepidium meyenii, having activity against Phytophthora infestans. 

Catalase is one of the main antioxidant enzymes; it catalyzes the dismutation of H2O2 into 

water and dioxygen. This enzyme is located in peroxisomes and glyoxisomes. Catalase activity 

is affected by abiotic stressors, like boron (Karabal et al. 2003), light and chilling (Gechev et 

al. 2003), and acid rain (Gabara et al. 2003). In sunflower, catalase activity was increased by 

UV-B radiation (Costa et al. 2002) and cadmium treatment (Azpilicueta et al. 2007). Niebel et 

al. (1995) demonstrated induction of catalase in potato upon nematode and bacterial infection 

as well. Several plants have multiple CAT isoenzymes. For example, in sunflower at least eight 

isoforms (CAT1-CAT8) have been described (Azpilicueta et al. 2007). 


The USDA sunflower inbred lines RHA 274, RHA 340 and HA 335, as well as Plasmopara 

halstedii pathotype 700 were used to get one compatible, and two incompatible combinations, 

respectively. While HA 335 is characterized by total resistance, RHA 340 exhibits HLI 

466

(hypocotyl-limited) resistant type (Virányi & Gulya 1996). 

Pre-germinated seeds were soaked in an aqueous solution of BTH for at least 6 hours (first 

day), followed by the inoculation with P. halstedii sporangia (50 000 sporangia/ml) using the 

whole seedling inoculation technique (Cohen & Sackston 1973). Germlings were subsequently 

planted into pots filled with a commercial soil mixture and grown in the greenhouse (18/24 ºC, 

60 % RH, 16h light) for 3 weeks. 

Samples were taken 3, 9, 13, 17 days after infection (dpi). The whole seedlings were frozen in 

liquid nitrogen and grounded with mortal and pestle. Total RNAs were extracted, and then the 

extracted RNA treated with RNase inhibitor to protect the extracted RNA and with DNase I to 

remove genomic DNA contamination. The extracted RNAs were measured with 

spectrophotometer and adjusted the RNA’s concentration of 1µg/µl. One µg of RNA was 

reverse transcribed using cDNA synthesis kit. 

Primers for PCR amplifications were applied according to Radwan et al. (2005) and 

Azpilicueta et al. (2007) as shown in Table 1. Twenty-five µl of the PCR reaction mixture 

contained 1µl cDNA, 1 unit of Taq DNA polymerase, 2,5 µl 10X Taq polymerase buffer, 1 µl 

2,5mM dNTP mix, 1,5 µl 25mM MgCl2, 2,5 µl 5 µM primers and 13,8 µl PCR water.The 

amplification program included an initial step at 94 °C for 3 min and 25-32 cycles of 15 sec at 

94 °C, 15 sec at Tm °C and 20sec at 72°C. 

The PCR products were electrophorized through 1% agarose gel, visualized with ethidium 

bromide and photographed in a molecular imager gel doc system. The signals from gels were 

quantified using a Quantity One program with molecular mass ruler, and normalized over the 

signals from Ha-EF1α. 

Table 1. Primer sequences and accession numbers used in this study 

Gene* Primer sequences Acession number 

Ha-EF-1α Forward 5’-AGGCGAGGTATGATGAAATTGTCA-3’ 

Reverse 5’-GTCTCTTGGGCTCATTGATTTGGT-3’ 

Ha-GST Forward 5’-CCTCAGGATGCTTACGAGAAGG-3’ 

Reverse 5’-GCAGAAATATCAACCAGGTTGATG-3’ 

Ha-PDF Forward 5’-ATGGCCAAAATTTCAGTTGCTTTCA-3’ 

Reverse 5’-AAGACTTGCACTGGTCATCACAG-3’ 

CATA2 Forward 5’-TTCCCGCTTGAATGTGAAG-3’ 

Reverse 5’-CCGATTACATAAACCCATCATC-3’ 

AAM19764 

AY667502 

AF364865 

AF243517 

*Ha-EF-1α: constitutive elongation factor 1α, Ha-GST: glutathione S-transferase, Ha-PDF: defensin, CATA2: 

catalase isoenzymes. 

467

a. 

b. 

468 

relative transcript accumulation 

relative transcript accumulation 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

Ha-GST 

3 9 13 17 

days after inoculation 

Ha-PDF 

3 9 13 17 


274 inoculated 

274 BTH+ inoculated 


335inoculated 




335inoculated

c. 

relative transcript accumulaion 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

CATA2 

3 9 13 17 





335inoculated 

Figure 1. Accumulation of gene transcripts in sunflower plants after BTH treatment and 

Plasmopara halstedii inoculation. a: glutation S-transferase (Ha-GST); b: 

defensin (Ha-PDF) and c: catalase (CATA2) gene expression in a susceptible 

(RHA 274), partially (HLI) resistant (RHA 340) and totally resistant (HA 335) 

sunflower line. Each value represents three replicates (±S.D.) 

RESULTS 

As part of our results Figure 1. shows the infected (one susceptible and two resistant), as well 

as the BTH treated susceptible plants transcript accumulation only. 

In general, Ha-GST transcript accumulation was higher in the untreated resistant sunflowers 

than in the susceptible ones. At 3 and 9 dpi the highest transcript accumulation was detected in 

the HA 335 plants. At 13 and 17 dpi, however, this accumulation was higher in the ‘HLI 

resistant’ RHA 340 plants as compared to HA 335. The BTH treatment increased Ha-GST 

transcript level in both the susceptible and totally resistant plants throughout the experiment. In 

case of ‘HLI resistant’ plants, the BTH-treatment decreased this gene activation. 

At 0 dpi we did not detect any Ha-PDF transcript accumulation. HA-PDF transcript 

accumulation was found to be higher in the resistant sunflower lines than in the susceptible 

one, similar to the HA-GST transcript accumulation. The positive effect of BTH treatment on 

PDF activity was detectable in both the susceptible and totally resistant sunflowers. There was 

no transcript accumulation in the untreated susceptible plants at 3 dpi. However, the BTHtreated 

susceptible plants showed similar transcript level, than the untreated ‘HLI resistant’ HA 

469

340 plants, and this enhanced transcript level remained throughout the experiment in the BTHtreated 

susceptible plants. The activator treatment enhanced the gene expression in the totally 

resistant plants, similarly to the susceptible ones. In case of ‘HLI resistant’ plants, the effect of 

BTH treatment was contradictory. 

As for catalase activity, both type of resistant sunflowers exhibited higher CATA2 transcript 

level, than did the susceptible one. In case of ‘HLI resistant’ plants, a continuous increase in 

transcript accumulation of CATA2 was found reaching its maximum at 17 dpi, and this 

sunflower genotype showed the highest transcript level. BTH-treatment considerably increased 

the level of CATA2 transcript in the susceptible sunflowers. In case of ‘HLI resistant’ plants, 

the BTH treatment decreased the catalase activity. There were no detected differences between 

BTH-treated and untreated totally resistant plants (Fig. 1). 

DISCUSSION 

In this study molecular changes of chemical activator-treated sunflowers were the subject of 

investigations associated with infection by P. halstedii. PCR was used in attempt to describe 

induced resistance events in different sunflower genotypes. 

Glutation S-transferase usually detoxifies xenobiotica in plant tissues. We found an increased 

level of GST activity in the activator treated, susceptible sunflower and this increased activity 

resembled that detected in the untreated ‘HLI resistant plants.’ Fodor et al. (1997) reported 

about similar results with tobacco either treated or non-treated with salicylic acid. In contrast, 

El-Zahaby et al. (1995) found a significantly higher level of GST activity in susceptible barley 

plants than in resistant ones after powdery mildew inoculation. They assumed that the fungus 

itself contained GST enzyme, so that both the host and the pathogen might contribute to this 

increases in GST activity. 

Defensins are a class of antimicrobial peptides found in several plants, including sunflower. In 

our experimental condition defensin gene expression was induced by BTH treatment in the 

susceptible sunflower plants, and this enhanced level resembled that was found in the untreated 

‘HLI-resistant’ plants. Similar to Radwan et al. (2005), Ha-PDF transcript accumulation was 

lower in the non-treated susceptible than in the resistant plants. 

Catalase is usually considered to be one of the most important antioxidant enzymes. In treated 

susceptible plants CATA2 transcript level increased, but this effect was not evident in the 

resistant sunflowers. 

In conclusion, the plant activator BTH had a positive effect on the natural defense system of 

sunflower by enhancing the expression of three genes that are considered to be associated with 

the chemically induced host resistance to P. halstedii. 

470

REFERENCES 

Albourie J M; Tuorvieille J; Labrouhe D T (1998). Resistance to metalaxyl in isolates of the 

sunflower pathogen Plasmopara halstedii. European Journal of Plant Pathology 104, 

235-242. 

Azpilicueta E C; Benavides P M; Tomaro L M; Gallego S M (2007). Mechanism of CATA3 

induction by cadmium in sunflower leaves. Plant Physiology and Biochemistry 45, 

589-595. 

Bán R; Virányi F; Körösi K; Nagy S (2004). Indukált rezisztencia a napraforgóperonoszpórával 

szemben. Növényvédelem 40, 545-550. 

Cohen Y; Niderman T; Mösinger E; Fluhr R (1994). β-aminobutyric acid induces the 

accumulation of pathogenesis-related proteins in tomato (Lycopersicon esculentum L.) 

plant and resistance to late blight infection caused by Phytophthora infestans. Plant 

Physiology 104, 59-66. 

Cohen Y; Sackston W E (1973). Factors affecting infections of sunflowers by Plasmopara 

halstedii. Canadian Journal of Botany 52, 15-22. 

Costa H; Gallego S M; Tomaro M L (2002). Effect of UV-B radiation on antioxidant defense 

system in sunflower cotyledons. Plant Science 162, 939-945. 

Dann E; Brian D; Byrum J; Hammerschmidt R (1998). Effect of treating soybean with 2,6dichloroisonicotinic 

acid (INA) and benzothiadiazole (BTH) on seed yields and the 

level of disease caused by Sclerotinia sclerotiorum in field and greenhouse studies. 

European Journal of Plant Pathology 104, 271-278. 

Dean J V; Gronwald J W; Eberlein C V (1990). Induction of glutation S-transferase 

isoenzymes in sorghum by herbicide antidotes. Plant Physiology 92, 467-473. 

El-Zahaby H M; Gullner G; Király Z (1995). Effects of powdery mildew infection of barley on 

the ascorbate- glutathione cycle and other antioxidants in different host-pathogen 

interaction. The American Phytopathological Society 85, 1225-1230. 

Fodor J; Gullner G; Ádám A L; Barna B; Kőmíves T; Király Z (1997). Local and systemic 

responses of antioxidants to tobacco mosaic virus infection and salicylic acid in 

tobacco. Plant Physiology 114, 1443-1451. 

Gabara B; Sklodowska M; Wyrwicka A; Glinska W; Gapinska M (2003). Changes in the 

ultrastructure of chloroplasts and mitochondria and antioxidant enzyme activity in 

Lycopersicon esculentum Mill. leaves sprayed with acid rain. Plant Science 164, 

507-516. 

Gechev T; Willekens H; Van Montagu M; Inzé D; Van Camp W; Toneva V; Minkov I (2003). 

Different responses of tobacco antioxidant enzymes to light and chilling stress. Journal 

of Plant Physiology 160, 509-515. 

Gulya T (2007). Distribution of Plasmopara halstedii races from sunflower around the world. 

In: Advances in Downy Mildew Research, Vol. 3, eds A Lebeda & P T N Spencer- 

Philips, pp. 121-134. Palacky University in Olomouc and JOLA, v.o.s.: Kostelec na 

Hané Czech Republic. 

Karabal E; Yücel M; Öktem H A (2003). Antioxidant responses of tolerant and sensitive barley 

cultivars to boron toxicity. Plant Science 164, 925-933. 

Kogel K-H; Beckhove U; Dreschers J; Münch S; Romme Y (1994). Acquired resistance in 

barley. Plant Physiology 106, 1296-1277. 

471

Niebel A; Heungens K; Barthels N; Inze D; Van Montagu M; Gheysen G (1995). 

Characterization of a pathogen-induced potato catalase and its systemic expression 

upon nematode and bacterial infection. Molecular Plant-Microbe Interactions 8, 

371-378. 

Pajot E; Le Corre D; Silué D (2001). Phytogard and DL-b-amino butyric acid (BABA) induces 

resistance to downy mildew (Bremia lactucae) in lettuce (Lactuca sativa L.). European 

Journal of Plant Pathology 107, 861-869. 

Radwan O; Mouzeyar S; Venisse J S; Nicolas P; Bouzidi M F (2005). Resistance of sunflower 

to the biotrophic oomycete Plasmopara halstedii is associated with a delayed 

hypersensitive response within the hypocotyls. Journal of Experimental Botany 56, 

1683-2693 

Sauerborn J; Buschmann H; Ghiasi K G; Kogel K-H (2002). Benzothiadiazole activates 

resistance in sunflower (Helianthus annuus) to the root-parasitic weed Orobanche 

cumana. Phytopathology 92, 59-64. 

Schweizer P; Schlagenhauf E; Schaffrath U; Dudler R (1999). Different patterns of host genes 

are induced in rice by Pseudomonas syringae, a biological inducer of resistance, and 

chemical inducer benzothiadiazole (BTH). European Journal of Plant Pathology 105, 

659-665. 

Solis J; Medrano G; Ghislain M (2007). Inhibitory effect of a defensin gene from the Andean 

crop maca (Lepidium meyenii ) against Phytophthora infestans. Journal of Plant 

Physiology 164, 1071-1082. 

Stadnik M J; Buchenauer H (1999). Control of wheat diseases by a benzothiadiazole-derivative 

and modern fungicides. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 106, 

466-475. 

Urdangarín M; Norero N S; Broekaert W F; de la Canal L (2000). A defensin gene expressed 

in sunflower inflorescence. Plant Physiology and Biochemistry 38, 253-258. 

Tosi L; Luigetti R; Zazzerini A (1998). Induced resistance against Plasmopara helianthi in 

sunflower plants by DL-beta-amino-n-butyric acid. Journal of Phytopathology 146, 

295-299. 

Virányi F; Gulya T (1996). Expression of resistance in Plasmopara halstedii- sunflower 

pathosystem. In: ISA Symposium I. Disease Tolerance in Sunflower, Bejing, 13 June 

1996, 14-21. 

472

Ruge-Wehling B, Thiele C, Eickmeyer F, Wehling P: Sources of Resistance and Development of Molecular 

Markers for Anthracnose Resistance in Narrow-Leafed Lupin (Lupinus angustifolius ). In: Feldmann F, Alford D 



10-4 Sources of Resistance and Development of Molecular Markers for 

Anthracnose Resistance in Narrow-Leafed Lupin (Lupinus 

angustifolius ) 

Ruge-Wehling B 1 , Thiele C 2 , Eickmeyer F 2 , Wehling P 1 

1 

Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding 

Research on Agricultural Crops, Quedlinburg/Groß Lüsewitz, Germany 

2 Saatzucht Steinach, Breeding Station of Bornhof, Bocksee, Germany 

Email: brigitte.ruge-wehling@jki.bund.de 

Abstract 

Different cultivars, breeding lines as well as genebank accessions of Lupinus 

angustifolius were screened for novel anthracnose resistances. A a reliable 

resistance test was used under controlled greehouse conditions. While all the 

German cultivars tested proved to be susceptible, two breeding lines and one 

genebank accession were identified which displayed strong resistance to 

Colletotrichum lupini. One of the breeding lines was subsequently tested in the field 

and strong resistance could be confirmed. F2 populations for the mapping of the 

potentially novel resistances have been developed. For mapping purposes, a set of 

gene-based markers derived from the genetic map of Lupinus angustifolius was 

used. Additionally, novel markers were established by using the sequence 

information from the model genomes of Medicago truncatula, Lotus japonicus and 

Pisum sativum and will used as potential resources for mapping the novel resistance 

genes. 

INTRODUCTION 

At present, the cultivation of lupins in Europe is negligibly small. In Germany where L. 

angustifolius has remained as the only lupin species grown to some extent, the acreage was less 

than 20,000 ha in 2008. As a home-grown legume rich in high-quality seed protein, though, 

narrow-leafed lupin currently is attracting increasing interest both as a feed crop and as a 

potential substitution for animal or soy protein in food products. 

One challenge in lupin breeding is the improvement of resistance to anthracnose , which is one 

of the most important lupin diseases worldwide. The disease, caused by Colletotrichum lupini, 

473

is present in most parts of the world where lupins are cultivated (Yang et al. 2004). To-date, 

the high-yielding resistant cv. Mandelup as well as the highly resistant cv. Tanjil have been 

used for breeding strategies in Australia to improve anthracnose resistance (Yang et al. 2004; 

Yang et al. 2008). Resistance of cv. Tanjil is inherited by a dominant gene which was 

designated Lanr1 (Yang et al. 2004) and which can be tracked in breeding programmes by use 

of a closley linked co-dominant molecular marker (You et al. 2005). We have started efforts to 

screen genetic resources of narrow-leafed lupin for novel resistances which are effective under 

the growing conditions met in Germany's agriculture, and to genetically analyse these 

resistances including the mapping relative to molecular markers. 


Plant material 

Screening for novel resistances 

A total of 13 cultivars (Arabella, Bolivio, Bora, Bordako, Boregine, Boruta, Borweta, Haagena, 

Haags Blaue, Mandelup, Polonez, Tanjil, Vitabor), 15 breeding lines as well as 26 genebank 

accessions were avaiable for resistance screening in the greenhouse. The breeding lines 

Bo7212 and Bo3533, the German cv. Arabella and the Australian cv. Tanjil were included in a 

field test for anthracnose infestation in 2007. 

Mapping populations 

Breeding lines Bo7212, Metel1 and the genebank accession JKI-1 were used as pollen parents 

in crosses with cvs. Arabella, Haags Blaue and Haagena. To increase marker polymorphism the 

two resitant breeding lines were also crossed with a susceptible genebank accession. One 

seed/pod was checked for their hybridity by using molecular markers (Fig. 2). 

Anthracnose strains 

Plants were inoculated in the greenhouse with strain BBA70358 of C. lupini var. setosum. 

Plants were inoculated in the field with a mixture of five different strains of C. lupini var. 

setosum (BBA70400, BBA70397, BBA70358, BBA70385, BBA71238). The strains belong 

the classification VG2 which is also used by Yang et al. (2004). The fungi were kindly 

provided by H. I. Nirenberg at the former Federal Biological Research Centre for Agriculture 

and Forestry. 

Methods 

Resistance testing 

Greenhouse resistance tests were performed according to Yang et al. (2004). Plants were 

inoculated by spraying with a conidial suspension (10 5 conidia per ml). The inoculated plants 

474

were incubated in the dark for 16 h. Disease was recorded 10-14 days after inoculation in a 

climate chamber. Plants with superficial scars were assessed as beeing resistant. Plants 

displaying collapsed spikes and/or lesions bearing pink conidial masses were regarded as 

susceptible. 

For field testing a randomized block design with two replications was used. Field testing was 

done in 2007 at the two locations of Bocksee and Groß Lüsewitz. For inoculation under field 

conditions, 5 infection rows per block were used. Each infection row comprised 15 seeds of cv. 

Arabella contaminated with conidia and sown when the test plants were at the 2-5 leaf stage. 

The seeds for infection rows had been prepared by immersing in a conidial suspension of 10 5 

conidia per ml for 4 h and subsequent drying overnight. Scoring was performed three times, 

i.e., at the 6-8-leaf stage, at flowering time and at the early-pod stage. 


Sequence information of PCR markers from Lupinus angustifolius was kindly provided by M. 

Nelson, Univ. of Western Australia, Perth. Medicago truncatula primers were used as 

recommended in the mtgenome database (http://mtgenome.ucdavis.edu/index.html). By using 

the NCBI database (http://www.ncbi.nlm.nih.gov/sites/) EST sequences from Lotus japonicus 

were transferred to the SSRIT software application (http://www.gramene.org/gramene/searches 

/ssrtool) for searching SSR motives. Primers for ESTs from Lotus japonicus were designed 

using the software package Prime3 (Rozen & Skaletsky 2000). Pisum sativum primers were 

drawn from the website http://bioweb.abc.hu/cgi-mt/pisprim/pisprim.pl. 

For PCR with the various primer pairs, 50-100 ng of genomic DNA was used in a solution 

containing 1x reaction buffer (Qiagen), 200 µM dNTPs, 5 pmol primers and 0.5 U of Taq DNA 

polymerase (Qiagen). CAPS markers were developed by cutting the PCR products with either 

Taq α I or BstNI. PCR products were separated in 2.5% agarose gels followed by ethidium 

bromide staining or in 10% PAGE followed by silver staining (Budowle et al. 1991). 


Search for potential resistance sources 

In the resistance test under greenhouse conditions, each of the 12-15 plants tested from 11 

European cultivars proved to be susceptible to anthracnose. In contrast, the Australian cvs. 

Tanjil and Mandelup were highly resistant. Of the 15 breeding lines tested, two breeding lines 

(Bo7212, Metel1) were found to be resistant while 12 entries were classified as susceptible. 

Genebank accession 070014 displayed also high resistance under controlled conditions. 

Breeding line Bo3533 displayed an intermediate reaction, with lesions considerably smaller 

than those observed with the susceptible entries. 

Resistance testing under field conditions gave slightly higher infestation rates with the Groß 

Lüsewitz (G.L.) location as compared with the location of Bocksee (Fig. 1). This might be due 

to the more humid conditions at the near-coastal location of G.L. in 2007. Despite these small 

475

differences the reaction patterns of entries were identical at both locations (Fig. 1). Breeding 

line Bo7212 was significantly less affected as compared with the remaining entries. Hence, the 

476 

Figure 1. Infestation of Lupin cultivars 

Figure 2. Seeds displayed both parental marker alleles 

strong resistance of Bo7212 observed under controlled conditions in the greenhouse could be 

confirmed under variable field conditions. While cv. Arabella as well as breeding line Bo3533 

became highly diseased, the cv. Tanjil displayed a somewhat intermediate reaction with a 

significantly lower percentage of diseased plants than Arabella and Bo3533 but significantly 

higher infestation as compared with Bo7212. Notably, cv. Tanjil displayed considerable 

infestation of the pods (Fig. 1). The difference between the earliest and latest flowering date 

among the four entries was 3-5 days, with cv. Arabella starting first and cv. Tanjil flowering 

somewhere in the center-field of the time window. Whether this small difference in flowering

dates might have caused the significantly differing reactions of cv. Tanjil vs. Bo7212 as shown 

in Fig. 1 remains to be clarified in further field trials. Alternatively, the quite dissimilar 

reactions of Bo7212 and cv. Tanjil might be due to the presence of different resistance genes. 

Further elaboration via molecular-marker analysis will be needed to draw final conclusions on 

this question. 

Mapping populations 

For genetic and molecular analysis crosses between susceptible cultivars and the novel 

resistance resources (Bo7212, Metel1, Genebank accession 070014) have been performed. F1 

plants were checked for hybridity using molecular markers. One seed from each pod was 

checked with 6 polymorphic markers. With the exception of one pod (Fig. 2, S) which turned 

out to be selfed offspring of the seed parent of cv. Arabella, all the other pods contained seeds 

displaying both parental marker alleles, i.e., one from either cv. Arabella, Haagena or Haags 

Blaue as the female parent and the other one coming from Bo7212 or Metel1, respectively, as 

the male parent (Fig. 2). F2 mapping populations with more than 100 individuals were obtained 

after selfing the F1 hybrids. A first screening of F2 individuals of mapping population JKI- 

1013 (Arabella x Bo7212) revealed a differentiation between completely susceptible plants and 

those with nearly any symptoms (Fig. 3). Segregation patterns indicate a dominant mode of 

inheritance. The genetic analysis of additional individuals hopefully will give evidence for the 

identification of a novel dominant resistance gene in L. angustifolius. 

Figure 3. Differentiation between completely susceptible plants and those with nearly 

any symptoms 

477


For mapping the novel resistance four different marker resources are avaiable: 

(i) 62 STS and CAPS markers derived from the genetic map of Lupinus angustifolius (Nelson 

et al. 2006), 

(ii) approximately 30 SSR markers from Medicago truncatula (mtgenome database), 

(iii) approximately 60 SSR markers derived from Lotus japonicus EST sequences (NCBI 

database) that carry SRR motives, 

(iiii) about 50 STS markers of Pisum sativum database. 

In general, markers drawn from these resources are able to distinguish between the resistant 

and suceptible breeding lines and the cultivars (Fig. 4). Currently eight resistant vs. susceptible 

genotypes of the mapping population JKI-1013 are screened for marker polymorphism. 

478 

Figure 4. Markers drawn from these resources are able to distinguish between the 

resistant and suceptible breeding lines and the cultivars 


This work is supported by the Innovation Programme of the Federal Ministry of Food, 

Agriculture and Consumer Protection (BMELV).

REFERENCES 

Boersma J G; Pallotta M; Chengdao M L I; Buirchell B J; Sivasithamparam K; Yang H (2005). 

Construction of a genetic linkage map using MFLP and identification of moleular 

markers linked to domestication genes in narrow-leafed lupins. Cellular & Molecular 

Biology Letters 10: 331-344. 

Budowle B; Chakraborty R; Giusti A M; Eisenberg A J; Allen R C (1991). Analysis of VNTR 

locus D1S80 by the PCR followed by high resolution PAGE. American Journal of 

Human Genetics 48, 137-144. 

Nelson M N; Phan H T T; Ellwood S R; Moolhuijzen P M; Hane J; Williams A; O'Lone C E; 

Fosu-Nyarko J; Scobie M; Cakir M; Jones M G K; Bellgard M; Ksiżkiewicz M; Wolko 

B; Barker S J; Oliver R P; Cowling W A (2006). The first gene-based map of Lupinus 

angustifolius L. – location of domestication genes and conserved synteny. Theoretical 

and Applied Genetics 113, 225-238. 

Rozen S; Skaletsky H J (2000). Primer3 on the WWW for general users and for biologist 

programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular 

Biology, eds S Krawetz & S Misener, pp 365-386. Humana Press: Totowa, NJ. 

Yang H, Boersma J G; You M; Buirchell B J; Sweetingham M W (2004). Development and 

implementation of a sequence-specific PCR marker linked to a gene conferring 

resistance to anthracnose disease in narrow-leafed lupin (Lupinus angustifolius L.). 

Molecular Breeding 14, 145-151. 

Yang H D; Renshaw D; Thomas G; Buirchell B, Sweetingham M (2008). A strategy to develop 

molecular markers applicable to a wide range of crosses for marker assisted selection in 

plant breeding: a case study on anthracnose disease resistance in lupin (Lupinus 

angustifolius L.). Molecular Breeding 21, 473-483. 

You M; Boersma G J; Buirchell B J; Sweetingham MW; Siddique K H M; Hang H (2005). A 

PCR-based molecular marker applicable for marker-assisted selection for anthracnose 

disease resistance in lupin breeding. Cellular & Molecular Biology Letters 10, 123-134. 

479

Thielert W, Boer B d: Combining Stress Shield Chemistry and Parp-Silencing to Improve Productivity in OSR. 

In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 480-488; ISBN 


10-5 Combining Stress Shield Chemistry and Parp-Silencing to Improve 

Productivity in OSR 

Thielert W 1 , Boer B d 2 

Bayer CropScience AG, Monheim, Germany 

Bayer BioScience N.V., Gent, Belgium. 

480 

ABSTRACT 

Long term evaluation of field trials data indicated that seed, soil or foliar applied 

Imidacloprid – a broad spectrum neonicotinoid insecticide for controlling major 

sucking/piercing and chewing insect pests - resulted in positive growth responses 

and higher yields in a wide range of annual and perennial crops, even in the absence 

of relevant pest species. Gene expression profiling of stressed plants treated with 

Imidacloprid confirmed an additional mode of action explaining the abiotic and 

biotic stress mitigation termed ‘Stress Shield’. Besides the mitigation of abiotic 

stress through chemistry, future varieties will contain stress tolerance traits. One 

promising way is to maintain energy homeostasis under stress conditions by 

silencing the activity of PARP – Poly (ADP-ribose) polymerase. Field trial results 

from Canada are presented showing genetically modified oil seed rape - containing 

hairpin (dsRNA) PARP1 and PARP2 silencing constructs, azygous control lines 

and checks (conventional lines and commercial hybrid) – in combination with an 

Imidacloprid Stress Shield seed treatment, resulting in a significant canopy height 

increase and yield enhancement compared to treatments without Imidacloprid under 

pest free conditions. 

INTRODUCTION 

Crop growth and productivity as well as the product quality are greatly influenced by 

environmental stresses. A major proportion of yield losses in crop plants is due to so-called 

abiotic stress factors caused by e.g. drought, hypoxia, heat, cold, excessive light, high salt 

concentration in soil or ozone in air. Additional losses from pathogens and herbivores attack 

are attributed to biotic stresses (Bray et al. 2000). Long term evaluation of field trials data

indicated that seed, soil or foliar applied Gaucho ® , Admire ® and Confidor ® – containing the 

broad spectrum neonicotinoid insecticide Imidacloprid for controlling major sucking/piercing 

and chewing insect pests - resulted in positive growth responses and higher yields in a wide 

range of annual and perennial crops, even in the absence of relevant pest species (Thielert 

2006; Zelinski et al. 2007; Zelinski & Thielert 2008; Gonias et al. 2008). Analyses of the 

growing conditions given pointed to environmental stress factors being involved. Gene 

expression profiling of stressed plants treated with Imidacloprid showed a reduced expression 

of dehydrins (drought stress marker genes) and an overexpression of specific PR-proteins 

(pathogenesis related proteins) indicating an additional ‘Stress Shield’ mode of action 

contributing to abiotic and biotic stress mitigation in crops (Thielert 2006). Beyond chemical 

options to manage plant stress, future varieties will contain improved stress tolerance traits 

transferred through genetic modification. One promising way is to maintain energy 

homeostasis under stress conditions by silencing the activity of PARP – Poly (ADP-ribose) 

polymerase a key cell repair enzyme system (De Block et al. 2005). 

In plants, PARP genes are structurally and functionally homologous to their mammalian 

counterparts (Babiychuk et al. 1998). PARP1 and PARP2 are activated by DNA damage 

caused for example by reactive oxygen species. Upon activation, polymers of ADP-ribose are 

synthesized on a range of nuclear enzymes using NAD + as substrate. In plants abiotic stress 

factors activate PARP causing NAD + breakdown and ATP consumption. When the PARP 

activity is reduced by gene silencing, cell death is inhibited and plants become tolerant to a 

broad range of abiotic stresses. Plant lines with low poly(ADP-ribosyl)ation activity maintain 

under stress conditions their energy homeostasis by reducing NAD + breakdown and 

consequently energy consumption. The higher energy-use efficiency avoids the need for a too 

intense mitochondrial respiration and consequently reduces the formation of reactive oxygen 

species. 

A field trial was conducted in Saskatchewan, Canada during 2007 to evaluate growth and seed 

yield of genetically modified oilseed rape - containing hairpin (dsRNA) PARP silencing 

constructs – in combination with Imidacloprid seed treatment to confirm synergistic growth 

effects observed under waterlogging conditions in greenhouse trials (unpublished internal 

report). 


Entries: 

Transgenic homozygous down-regulated hpPARP1 line (P1), transgenic heterozygous downregulated 

hpPARP1 line (P1 x Simon) and transgenic heterozygous hpPARP1 + hpPARP2 line 

(P1 x P2) were compared to their azygous control equivalents and checks. 

The azygous control for P1 x Simon was obtained by crossing the azygous control of P1 with 

Simon and the azygous control of P1 x P2 was obtained by crossing the azygous control of P1 

x the azygous control of P2. Checks included: N90-740, Simon (doubled haploid derived from 

481

N90-740) and a hybrid, InVigor 5020. All entries were grown under unspecified, natural 

abiotic stress conditions, i.e. cold temperatures after sowing and seed emergence in May and 

drought and heat conditions during the flowering period June/July 2007. 

Treatments: 

Half of the plots received a seed treatment with Antarc FS 500 ® at a rate of 25 ml/kg seed 

equivalent to 10,5 g Imidacloprid + 2 g Beta-Cyfluthrin/kg seed. At planting, all plots received 

additionally an in furrow application of corn cob grits at a rate of 8 kg/ha (not directly on the 

seed) treated with Prosper FS 300 ® (326 ml/ha), a systemic insecticide and fungicide to ensure 

that a potential growth and yield effect were not due to potential insect pressure but to the 

Imidacloprid treatment. Prosper ® is a combination the insecticide Clothianidin 120 g/l and 

fungicides Carbathiin 56 g/l, thiram 120 g/l and metalaxyl 4 g/l for the control of flea beetles, 

seed rot, damping off, seedling blight and early season root rot caused by Pythium, 

Rhizoctonia, Fusarium, Alternaria spp. and seedborne Phoma. A post emergence insecticide 

Sevin XLR ® (Carbaryl) for flea beetle control was applied once prior to flowering BBCH 13- 

14 at a rate of 494 ml/ha. 

Location: 

Vanscoy, Saskatchewan, Canada; sandy soil. 

Trial Design: 

Split-plot, 3 replicates, plot size: 1.5 m x 5 m (7.5 m²) 

Evaluation: 

Height in cm and seed yield per plot in g. 

RESULTS 

Plots treated with Imidacloprid displayed a greater crop canopy height compared to plots 

treated without Imidacloprid (Tab. 1). All canopies of transgenic lines treated with 

Imidacloprid consistently responded with a significant height increase of 10 cm compared to 

equivalent azygous Imidacloprid-treated control lines (Tab. 2). Over all transgenic entries 

homozygous hpPARP1 + Imidacloprid showed the highest absolute canopy height 

development followed by heterozygous hpPARP1 +_ heterozygous hpPARP2 + Imidacloprid 

with the highest relative height increase compared to its equivalent azygous control line 

without Imidacloprid treatment (+17.2%). However, increased canopy height does not 

necessarily coincide with higher yields as indicated by the hybrid line InVigor 5020 which 

482

achieved the highest yield (Tab. 3) with a canopy height of 13.3 cm below homozygous 

hpPARP1 + Imidacloprid (Tab. 2). 

The average yield increase of all plots with Imidacloprid treatments reached 13.9% (Tab. 1). 

Table 1. Plot analysis comparing the effect of seed treatment Imidacloprid on height 

and yield over all entries 

TREATMENT Canopy 

Height in 

cm 

% Canopy Height 

Difference vs. 

‘Without 

Imidacloprid’ 

Mean Seed 

Yield g/plot 

% Yield Difference 

vs. ‘Without 

Imidacloprid’ 

Seed Treated with 

Imidacloprid 118,3 103,9 1563,9 113,9 

Seed Treated without 

Imidacloprid 113,8 100 1373,3 100 

Coefficient of 

Variation 3,6% 5,2% 

LSD 4,2 73,6 5,4 

Table 2. Plot analysis comparing the effect of seed treatment Imidacloprid on plant 

height of individual entries 

Canopy Height in cm 

PEDIGREE Imidacloprid without Imidacloprid 

P1-hom 130,0 123,3 

azygous control 120,0 116,7 

P1-het 121,7 118,3 

azygous control 1 111,7 116,7 

P1-het + P2-het 125,0 118,3 

azygous control 2 115,0 106,7 

SIMON 121,7 111,7 

N90-740 118,3 115,0 

InVigor 5020 116,7 111,7 

Coefficient of Variation 3,6% 

LSD 5,9 

P1-hom= homozygous hpPARP1 

P1-het= heterozygous hpPARP1 (P1-hom x Simon) 

P1-het + P2-het = heterozygous hpPARP1 + heterozygous hpPARP2 (P1-hom x P2-hom) 

1: azygous control P1 x Simon 

2: azygous control P1 x azygous control P2 

483

484 

Table 3. Plot analysis comparing the effect of seed treatment Imidacloprid on seed yield 

of individual entries 

PEDIGREE With 

Imidacloprid 

Seed Yield in g/plot 

Without 

Imidacloprid 

Yield Difference vs. 

‘Without 

Imidacloprid’ in % 

LSD % 

P1-hom 1549,6 1488,1 104,1 7,2 

azygous control 1132,9 958,9 118,2 11,2 

P1-het 1683,8 1457,2 115,5 7,4 

azygous control 1 

1492,6 1338,9 111,5 8,0 

P1-het + P2-het 1654,7 1367,1 121,0 7,9 

azygous control 2 1435,3 1260,2 113,9 8,6 

SIMON 1483,8 1335,9 111,1 8,1 

N90-740 1351,9 1136,3 119,0 9,5 

InVigor 5020 2192,5 1854,4 118,2 5,8 

Coefficient of Variation 5,2% 

LSD 107,7 

P1-hom= homozygous hpPARP1 

P1-het= heterozygous hpPARP1 (P1-hom x Simon) 

P1-het + P2-het = heterozygous hpPARP1 + heterozygous hpPARP2 (P1-hom x P2-hom) 

1: azygous control P1 x Simon 

2: azygous control P1 x azygous control P2 

seed seed yield yield 

(g/plot) 

2500 

2000 

1500 

1000 

500 

0 

9% 19% 17% 

ns 

ns * 

hpPARP1 hpPARP1 

hpPARP1 hpPARP1 x x Simon Simon 

hpPARP1 hpPARP1 x x hpPARP2 hpPARP2 

Simon Simon 

N90-740 N90-740 

Invigor Invigor 5020 5020 

entry 

55% 

*** 

transgenic 

azygous control 

checks 

*

seed yield 

(g/plot) 

2000 

1500 

1000 

500 

0 

20% 18% 11% 

* 

ns ns 

hpPARP1 hpPARP1 

hpPARP1 hpPARP1 x x Simon Simon 

hpPARP1 hpPARP1 x x hpPARP2 hpPARP2 

Simon Simon 

N90-740 N90-740 

Invigor Invigor 5020 5020 

entry 

50% 

** 

transgenic 

azygous control 

checks 

*

Overall, Imidacloprid plots out-yielded plots without Imidacloprid except for homozygous 

hpPARP1 and N90-740 where no statistical difference was noted at P

for the production of the energy rich molecules ATP in the respiration chain , however, in 

stressed plants NAD + is also consumed with priority as a substrate by various cell repair 

enzymes like for instance PARP (poly-ADP-ribose-polymerase). The cell repair enzymes 

rapidly deplete the NAD + pool under stress and form nicotinamide. Similar to bacteria and 

yeast, plants convert nicotinamide back into NAD + in a four-step salvage pathway (Wang & 

Pichersky 2007). Interestingly, between step 1 and 2 nicotinic acid is formed and this is 

possibly the entrance for 6-chloro-nicotinic acid delivering additional building blocks for an 

enhanced recycling of nicotinamide to NAD + . As a result, more NAD + is presumably available 

for ATP production enabling enhanced plant growth under stress! 


Many thanks to Tim Darragh, Bayer BioScience Inc. Canada, for conducting and evaluating 

the field trial at Vanscoy, Canada. 

REFERENCES 

Babiychuk E; Cottrill P B; Storozhenko S; Fuangthong M; Chen Y; O’Farrell M K; Van 

Montagu M; Inzé D; Kushnir S (1998). Higher plants possess 2 structurally different 

poly (ADP-ribose) polymerases. The Plant Journal 15 (5), 636-645. 

Bray E A; Bailey-Serres J; Weretilnyk E (2000). Responses to abiotic stress. In: Biochemistry 

and Molecular Biology of Plants, eds B Buchanan; W Gruissem; R L Jones, pp. 

1159-1160. American Society of Plant Physiologists. 

Brown R S; Oosterhuis D M; Gonias E D (2004). Efficacy of foliar applications of Trimax 

insecticide during water-deficit stress on the physiology and yield of cotton. 

Proceedings Beltwide Cotton Conferences, San Antonio, TX. 

Colby S R (1967). Calculating synergistic and antagonistic responses of herbicide 

combinations. Weeds 15, 20-22. 

De Block M; Verduyn C; De Brouwer D; Cornelissen M (2005). Poly(ADP-ribose) polymerase 

in plants affects energy homeostasis, cell death and stress tolerance. The Plant Journal 

41, 95-16. 

Gonias E D; Oosterhuis D M; Brown R S (2004). Effect of Trimax insecticide on the 

physiology, growth and yield of cotton. Proceedings Beltwide Cotton Conferences, San 

Antonio, TX. 

Gonias E D; Oosterhuis D M; Androniki C B (2008): Physiologic response of cotton to the 

insecticide Imidacloprid under high-temperature stress. Journal of Plant Growth 

Regulation 27 (1), 77-82. 

Hundley L; Cothren J T (2004). Cotton response to Trimax insecticide. Proceedings Beltwide 

Cotton Conferences, San Antonio, TX. 

Oosterhuis D M; Brown R S (2003). Effects of Trimax on the physiology, growth and yield of 

cotton. Proceedings Beltwide Cotton Conferences, Nashville, TN. 

Palrang D; Cagle J (2003). Plant growth and yield response to Trimax insecticide in Texas and 

Oklahoma. Proceedings Beltwide Cotton Conferences, Nashville, TN. 

487

Thielert W (2006). A unique product: the story of the imidacloprid stress shield. 

Pflanzenschutz-Nachrichten Bayer 59, 73-86. 

Wall L; Cothren J T; Witten T K (2003). Physiological assessment on foliar Trimax treatments 

on cotton. Proceedings Beltwide Cotton Conferences, Nashville, TN. 

Wang G; Pichersky E (2007). Nicotinamidase participates in the salvage pathway of NAD 

biosynthesis in Arabidopsis. The Plant Journal 49, 1020-1029. 

Young H S; Holder J (2003). Plant growth and yield response to Trimax insecticide in Georgia. 

Proceedings Beltwide Cotton Conferences, Nashville, TN. 

Zelinski L; Thielert W; Rogers D (2007). The use of remote sensing to identify Provado 

yield response at differing cotton stress levels. Proceedings Beltwide Cotton 

Conferences, Nashville, TN. 

Zelinski L; Thielert W (2008). Response of cotton at different stress levels to Trimax Pro. 

Proceedings Beltwide Cotton Conferences, Nashville, TN. 

488

Bothe H: Arbuscular Mycorrhizal Fungi and Their Roles in Relieving Abiotic Stress. In: Feldmann F, Alford D V, 



11-1 Arbuscular Mycorrhizal Fungi and Their Roles in Relieving Abiotic 

Stress 

Bothe H 

Botanical Institute, The University of Cologne, Gyrhofstr.15, D-50923 Köln, Germany 

GENERAL PROPERTIES OF ARBUSCULAR MYCORRHIZAL FUNGI (AMF) 

More than 80% of all higher plants form a symbiosis with AMF (Smith & Read 2007). The 

fine hyphae of these AM fungi better exploit minerals and water from the soil particles than 

plant roots. An efficient transfer from soils to plants has been demonstrated for phosphorus, 

nitrogen, Fe, Zn, Mo and others. In addition, the fungi make the plants more tolerant to heavy 

metals, salinity and acidity of soils, and to attack by pathogenic microorganisms, possibly even 

by exerting systemic effects on the plants. In greenhouse experiments, plants are not AMFcolonized 

when fertilization is optimized. Plants then save the expenditure of some 20% of 

their fixed carbon which they otherwise must deliver to feed their fungal symbiotic partner. In 

Nature, mycorrhizal plants always seem to be colonized. Observations in the Braunschweig- 

Magdeburger Börde (Chernozem soil) showed a strong colonization of the plants investigated 

indicating that non-identified factor(s) might be growth-limiting even in the best soil-types. 

The life cycle of an AM fungus is simple (Fig. 1). A spore in a soil germinates and its hypha 

forms an appressorium when reaching the surface (rhizodermis) of the plant roots. The hypha 

then squeezes into the interior of the roots by dissolving the walls of two neighbouring plant 

cells. The fungus reaches the inner cortical cells by forming intraradical hyphae. These can 

differentiate into tree-like structures, the arbuscules. They are entrapped by the plant 

cytoplasmic membrane (plasmalemma). The plant cells also undergo drastic changes. Several 

small vacuoles are formed instead of one large central one and the cytoplasm is rich in 

mitochondria. Experiments with isotopes and antibodies showed that the two membranes 

(periarbuscular membrane of the plant cells and cytoplasmic membrane of the fungus) are the 

sites of extensive metabolite exchange between both symbiotic partners. The fungi receive 

their carbon as glucose. Arbuscules have a life time of some two weeks before being degraded. 

Most AMF also form other structures within the plant roots cortex, the vesicles. These are 

compartments for the storage of lipids which are often discernible as droplets. However, not all 

AMF form vesicles, for example not the genus Gigaspora. Therefore the former term vesicular 

arbuscular mycorrhiza was replaced by arbuscular mycorrhiza in more recent years. 

489

490 

Figure 1 The life cycle of an arbuscular mycorrhizal fungus. For explanations see text. 

BAS = branched absorbing structures. 

Outside of the roots AMF form extraradical hyphae which can extensively ramify to branched 

absorbing structures (BAS) that likely are most active in acquiring water and minerals. Spores 

are formed both outside and within the roots. Sexual states are not known with AMF. All AMF 

cells are large and can contain more that 1000 nuclei. The nuclei differ (slightly) in their gene 

content. The extent of these DNA variations is much debated among experts. tRFL- banding 

pattern show that the variations are small with different DNA isolations from one AMF 

species (e.g. Wilde et al. 2009). Fungal species can be identified by DNA sequencing or by 

electrophoretic methods and also can be kept stable in culture collections over years. 

The AMF symbiosis evolved with the formation of the first land plants and is thus very old 

(Redecker et al. 2000). As the Rhizobium-legume symbiosis the AMF-plant partnership might 

have developed from pathogenic interactions. AMF form a separate phylum, the 

Glomeromycota, which are unrelated to other fungi (Schüßler et al. 2001). This might be the 

reason why the molecular identification of fungal genes by heterologous probing or by PCR– 

based techniques was so difficult in the past. An AMF has not totally been sequenced as yet. 

The fungi cannot be grown independently of a symbiotic partner. However, plants can be 

substituted by bacteria of the genus Paenibacillus (Hildebrandt et al. 2006). When grown in the

presence of these bacteria the AMF Glomus intraradices develops until the formation of new, 

fertile spores. 

Most plant species, even ferns and mosses, form a symbiosis with AMF. Members of the 

families Brassicaceae, Caryophyllaceae, Cyperaceae and among the Fabaceae the genus 

Lupinus are not or at best poorly AMF-colonized. The reason(s) for this inability is unclear as 

yet. Experiments with the Brassicaceae Biscutella laevagita (Orłowska et al. 2002) and Thlaspi 

sp. (Regvar et al. 2003) indicated that the roots are poorly colonized (less than 3% of all roots 

show a mycorrhizal structure). However, all fungal structures, intraradical hyphae, arbuscules 

or vesicles are discernible particularly at the flowering state. Thus the total program for 

establishing an AMF symbiosis can be detected in the roots of these plants, but the structures 

are insufficiently formed. It should, however, be stated that there is no apparent correction 

between the intensity of fungal structures within roots and the effectiveness of the symbiosis. A 

plant where almost all roots are AMF colonized must not be particularly active in metabolizing 

nutrients from soils. 

AMF could have enormous potential applications. This may not be so much in farming, since 

the added fungi are often out–competed by inborn ones that are better adapted to the soil and 

climate conditions. However, the growth of ornamental plants in greenhouses can be enhanced 

by adding AMF and also the risk of the attack by pathogenic fungi may be diminished (Fig. 2) 

Small companies have come into existence that sell AMF inocula for supporting growth of 

plants in houses. The main problem is to produce fungal inocula that show sustainable effects 

in application. Since the fungi cannot be propagated without a symbiotic partner as yet, inocula 

are produced in co-culture with plants such as broad bean (Vicia faba) with all the risk of 

potential contaminations. 

SOME GENERAL FEATURE OF HEAVY METAL TOXICITY AND TOLERANCE 

Many sites exist worldwide that are contaminated by heavy metals such as Zn, Pb, Ni, Fe, Cu, 

Cd and others. They carry a rather specific flora with particularly adapted plants, the heavy 

metal plants or metalophytes. Heavy metal soils differ in their plant coverage depending on the 

prevailing heavy metal. Metalophytes in turn belong to different, mainly totally unrelated plant 

families. These observations already indicate that not one or few mechanisms have been 

developed that enable metalophytes to thrive on heavy metal soils. Instead, each heavy metal 

plant has its own strategies to cope with the adverse affects of heavy metals. All heavy metal 

plants of Central Europe grow better in non-polluted garden soil. The poor competitiveness of 

heavy metal plants on non- polluted soils has forced heavy metal plants to find an ecological 

niche on heavy metal polluted soils. 

Heavy metals react in cell metabolism by blocking essential, functional SH-groups of enzymes. 

In addition, they may enhance the production of ROS (reactive oxygen species) such as O2 .- , 

H2O2, OH . , 1 O2 generated in the Fenton or Haber-Weiss reaction (Bothe et al. 2009). Plants 

may be respond to these toxicities by a) excreting siderophores to the soil that bind heavy 

metals there, b) forming metallothioneins and/or phytochelatins in the cells that bind the heavy 

491

492 

Figure 2. Arbuscular mycorrhizal fungi support plant growth and suppress the actions of 

pathogenic fungi as shown here for the AMF Glomus versiforme. It enhances 

growth of cyclamens (upper row in the figure) and partly relieves the effect of 

the pathogenic Fusarium oxysporum. Experiment of Dr. H. Baltruschat, D- 

Gießen, who kindly provided the photo. 

metals at their abundant SH-groups, c) by using heavy metal transporters that catalyze the 

excretion of the heavy metals out of the cells across the plasmalemma membrane. Such 

transporters comprise a huge class of proteins including CPx-ATPases for the transport of Cu 

or Cd, ABC-transporters for Cd-transport into the vacuole, ZIP-transporters (ZRT-, IRT-related 

proteins) for Fe or Zn, Nramp transporters for a broad range of heavy metals and others. The 

study of these transporters is complicated by the fact that every transporter is generally 

multigenic. Thus several isoforms for any transporter exist which may reside in different 

tissues of the plants. 

Several of the heavy metals such as Zn, Fe, Mo or Mn are indispensable for the growth of 

plants in lower concentration. Elements like Ni, V or Co are needed for few enzymes or few 

plants. Others like Hg and Cd are always toxic to plants. All heavy metals become toxic to 

plants at a certain threshold value which is different for each heavy metal and plant species or 

even individual. Plants may even vary in their response to heavy metals depending on different 

growth states. Thus a general heavy metal tolerance of plants does not exist. For plant species, 

there is a gradual increase from extreme sensitivity to the capability to endure concentration of 

heavy metals. Plants exist that can accumulate high amounts of heavy metals, like Thlaspi sp. 

or Minuartia verna and are therefore called hyperaccumulators. A separation into strict 

categories “metal hypotolerant”, “basal metal tolerant” and “metal hypertolerant” (Ernst et al. 

2008) neglects the complexity and the graduations between species for each specific heavy 

metal.

HEAVY METAL TOLERANT PLANTS AND ARBUSCULAR MYCORRHIZA 

The South-African Asteraceae Burkheya coddii is strongly colonized on heavy metal soils 

(Orlowska et al. 2002) and may be a good candidate for phytoremediation purposes. Other of 

the world-listed metalophytes (Prasad & Hagemeyer 1999) might also be mycorrhizal. In 

Central Europe, however, heavy metal plants mainly belong to non AMF - plant families and 

are therefore at best poorly colonized. This is the case for the already mentioned pennycress 

species (Thlaspi coerulescens, calaminare, goesingense) or for Cardaminopsis (Arabidopsis) 

halleri of the Brassicaceae, Armeria maritima ssp. halleri of the Plumbaginaceae and 

Minuartia verna and Silene vulgaris var. humilis of the Caryophyllaceae. In contrast, the zinc 

violets are strongly AMF-colonized and the degree of mycorrhizal colonization apparently 

increases in parallel with the raise of the heavy metal content in the soil (Hildebrandt et al. 

1999). The zinc violets have a very restricted, endemic distribution in Central Europe. The 

yellow zinc violet (Viola lutea ssp. calaminaria) occurs on heavy metal heaps in the Aachen- 

Liėge area, and the form (V. lutea ssp. westfalica) in the Pb-ditch and surrounding heap of 

Blankenrode, Eastern Westfalia. Both violets are descendants of the alpine Viola lutea 

(Hildebrandt et al. 2006). The colonization of the roots by AMF is even visible by eye, by the 

strong yellow colourization of the roots from the yellow pigment, a C-14 carotenoid termed 

mycorradicin (Klingner et al. 1995). 

Heavy metal soils contain mycorrhizal fungi (spores) though not as much abundant as at non 

polluted sites (Hildebrandt et al. 1999; Tonin et al. 2001). A Glomus intraradices isolate 

(termed Br1) was obtained from roots of the yellow zinc violet from the Breinigerberg site 

close to Stolberg near Aachen, Germany. This fungus consistently conferred heavy metal 

tolerance to plants provided the fertilization was optimized in the pot experiments performed in 

the greenhouse (Hildebrandt et al. 1999; Kaldorf et al. 1999). Diverse plants such as maize, 

barley, alfalfa or rye grass were grown in diverse heavy metal soils supplemented with AMF, 

where G. iintraradices Br1 proved better than other fungi from non-polluted sites. Such 

isolates might offer good perspectives in phytoremediation projects, particularly when applied 

as combinations of different AM fungi. 

BIOCHEMISTRY AND MOLECULAR BIOLOGY OF HEAVY METAL 

TOLERANCE CONFERRED BY AMF 

As shown by biophysical methods such as EDXA, SIMS, LAMMA (Kaldorf et al. 1999) or 

PIXE (Vogel-Mikuš et al. 2009) maize colonized by Glomus intraradices shows distinctly less 

heavy metals in both roots and shoots than non-colonized plants. In contrast, essential elements 

like Mg, Ca and P are enriched in AMF-colonized plants. Those heavy metals that are 

inevitably taken up by the roots are mainly detectable in the region of the inner cortical cells 

where most of the AMF structures reside. The biophysical techniques do not discriminate a 

deposition of the heavy metals between plant and fungal cells. Supposedly, the heavy metals 

are mainly deposited in the cell walls and the vacuoles of the fungal cells where they cannot 

exert toxic effects. 

493

As said, a completely sequenced genome of an AMF is not available as yet. Therefore it is 

difficult to get assess to genes and their products that are involved in heavy metal tolerance of 

the fungi. A metallothionein gene of Gigaspora margarita (Lanfranco et al. 2002) and of G. 

intraradices (Gonzales-Guerrero et al. 2007) and a Zn-transporter of the latter organism 

(Gonzales-Guerrero et al. 2005) were shown to be upregulated by heavy metals or oxidative 

stress. However, these are single genes that respond to specific stress events. To get a more 

comprehensive view on the genes expressed upon heavy metal stress, different display 

approaches were performed with tomato colonized by Glomus intraradices (Ouziad et al. 

2005). The sequences deposited in Genbank were employed to screen for conserved motifs and 

to use them for the synthesis of oligonucleotide primers from tomato. Using these, about 500 

bp segments were obtained by PCR of the following genes: four metallothioneins, one 

phytochelatin synthase and three Nramp transporters. Northern analyses, real-time PCR 

experiments and in situ hybridizations then showed that the expression of some of the genes 

(Nramp transporter 1 and 3 and metallothionein 2) were down-regulated in AMF colonized 

tomato but not in control plants, both grown in a heavy metal soil. Transcript levels of the 

mRNA of the other genes remained unimpaired. The data were interpreted to mean that AMF 

lower the concentration of the heavy metals in the roots to amounts that do not require anymore 

the maximal expression of these detoxifying genes in tomato. 

An upregulation of the counterpart genes in fungi was expected. However, a suppression 

subtractive hybridization cDNA library from hyphae of Glomus intraradices grown in the 

presence of high or low Zn concentrations did not show the upregulation of any of the genes 

just mentioned (Ouziad et al. 2005). In contrast, the library contained several EST sequences of 

genes coding for enzymes involved in the detoxification of reactive oxygen species: 

glutathione-S-transferase, superoxide dismutase, cytochrome P450, thioredoxin. Their 

enhanced expression upon exposure to high Zn-amounts was confirmed by reverse Northern 

analysis. The heavy metals reaching the cells of the fungi might particularly cause oxidative 

stress (might generate reactive oxygen species) and the fungi respond by producing the 

detoxifying enzymes. Such an interpretation was corroborated by an expression study of five 

genes with products potentially involved in fungal heavy metal tolerance (glutathione-Stransferase, 

HSP 90, a stress induced chaperone, a metallothionein and a Zn transporter of the 

ZIP family. For further details the reader is referred to (Hildebrandt et al. 2007). 

ARBUSCULAR MYCORRHIZA AND SALT TOLERANCE 

The literature on AMF and salt tolerance is somewhat controversial. Salt was reported to 

inhibit germination of spores, hyphal growth and colonization of the plants (Juniper & Abbott 

1993; 2006) Many plants of saline habitats (the halophytes), indeed, belong to Cyperaceae, 

Juncaceae and other non-mycorrhizal plant families. On the other hand, plants like the sea 

plantains are strongly mycorrhizal, and the salt aster, Aster tripolium, is one of the highest 

colonized plant with almost all roots showing AMF structures (Hildebrandt et al. 2001). 

Diverse salt marshes of Central Europe, irrespectively of the salt type (NaCl, Na2SO4, Na2CO3, 

494

K2CO3) show fairly high amounts of AMF spores, among which up to 80% belong to one 

species, Glomus geosporum (Hildebrandt et al. 2001; Landwehr et al. 2002; Carvalho et al. 

2001; 2004). The size of the G. geosporum spores in salt marshes is rather variable. This 

fungus may be forced to produce many spores under the harsh saline conditions (Wilde et al. 

2009). The recent molecular characterization of the fungi within the roots of halophytes (Wilde 

et al. 2009) showed that G. geosporum can be detected there. However, other fungi prevailed. 

Their sequences matched to those of uncultured fungi with distant relatedness to Glomus 

intraradices or were completely new. All attempts to cultivate these or to find their 

corresponding spores consistently failed (Wilde et al. 2009). 

These fungi may be the organisms that confer salt tolerance to plants, thus may enable plants to 

grow under such adverse conditions. In the past, our repeated attempts with G. geosporum to 

confer salt tolerance to plants in greenhouse experiments consistently failed (Füzy et al. 2008), 

possibly due to the use of this wrong fungus. It should, however, be mentioned that others 

claimed to be more successful in using AMF for conferring salt tolerance to plants (Al-Karaki 

2000; Tian & Feng 2004 and others). In greenhouse experiments, salt is easily drained out with 

watering the plants. Therefore care has to be taken to monitor the salt concentration and to 

keep it constant during the course of the experiment. 

The dimension for the application of AMF in salt tolerance is much higher than in heavy metal 

pollution. About 7% of the land surface is affected by salt and therefore not amenable for 

farming. The idea is to develop an isolate or a mixture of AMF that allows crops to be grown in 

saline habitats and even to use sea water for irrigation. Such a perspective is, however, only a 

dream at present. 

BIOCHEMICAL AND MOLECULAR ASPECTS OF AMF AND HALOPHYTES 

Elemental studies of Na + and Cl - are not so easily performed as with heavy metals, since these 

salts are easily washed out or artificially translocated with the cutting of the tissues. Despite 

this, an attempt was made with AMF colonized roots of Aster tripolium (Scheloske et al. 

2004). Roots of this plant contain a lot of aerenchyma. Their structure is altered upon 

colonization by AMF. Instead of few, large arenchyma many of small size are formed and the 

cortical cells are more densely packed. Element localization studies by PIXE indicated that 

AMF roots contain more Na + and Cl - than controls. The exact location of these two ions is 

difficult to demonstrate since the roots are really fragile (Scheloske et al. 2004). 

In soils and water, NaCl is dissociated, and these ions strongly bind water. In saline soils with 

extremely low water potentials, plants have to cope not only with toxic effects caused by Na + 

and Cl - but also with the problem to acquire sufficient water to avoid wilting particularly in 

drought periods. AMF could be of help here to minimize the water problem. A recent study in 

the Hungarian plain (Füzy et al. 2008) indicated that AMF plants from saline sites, indeed, 

respond to rainfall. There was an inverse correlation between the intensity of rainfall and the 

number of arbuscules formed during the course of the year. Halophytes formed many 

arbuscules in periods of droughts but few when rainfall was sufficient. 

495

Growth of plants in saline habitats requires osmotic adjustments within the cells in order to 

avoid wilting. Osmolyticants can be proline, glycine, betaines, glycerol, polyols and other and 

are plant specific. Excess of Na + may be removed out of the cells or deposited in the vacuole by 

Na + /H + transporters and water may be transported to the plants by aquaporins, residing at the 

plasmalemma and/or the tonoplast. The sequences available in the databanks were used to 

develop specific probes for tomato genes with products possibly involved in salt tolerance: two 

for plasmalemma aquaporins (abbreviated PIPs), one for a tonoplast aquaporin (TIP) and two 

for Na + /H + transporters (Ouziad et al. 2006). Studies on their differential expression in salt 

stressed tomato colonized by AMF (Ouziad et al. 2006) showed that the expression of both 

Na + /H + transporters was not significantly affected by salt or AMF colonization. In contrast, 

both Northern analyses and in situ hybridizations indicated that the expression of two 

aquaporins (one of the PIPs and the TIP) was reduced by salt stress and that this effect was 

enhanced by AMF colonization of the plants under the same salt stress. In leaves, the mRNA 

transcript concentrations of all three genes were higher in AMF colonized tomato than in nonmycorrhizal 

plants under salt stress. 

Among 384 differentially expressed clone (ESTs) of a SSH clone library (obtained from 

extraradical hyphae of Glomus intraradices stressed with 0.7% NaCl minus non-treated 

controls) the following appeared to be noteworthy: 90 KDa heat shock protein, thioredoxin 

peroxidase, glutathione reductase, glutathione-S-transferase, Cu/Zn superoxide dismutase, 

DNA repair protein, peptidyl-prolyl isomerase, vacuolar H + -ATP sythase (V-ATPase, subunit 

C) and calcium transporting ATPase (P-type IIA ATPase). An aquaporin and a Na + /H + 

transporter were not detected among these ESTs. It contained genes coding for enzymes that 

serve in protecting against oxidative stress similarly as noted also under heavy metal stress. 

Thus salt, drought and heavy metal stress might generate reactive oxygen radicals in the cells 

which must be detoxified by such enzymes. 

Attempts have been forwarded to engineer salt tolerant plants by overexpressing Na + /H + 

transporters (Yamaguchi & Blumwald 2005, Apse & Blumwald 2007). The experiments 

reported here from the own laboratory are only a start on the molecular biology of salt stress of 

plants and their potential alleviation by AMF as yet. They might already indicate that toxic 

effects of Na + and Cl - are not the major problem imposed on plants. The prevention of drought 

and destruction by oxygen radicals may be the major concern for the plants, and AMF might be 

beneficial in coping with these problems. 

REFERENCES 

Al-Karaki G N (2000). Growth of mycorrhizal tomato and mineral acquisition under salt stress. 

Mycorrhiza 10, 51-54. 

Apse M P; Blumwald E (2007) Na+ transport in plants. FEBS Letters 581, 247-254. 

Bothe H; Regvar M; Turnau K (2009; in press). Arbuscular mycorrhiza, heavy metal and salt 

tolerance. In: Soil heavy metals, Chapter V. eds I Sherameti & A Varma. Springer: 

Heidelberg, Berlin, New York. 

496

Carvalho L M; Caçador I; Martins-Loução M A (2001). Temporal and spatial variation of 

arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal). Mycorrhiza 

11, 303-309. 

Carvalho L M; Correia P M; Martins-Loução M A (2004). Arbuscular mycorrhizal fungal 

propagules in a salt marsh. Mycorrhiza 14, 165-170. 

Ernst W H O; Krauss G J; Verkleij J A C; Wesenberg D (2008). Interaction of heavy metals 

with sulphur metabolism in angiosperms from an ecological point of view. Plant Cell 


Füzy A; Tóth T; Hildebrandt U; Biró B; Bothe H (2008). Drought, but not salinity, determines 

the apparent effectiveness of halophytes colonized by arbuscular mycorrhizal fungi. 

J Plant Physiology 165, 1181-1192. 

Gonzales-Guerrero M; Cano C; Azcon-Aguilar C; Ferrol N (2007). GintMT1 encodes a 

functional metallothionein in Glomus intraradices that responds to oxidative stress. 


Gonzales-Guerrero M; Azcon-Aguilar C; Mooney M; Valderas A; MacDarmid C W; Eide D J; 

Ferrol N (2005). Characterization of a Glomus intraradices gene encoding a putative Zn 

transporter of the cation diffusion facilitator family. Fungal Genet Biol 42,130-140. 

Hildebrandt U; Kaldorf M; Bothe H (1999). The zinc violet and its colonisation by arbuscular 

mycorrhizal fungi. J Plant Physiol 154, 709-717. 

Hildebrandt U; Regvar M; Bothe H. (2007). Arbuscular mycorrhiza and heavy metal tolerance. 

Phytochemistry 68, 139-146. 

Hildebrandt U; Ouziad F; Marner F J; Bothe H (2006). The bacterium Paenibacillus validus 

stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the 

formation of fertile spores. FEMS Microbiol Lett 254, 258-267. 

Hildebrandt U; Janetta K; Ouziad F; Renne B; Nawrath K; Bothe H (2001). Arbuscular 

mycorrhizal colonisation of halophytes in Central European salt marshes. Mycorrhiza 

10, 175-183. 

Juniper S; Abbott L K (1993). Vesicular-arbuscular mycorrhizas and soil salinity. Mycorrhiza 

4, 45-57. 

Juniper S; Abbott L K (2006) Soil salinity delays germination and limits growth of hyphae 

from propagules of arbuscular mycorrhizal fungi. Mycorrhiza 16, 371-379. 

Kaldorf M O; Kuhn A J; Schröder W H; Hildebrandt U; Bothe H (1999). Selective element 

deposits in maize colonized by a heavy metal tolerance conferring arbuscular 

mycorrhizal fungus. J Plant Physiol 154, 718-728. 

Klingner A; Bothe H; Wray V; Marner F J (1995). Identification of a yellow pigment formed 

in maize roots upon mycorrhizal colonization. Phytochemistry 38, 53-55. 

Landwehr M; Hildebrandt U; Wilde P; Nawrath K; Tóth T; Biró B; Bothe H (2002). The 

arbuscular mycorrhizal fungus Glomus geosporum in European saline, sodic and 

gypsum soils. Mycorrhiza 12, 199-211. 

Lanfranco L; Bolchi A; Ros E C; Ottonello S; Bonfante P (2002). Differential expression of a 

metallothionein gene during the presymbiotic versus the symbiotic phase of an 

arbuscular mycorrhizal fungus. Plant Physiol 130, 58-67. 

Orłowska E; Zubek S; Jurkiewicz A; Szarek-Lukaszewska G; Turnau K (2002). Influence of 

restoration on arbuscular mycorrhiza of Biscutella laevigata L. (Brassicaceae) and 

Plantago lanceolata L. (Plantaginaceae) from calamine spoil mounds. Mycorrhiza 12, 

153-160. 

497

Ouziad F; Hildebrandt U; Schmelzer E; Bothe H (2005). Differential gene expressions in 

arbuscular mycorrhizal-colonized tomato grwon under heavy metal stress. J Plant 

Physiol 162, 634-649. 

Ouziad F; Wilde P; Schmelzer E; Hildebrandt U; Bothe H (2006). Analysis of expression of 

aquaporins and Na+/H+ transporters in tomato colonized by arbuscular mycorrhizal 

fungi and affected by salt stress. Environ Exp Bot 57, 177-186. 

Prasad M N V; Hagemeyer J (eds.) (1999). Heavy metal stress in plants -from molecules to 

ecosystens. Springer: Berlin, Heidelberg, New York. 

Redecker D; Kodner R; Graham L E (2000). Glomalean Fungi from the Ordovician. Science 

289, 1920-1921. 

Regvar M; Vogel-Mikuš K; Irgel N; Wraber T; Hildebrandt U; Wilde P; Bothe H (2003). 

Colonization of pennycresses Thlaspi sp. of the Brassicaceae by arbuscular mycorrhizal 

fungi. J Plant Physiol 160, 615-626. 

Scheloske S; Maetz M; Schneider T; Hildebrandt U; Bothe H; Povh B (2004). Element 

distribution in mycorrhizal and nonmycorrhizal roots of the halophyte Aster tripolium 

determined by proton induced X-ray emission. Protoplasma 223, 183-189. 

Schüßler A; Schwarzott D; Walker C (2001). A new fungal phylum, the Glomeromycota: 

phylogeny and evolution. Mycol Res 105, 1413-1421. 

Smith S E; Read D J (2007). Mycorrhizal Symbiosis. 3. Ed. Academic Press: San Diego, USA. 

Tian C Y; Feng G (2004). Different effects of arbuscular mycorrhizal fungal isolates from 

saline or non-saline soil on salinity tolerance of plants. Appl Soil Ecol 26, 143-148. 

Tonin C; Vandenkoornhuyse P; Joner E J; Strczek J; Leyval C (2001). Assessment of 

arbuscular mycorrhizal fungi diversity in the rhizosphere of Viola calaminaria and 

effect of these fungi on heavy metal uptake by clover. Mycorrhiza 10, 161-168. 

Vogel-Mikuš K; Pongrač P; Pelicon P; Vapetic P; Povh B; Bothe H; Regvar M (2009). Micro- 

PIXE analysis for localisation and quantification of elements in roots of mycorrhizal 

metal tolerant plants. In: Symbiotic Fungi: Principles and Practice, eds A Varma & A 

Kharkwal. Springer: Berlin, Heidelberg, New York. 

Wilde P; Manal A; Stodden M; Sieverding M; Hildebrandt U; Bothe H (2009). Biodiversity of 

arbuscular mycorrhizal fungi in roots and soils of two salt marshes. Environ Microbiol 

in press. 

Yamaguchi T; Blumwald E (2005). Developing salt-tolerant plants: challenges and 

opportunities. Trends Plant Science 10, 615-620. 

498

Verticillium wilt pathogen: Verticillium dahliae. 

Soil Using ordinary nutritious soil after 60Co sterilization, 500mL per pot. 

Pot Filling soil in plastic barrel-shaped belt of 200mm-perimeter to make pot without 

upper and lower lids. 

Method 

Trial condition:greenhouse with additional light system 

Trial design: 6 treatments is following, repetition is 10. 

CK: the control (without any inoculation) 

Vd: inoculate with Verticillium dahliae 30 days after sowing 

Gm: inoculate with Gl. mosseae when sowing 

Ge: inoculate with Gl. etunicatum when sowing 

Gm+Vd: inoculate with Gl. mosseae when sowing and inoculate with V. dahliae 

30 days after: 

Ge+Vd: Inoculate dwith Gl. etunicatum when sowing and inoculate with V. 

dahliae 30 days after 

Inoculation 

− with AMF: first mixture AMF inoculum with soil according to the inoculation demand of 

500 spores per pot, then fill the pot and sow. The treatments of CK and Vd add the same 

amount of inoculum after sterilization. 

− with pathogen: inoculate by root-cut method 30 days after sowing with 20mL of V. 

dahliae conidiophore suspend solution (10 7 spores/mL). The treatments of CK, Gm and 

Ge use the same amount of sterilized water. 

Investigations 

− Cotton growth and AMF colonization: investigate shoot length, shoot weight, root 

weight, leaf chlorophyll content and AMF conolization frequency 40 days after seedling 

coming out. 

− Verticillium wilt occurrence and development: investigate the disease rate and index on 

the 11 th and 18 th day after V. dahliae inoculation. 

− Root defensive enzymes activities: investigate chitinase (by n-acetyl glucosamine 

colorimetric method) and PAL ( by trans-cinnamic acid method) activities everyday in 

the first week after V. dahliae inoculation, and investigate POD activity (by guaiacol 

method) every two day in the first 11 days after V. dahliae inoculation. 

500

502 

Table 2. Verticillium wilt development in different treatments 

Treatment Disease rate(%) 

(11 th day) 

Disease index(%) 

(11 th day) 

Disease rate(%) 

(18 th day) 

Disease index(%) 

(18 th day) 

CK 0 0 0 0 

Vd 42.5 12 56 26 

Gm 0 0 0 0 

Ge 0 0 0 0 

Gm+Vd 25 6.5 32 11 

Ge+Vd 18.7 5.2 26.7 6.7 

Root defensive enzymes activities 

Chitinase 

Chitinase is one of the most important anti-fungi enzymes produced by plant which target on 

chitin, the main component of fungi cell wall. The trend of root chitinase activities in 7 days 

after V. dahliae inoculation in treatments shows in Fig. 1. 

Chitinase activeties(U.g -1 ) 

70 

60 

50 

40 

30 

20 

10 

0 

1 2 3 4 5 6 7 

Days after V. dahliae inoculation 

CK 

Vd 

Gm 

Ge 

Gm+Vd 

Ge+Vd 

Figure 1. The trend of root chitinase activities under V. dahliae stress 

Phenylalanine ammonialyase (PAL) 

PAL is a critical enzyme in the plant secondary metabolism especially in the phenylpropane 

passway, which is directly related disease resistance of the plant. The trend of root PAL 

activities in 7 days after V. dahliae inoculation in treatments shows in Fig. 2.

Root resistance-related substances contents 

Malondialdehyde (MDA) 

MDA is the final decomposition product of peroxidation of membrane lipids which occurs to 

plant cells when aging or under stresses, thus MDA could be an indicator of the damage degree 

the plant suffered. The trend of root MDA contents in 11 days after V. dahliae inoculation in 

treatments shows in Fig. 4. 

Total phenol 

504 

MDA content(umol.g-1) 

0.16 

0.14 

0.12 

0.1 

0.08 

0.06 

0.04 

0.02 

0 

0 1 3 5 7 9 11 

Days after V. dahliae inoculation 

CK 

V.d 

Gm 

Ge 

Gm+Vd 

Ge+Vd 

Figure 4. The trend of root MDA content under V. dahliae stress 

Phenol is a kind of plant secondary metabolism products which is related to disease resistance. 

The root total phenol contents of treatments 11 days after V. dahliae inoculation shows in 

Figure 5. 

Phenol content (ug.g -1 ) 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

CK Vd Gm Ge Gm+Vd Ge+Vd 

Figure 5. Root total phenol contents of treatments

Fig 4 and 5 show that under V. dahliae stress, the AMF-cotton symbiont on one hand reduced 

root MDA content which indicates the damage degree of plant cell, on the other hand increased 

the defensive substance content of total phenol. 

Root cell ultramicrostruture 

In the pictures in Figure 5, the cells of CK are observed to be normal with normal structure, 

uniform nucleoplasm and cytoplasm; in Gm and Ge, some changes occurred to the cells 

structure, such as cell deformation and pyknosis, decrease in vacuole number and xylem 

expanded; some severe damages occurred to the cells of Vd, including severe distortion and 

deformation of cell shape, almost none cell organelles exist and severe cell wall damage; in 

(Gm+Vd) and (Ge+Vd), the color became darker, palisade tissues and vessels deformed, the 

cell walls became thicker obviously and lignified, material deposition occurred on cell walls. 

Figure 5. Pictures taken by transmission electron microscope. 

505

DISSCUSSION 

Data for three kinds of enzymes (chitinase, PAL, POD) and MDA, indicate that the trends are 

similar. However, the peaks of chitinase and PAL activities appear 4 days after V. dahliae 

inoculation, 5 days for POD and 7 days for MDA, and the co-relationships between treatments 

are also similar. In the two kinds of AMF, Gl. etunicatum performed better than Gl. mosseae as 

a whole. 

It could be assumed from the above that the formation of symbiosis relationship between 

cotton root and AMF certainly introduces changes from root cell structure to physiology and 

biochemistry, the changes therefore are prepared to produce multiple stress responses when the 

symbiont exposed to the stresses such as V. dahliae in this case and to enhance resistance and 

alleviate damage. However, the molecular or genic mechanism of such changes needs further 

study. 

REFERENCES 

Long X; Wang J; Chen J; Cui W H; Yang R; Lu J; Feldmann F (2007). Effect of Inoculation 

with AMF Inoculum on Disease Resistance and Yield of Cotton Field, Xinjiang. 

Agricultural Sciences 44(4), 457-460. 

506

Feldmann F, Gillessen M, Hutter I Schneider C: Should we breed for effective mycorrhiza symbioses?. In: 



11-3 Should we breed for effective mycorrhiza symbioses? 

Feldmann F, Gillessen M, Hutter I 2 Schneider C 

1 

Julius Kühn-Institute, Messeweg 11-12, 38104 Braunschweig, Germany 

2 Institut für Pflanzenkultur, Solkau 2, D-29465 Schnega, Germany 

Email: Falko.Feldmann@jki.bund.de 

Abstract 

Most species of useful plants are able to develop symbioses with arbuscular 

mycorrhizal fungi (AMF). The partnership between these fungi and host roots can 

lead to an enhanced tolerance of plants to abiotic and biotic stresses. The 

mycorrhizal technology developed in the last few years provides agricultural and 

horticultural practice with suitable commercial mycorrhizal inoculum for the 

inoculation of annual vegetables, ornamentals, perennial herbs, shrubs or trees. New 

inoculation methods for already established plants and the flexibility of modern 

inoculum products allow the inclusion of mycorrhizal technology within integrated 

plant production systems as important biological phytosanitary factors. 

Unfortunately the variability of host dependency on mycorrhizal fungi varies 

between cultivars. Because of a lack of suitable breeding markers it is discussed 

whether the degree of mycorrhizal root colonization and the character “mycorrhizal 

dependency” should be used as breeding markers. It is recommended to collect data 

about mycorrhiza formation of new cultivars during breeding processes. 

INTRODUCTION 

An effective mycorrhiza is a pre-requisite for the maximum exploration of resources by 

mycotrophic plants in their artifical or natural environment. Nutrient deficiency is one of the 

most important stresses which can be overcome by mycorrhiza (Bethlenfalvay, 1992), resulting 

in practical applications concerning recultivation of marginal and degraded agricultural sites 

(Feldmann et al., 1995). The negative influence of water logging on one hand (Khan & Belik, 

1995) and water deficiency on the other (Auge & Stodola, 1990) can be reduced by 

mycorrhization. Reduction of salt-induced "physiological drought" (Rosendahl & Rosendahl, 

1991) is another important effect, e.g. in anthropogenic salty environments such as urban tree 

stands. Furthermore, the phytoremediation of areas polluted by heavy metals is more effective 

with mycorrhizal plants (Leyval et al., 2002). Estaun et al. (2008) rehabilitate limestone 

507

quarries only with mycorrhiza; Takacs et al. (2008) recommend application of mycorrhiza for 

successful phytoremediation and Schmid et al. (2008) for High Alpine revegetation. Tschirner 

et al. (2008) use the symbiosis for stabilization of roadsides, Dag et al. (2008) and Pivonia et 

al. (2008) apply mycorrhiza under arid conditions for tree or vegetable production. Even the 

weaning stage of in vitro cultivated plants can be favoured by mycorrhizal fungi (Schneider et 

al., 2008; Cheng et al., 2008). 

Besides reduction of symptoms of abiotic stress, the interrelationship between plant host, 

fungal symbiont and parasites have been studied (Dehne, 1982) and the phytomedical potential 

of mycorrhizal fungi recognized (Schönbeck, 1987) for decades. The damage of soil borne 

fungal pathogens causing root rot or vascular damage, e.g. Phytophtora parasitica (Cordier et 

al., 1996), Aphanomyces euteiches (Slezack et al., 2000), Fusarium spp., Verticillium spp, 

Sclerotium spp (Hooker et al., 1994; Azcon-Aguilar & Barea, 1996), as well as plantpathogenic 

nematodes causing root galls and root lesions (Meloidogyne spp, Pratylenchus spp 

and Radophulus spp, Pinochet, 1996), was reduced in presence of AMF. Feldmann et al. 

(2008) therefore use mycorrhizal fungi as a regulative against nematodes in horticultural 

production systems under greenhouse condition; Long et al. (2008) applied AMF as plant 

strengtheners under field conditions against pathogens of cotton and ornamentals. Leaf 

pathogens such as powdery mildew can be supported by AMF (Schönbeck & Dehne, 1979) 

and other leaf blight fungi repressed (Feldmann et al., 1989). Interactions with root-pathogenic 

bacteria are known to protect tomato plants against Erwinia carotovora and Pseudomonas 

syringae (Garcia-Garrido & Ocampo, 1988, 1989). 

Mechanisms underlying such bio-protective effects are: (i) improvement of plant nutrient 

status/damage compensation (Trotta et al., 1996), (ii) competition for host carbohydrates and 

colonization sites (Schönbeck, 1987; Feldmann and Boyle, 1998), (iii) changes in anatomy and 

architecture and function of the root system (Forbes et al., 1996), (iv) microbial changes in the 

rhizosphere (Linderman and Paulitz, 1990), (v) activation of plant defense mechanisms (Pozo 

et al., 2002), and (vi) systemic effects of AMF colonization (Cordier et al., 1996). 

Bio-protective effects depend on many factors, influencing the effect of the symbiosis. The 

most important factors are: (i) the AMF strain, (ii) the pathogen, concerning virulence and 

inoculum potential, (iii) the growing substrate, (iv) the prevailing environmental conditions 

(Azcon-Aguilar et al., 2002), the host plant cultivar which can be characterized by specific host 

dependency and responsiveness to mycorrhiza, and (vi) a degree of root colonization which 

exceeds thresholds to induce plant responses. This paper will focus on the question whether the 

degree of root colonization and the character “dependency” should be used as breeding 

markers. 

SHOULD WE BREED FOR BETTER MYCORRHIZAL COLONIZATION? 

Although most species of vascular plants are potential symbiotic partners, variations in the 

degree of colonization exist between and within species of genera which are thought to be 

mycotrophic (e.g. genus Viola, see Table 1). 

508

Table 1. Range of mycorrhiza frequency in roots of Viola species. The plants were 

collected between 1989 and 2009 as seed material at natural sites or botanical 

gardens or bought from different seed traders and then inoculated with Glomus 

etunicatum. Minimally, three plants were tested per species.(Feldmann, 

unpublished). 

Range of mycorrhizal frequency [%] 

0-20 21-50 51-100 

V. acuminata V. aetolica V. beckwithii 

V. adunca V. arvensis ssp. arvensis V. biflora 

V. alba V. bakeri V. calcarata 

V. alba ssp. alba V. canina V. calaminaria 

V. altaica V. cheiranthifolia V. elatior 

V. ambigua V. corsica ssp. limbarae V. guestphalica 

V. anagae V. douglasii V. hirta 

V. canadensis V. epipsila V. jordanii 

V. cazorlensis V. glabella V. mirabilis 

V. collina V. gracilis V. obliqua 

V. cornuta V. jooi V. odorata 

V. dubyana V. nuttallii V. pubescens 

V. hallii V. palmensis V. reichenbachiana 

V. kitaibeliana V. patrinii V. tricolor var. maritima 

V. lutea V. persicifolia 

V. palustris V. purpurea var. purpurea 

V. pedata V. rupestris 

V. pumila V. trinervata 

V. pyrenaica V. uliginosa 

V. riviniana 

V. suavis 

V. tricolor ssp. eutricolor 

V. tricolor ssp. tricolor 

V. × bavarica 

V. × scabra 

V. × wittrockiana 

Species such as V. calaminaria are colonized up to 100% (mycorrhizal frequency collected at 

their natural, zinc-polluted sites). Others, such as Viola tricolor, V. lutea and V. cornuta are 

509

weakly colonized. Together with V. altaica they are the basis for crossings, resulting in V. × 

wittrockiana hybrids – which colonize badly after inoculation with AMF suitable for other 

Viola species. 

To investigate the importance of the plant genome, a large number of cultivars were tested for 

variation in colonization levels (e.g. Figure 1, Kalanchoe blossfeldiana). In K. blossfeldiana 

the range of mycorrhiza formation ranged from 0 to 100% of the root system colonized. We are 

recently analyzing whether clusters of certain proveniences are more colonized than others. 

510 

Mycorrhizal Frequency [%] 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 10 20 30 40 50 60 70 80 

Cultivar number 

Figure 1. Mycorrhiza formation of 88 Kalanchoe blossfeldiana cultivars under uniform 

greenhouse conditions. Plants were incoculated with the commercial 

mycorrhiza inoculum “INOQ Hobby” (Feldmann, Gillesen, Brielmeier- 

Liebetanz, Schneider, unpublished). 

Differences of mycorrhization between these cultivars of K. blossfeldiana were not due to the 

influence of environmental differences or variation of the fungal inoculum. Instead, different 

degrees of root colonization of the cultivars were apparently induced by breeding. 

Much research on the genetic control of mycorrhiza colonization has involved Triticum and the 

ancestral Aegilops complex because of information available on the genetics of this group 

(Kapulnik and Kushnir 1991; Hetrick et al. 1992). A detailed comparison of wheat ancestors, 

primitive wheat lines and modern cultivars has shown a strong genetic basis for colonization 

ability. Most modern wheat cultivars, all landraces from Asian collections and all early United 

States cultivars showed mycorrhiza dependency (Hetrick et al. 1992). Wheat ancestors (except

Aegilops speltoides) carrying the AA and BB genomes benefitted from mycorrhizal 

colonization whereas ancestors of the DD genome, tetraploid wheat cultivars carrying the 

AABB or AAGG genome, or the hexaploid ancestor Triticum zhukovskyi with the AAAAGG 

genome, did not. Using differential RNA display, Martin-Laurent et al. (1997) cloned one of 

the plant genes involved in early events leading to a successful colonization of pea roots by 

Glomus mosseae. Expression of that gene was independent of rhizobial bacteria. Knowledge of 

the genetics of the colonization process will be fundamental for development of screening 

procedures and molecular markers to breed genotypes for more efficient mycorrhizal 

symbiosis. Mutants unable to sustain mycorrhizal colonization (e.g., in pea, Balaji et al., 1995; 

in tomato, Barker et al., 1998) are important to increase such knowledge. 

Mutants resistant to arbuscular mycorrhizal colonization were introduced in pea and Medicago 

truncatula (Rengel 2002) (for references see Gianinazzi-Pearson, 1996). The Myc mutation is 

recessive, genetically stable and controlled by the same single gene as Nod- (Duc et al., 1989). 

The product of the wild-type alleles of Myc-mutated loci may be involved in the biosynthesis 

of a plant susceptibility factor that negatively regulates the defence response (Gianinazzi- 

Pearson, 1996). So, without this susceptibility factor, plant root cells of Myc mutants have 

thick, reinforced cell walls loaded with detence-related molecules, thus preventing AM 

colonization of such cells. In contrast to Myc- mutation, Myc2- loci may be involved in 

metabolic specialisation of the AM-containing cells (Gianinazzi-Pearson et al., 1995). 

It would be misleading to enhance symbiosis by down-regulating the plant defence response, as 

susceptibility to pathogen attack might increase, even though Myc- and Nod- mutants 

described so far did not suffer from increased susceptibility to the pathogen attack. This 

indicates a considerable specificity in the infection pattern and changes in plant defence 

responses. In contrast, the fact that Myc mutants are also Nod- mutants (at least in pea) 

indicates that there are common mechanisms regulating the plant-microbe interactions in the 

two symbioses (Gianinazzi-Pearson, 1996), thus raising the possibility that breeding efforts to 

improve one symbiosis may fortuitously result in improvements to the other. For instance, 

Mercy et al. (1990) showed that in Vigna unguiculata there is a high variability among 

genotypes for mycorrhiza colonization, indicating the possibility of using this character in 

selection and breeding programmes (see Fig 1). Manske (1989), in studies of colonization of a 

high and low mycorrhiza-colonizing cultivars of Triticum aestivum rotundatum and the Fl of 

reciprocal crosses, concluded that both chromosomal and cytoplasmic genes are involved. 

Bertheau et al. (1980) observed that in three lines of the wheat (T. aestivum) cultivar Centana, 

isogenic except for dwarfing genes from the cultivar Norin 10, the dwarf line (Rht1 Rht2) had 

the highest mycorrhiza colonization level, while the semi-dwarf line (Rhtl) showed the greatest 

yield response to colonization. 

The expressions “plant response” and “dependency” are related to effectiveness of the 

symbiosis under certain colonization values. Significant differences in colonization levels 

occur among genotypes within a species, but these differences are generally based on % root 

length colonized by all fungal structures combined (i.e. hyphae, arbuscules, vesicles) and not 

511

nutrient exchange structure, the arbuscule, alone (reviewed by Peterson and Bradbury (1995). 

Exceptions are also reported (Toth et al. 1984; Blair 1987). 

There is consensus that no positive correlation between colonization level and plant growth can 

be expected (Estaun et al. 1987; Manske 1990; Kapulnik and Kushnir 1991; Vierheilig and 

Ocampo 1991a; Hetrick et al. 1992). Peterson and Bradbury (1995) consider that this lack of 

correlation could occur if the fungal species used are normally not associated with the chosen 

plant species. Hetrick et al. (1992) attempted to alleviate this problem by using fungi known to 

colonize their experimental plants. Differences in mycorrhizal colonization among intraspecific 

variants might also depend on the stage of plant growth (Stöppler et al. 1990), 

Focussing on the fungal partner in other experiments we observed that “plant dependency” 

may not explain differences in effectiveness, if different inocula are tested over four years 

under standard greenhouse conditons (Table 2). Effectiveness of the single inocula differed 

between years. At the same time responsiveness of hosts to different fungi is very heterogenous 

within the same year as well. At the same time, significant effectiveness was not correlated 

with the degree of root colonization. But no positive effectiveness was observed below a 

degree of colonization of 23% indicating a threshold level necessary for posistve response. 

Therefore, cultivar characteristics and fungal specifity have both an influence (see Boyetschko 

and Tewari, 1995). 

512 

Table 2: Mycorrhizal Effectiveness Index (MEI [%]) of three AMF inocula of Glomus 

on different host plants over four years (The inoculum was produced on Zea mays var. 

Felix, values printed bold are significantly different from control values: 95% probability; 

Feldmann, 1998a). 

G. etunicatum HH6 G. etunicatum HH13 G. intraradices 267 

Year of experiment 1. 2. 3. 4. 1. 2. 3. 4. 1. 2. 3. 4. 

Zea mays var. Felix 31 20 15 7 43 37 14 14 27 24 4 5 

Pelargonium zonale 26 - 49 30 28 - 56 25 20 - 23 28 

Trifolium repens - 12 20 - - 30 -1 - - 13 -1 - 

Petroselinum crispum - 9 13 21 - 11 -8 -4 - 17 -5 21 

Baptisia tinctoria - 7 - 18 - 5 - 20 - -2 - 5 

Helianthus annuus 1 -3 - - -4 2 - - 5 5 - - 

Triticum aestivum 1 4 -2 - -16 -9 -15 - 9 19 -1 - 

Genetic variability of AM colonization capacity was investigated in various genotypes of host 

species (e.g., bell pepper and tomato, Nemec and Datnoff, 1993; barley, Boyetchko and 

Tewari, 1995; grapevine, Karagiannidis et al., 1995). Hyphal growth and thus competitive

ability also varies widely in populations of mycorrhizal fungi (De la Bastide et al., 1995). This 

was studied in Zea mays cv. Felix. In Table 2 a continous decrease of effectiveness of all three 

inocula occurred on that cultivar. This decrease of effectiveness was not observed in cv. 

Badischer Landmais (Table 3). Exchanging the inoculum after four multiplication cycles 

increased the effectiveness of inoculum only on cv. Badischer Landmais whereas a decrease 

was investigated on cv. Felix. Badischer Landmais is an old corn cultivar and cv. Felix a 

modern hybrid. The maintenance of higher effectiveness obviously correlates with higher 

genetic heterogeneity of the host. Whether it is the “cause“ could not be proved. However, if a 

host’s genetic heterogeneity could guarantee stable effectiveness of AMF, the influence of biodiversity 

of host and fungal communities would have an important impact on symbiontal 

relevance in natural ecosystems and production systems. The experiment highlights that 

cultivar characteristics are relevant for the effectiveness of symbiosis. The same was shown 

among wheat cultivars where significant differences disappeared when two inoculations were 

used (Vierheilig and Ocampo 1991b). 

Table 3. Mycorrhizal Effectiveness Index (MEI [%]) of Glomus etunicatum on different 

host plant varieties over seven years of subsequent inoculation. (The inoculum 

was produced on each host cultivar and exchanged after the fourth year) (Feldmann et al., 1999) 

Multiplication cycle 

Zea mays cultivar 

Badischer Landmais Felix 

I 35 41 

II 30 38 

III 31 16 

IV 36 9 

Exchange of host/inoculum 

V 20 25 

VI 44 17 

VII 36 3 

Wheat genotypes differ in their capacity to sustain mycorrhiza, with yield responses varying 

from zero to positive or negative (Xavier and Germida, 1998). Benefits arising from the 

mycorrhizal symbiosis are not proportional to the extent of the root colonization, as genotypes 

vary in their dependence on mycorrhiza (Al-Karaki and Al-Raddad, 1997). Similar results have 

been obtained for barley genotypes varying in P efficiency (Baon et al., 1993). 

Graham et al. (1991) stress that breeding plants for greater colonization by vesicular-arbuscular 

mycorrhizal fungi must consider the cost/benefit of the association for the specific crop, soil 

and environmental conditions. A very high degree of colonization might be disadvantageous. 

Finally, Graham et al. (1991) used a number of rootstock genotypes of Citrus, Poncirus or 

513

Citrus X Poncirus, known to differ in mycorrhizal dependency. These rootstocks were grafted 

onto a single scion genotype (Citrus sinensis) in experiments to determine the relationship 

between mycorrhizal dependency and colonization by mycorrhizal fungi. Results showed that, 

in P-deficient soils, colonization level was positively correlated with mycorrhiza dependency 

(cited by Peterson and Bradbury, 1995). 

Does it make sense to breed for better colonization in breeding programmes? The pre-requisit 

for an effective symbiosis is colonization of the roots. Following our experience, even without 

a strong relationship between degree of colonization and effectiveness, a threshold value of 

probably 20-30% root colonization should be realized in cultivars (compare Table 2) otherwise 

the plant will not or rarely benefit from the mycorrhizal fungi. The mycorrhizal frequency is 

normally not correlated with the effectiveness. Therefore, it is recently recommended, not to 

breed for higher colonization but to take care that plant genotypes are selected during the 

breeding process with mycorrhizal formation capacity above this threshold. 

SHOULD WE BREED FOR HIGHER MYCORRHIZAL DEPENDENCY OF USEFUL 

PLANTS? 

As demonstrated above, mycorrhizal dependency of a host is genetically fixed (Azcon & 

Ocampo, 1981). The degree of mycorrhizal dependency is a gradient of the host’s ecological 

niche and environmental conditions. If a plant cannot explore its resources without mycorrhiza 

MacMahon et al. (1981) call it obligate mycotrophic. They assume that in this case 

effectiveness is always positive. In their opinion facultative mycotrophy leads to a shift of 

actual niche characteristics, if mycorrhiza is developed: in a facultative symbiosis mycorrhizae 

would either increase the acquisition of a limiting resource (e.g. P) or decrease it (e.g. carbon 

gain under low light). Allen (1991) supports this hypothesis. 

From our point of view, the hypothesis of MacMahon et al. (1981) is applicable in plant 

production with some considerations to be kept in mind. First, advanced industrial plant 

production is based on long selection processes of plant cultivars grown without consideration 

of mycorrhizal fungi. They are selected to be independent on mycorrhiza. Furthermore, longterm 

experiences normally led to optimized procedures and growing conditions during plant 

cultivation. The actual ecological niche of a cultivated intensively screened and selected plant 

cultivar therefore uses resources without any mycorrhiza. This circumstance leads to the 

impression that it is stress in production systems which leads to some dependency of useful 

plants. Deviations from the optimal growing system, suboptimal periods, and temporary 

depletion of resources, upcoming diseases and unknown limiting factors open the window for 

use of mycorrhiza in industrial plant production. If primary selections are used, wild 

collections tested or even plant cultivars with well documented host characteristics are 

produced, at least facultative mycotrophy should be assumed. 

A host’s classification as a facultative mycotrophic or an obligate symbiont is complicated by 

the multifactorial nature of an ecological niche. Important effects of mycorrhiza might be 

masked because of uninfluenced limiting factors in its cultivation system (Fig. 2): A host might 

514

e e.g. obligately dependent on mycorrhiza with respect to the survival under heavy metal 

stress, but because of light deficiency it might not grow better than non-mycorrhizal plants. 

[%] 

100 

80 

60 

40 

20 

0 

apparent effect hidden effect no effect 

A B C D E A B C D E A B C D E 

Ecofactors 

ecofactor strength mycorrhizal effect 

Figure 2. Relationship of limiting factors (lowest grey column) and apparent, hidden and 

no effect of AM. Mycorrhizal effects might be hidden by not influenced 

limiting factors. 

Our ability to predict mycorrhizal dependency of a host under specific conditions depends on 

the knowledge of stress tolerance characteristics and growth limiting factors of that host. The 

more experience a grower has, the better he can predict success of mycorrhizal application 

because the difference between actual niche and natural host niche defines the maximal 

mycorrhizal effectiveness. 

This stress definition follows Tsimilli-Michael and Strasser (2002). Stress should have a 

relative meaning, with non-stress as the reference condition, i.e. they consider stress as a 

deviation from non-stress situations. Stress adaptation is hence defined as a sequence of 

optimazation processes. Different adaptive strategies are employed to regulate different 

functional and structural parameters of the system. However, the environmental conditions 

never cease to manifest alterations and, thus, the system is perpetually undergoing stress - 

stress adaptation processes, searching and approaching harmony with its environment (Strasser, 

1988). 

Considering this stress concept it makes sense to take mycorrhiza into account when breeding 

useful plants for better stress tolerance. The model of Fig 2 indicates that mycorrhiza action is 

a multivariate trait and is involved in several pathways at the same time. Considering 

mycorrhiza in breeding activities should mean to speculate on synergistic effects: we should 

breed on limiting factors which cannot be overcome by the symbionts (Fig 2, “hidden effect” 

factor B) and we should only stop considering mycorrhiza when the status “no effect” (Fig 2) is 

515

eached. So, we should not breed for more mycorrhizal dependency, but we should integrate 

mycorrhiza until we know that the plant is independent of the symbionts, which – looking into 

the stress concept – is a long way to go. 

DISCUSSION 

Modern gardening and up-to-date plant production shows that the introduction of mycorrhizal 

inoculum is very useful (Feldmann, 2003). Green areas, gardens or parks and long-term 

conservation of artificial, man-made plant sociological formations can lead to AMF 

communities which are patchy distributed and of low diversity and low effectiveness 

(Feldmann, 1997). Growing media for roof tops and all substrates used for production of 

ornamentals, seedlings and cuttings are sterile and, therefore, free from mycorrhizal fungi. 

Overall, production and use of plants is characterized by a latent deficiency in symbioses 

leading to a higher stress susceptibility of facultative or obligate mycorrhiza dependent host 

plants. 

Mycorrhiza products are bound to carrier materials, mixed into growing media or fertilizers or 

encapsuled with seeds. This acceptance of the market reflects that breeding provided us with 

plants which are not well adapted to their subsequent environment. Of course, the expectation 

that breeding can adapt plants to thousands of variable conditions is unrealistic. However, in 

future the challenges of climate change and the need for yield increase require the use of all 

sources. Therefore, breeders have to recognize the importance of symbioses for their products. 

Breeders and inoculum producers follow the same approach. Breeders breed for better nutrient 

uptake and more stress tolerance of plants; inoculum producers develop mycorrhizal inoculum 

for increase of biomass as well as stress tolerance. Breeders still do not accept that mycorrhizal 

technology can help them to any great extent. However, it would be helpful if they were to 

provide simple information routinely: e.g. whether new plant cultivars form mycorrhiza in 

more than 20% of root length after inoculation. The costs for this analysis are low and the 

service could be out-sourced. The data could be collected in a freely accessible data bank to 

provide the community with such information. This would allow consultants to direct 

mycorrhizal use without time loss. Such basic information is missing in advisory services (Fig. 

3). 

In Fig. 3 relevant factors influencing mycorrhizal effectiveness are cited from various authors 

(see Allen, 1991). Genotypes of host and fungus form the mycorrhizal phenotype under the 

influence of concurrent environmental conditions. The mycorrhizal phenotype in relation to 

non-mycorrhizal host plants reflects the mycorrhizal effectiveness with regard to the evaluated 

effect. Roughly summarized, variability of environmental factors causes variability of 

effectiveness via qualitative and quantitative changes of inoculum characteristics, root 

characteristics or root colonization (see Feldmann, 1998b). 

For the micro-symbiont there are two quantitative aspects of major importance: the inoculum 

potential, i.e. the number of propagules or potential “colonising units“ of AMF inoculum and 

516

the AMF population composition. These two parameters of inoculum are influenced during 

technical inoculum production. The other parameters cited in Fig. 3 are processing factors 

realising the desired coincidence of the right developmental stage of roots/plants and infective 

micro-symbionts. High quality inoculum has to provide sufficient fungal material leading to 

desired effects with commercially reasonable effectiveness and is technologically no real 

problem (Feldmann and Grotkass, 2002; Feldmann and Schneider, 2008). 

host genotype 

resistance 

reactions 

root 

morphology 

developmental 

stage 

mycorrhizal 

dependency 

abiotic & biotic 

environmental factors 

root 

colonisation 

mycorrhizal 

effectiveness 

colonisation 

capacity 

coincidence 

basic AMF 

characteristics 

AMF genotype 

competition 

strenght 

inoculum 

potential 

AMF population 

composition 

Figure 3. Factors for the advisory service to meet maximum mycorrhizal effectiveness 

in practice: information on host genotype is a basic requirement 

Mycorrhizal technology developed over the past few years provides plant production with 

suitable commercial mycorrhizal inoculum for the inoculation of annual vegetables, 

ornamentals, perennial herbs, shrubs or trees (Feldmann and Schneider, 2008). Furthermore, 

inoculation methods for already established plants are now available and offer the possibility to 

include AMF into the design of integrated plant protection procedures (Feldmann, 2008). The 

flexibility of modern inocula allows the inclusion of the mycorrhizal technology to integrated 

plant protection systems as important biological phytosanitary factors (Feldmann et al., 2008). 

The two recently parallely co-existing research lines “breeding” and “mycorrhizal technology” 

should provide plant producers with easier access to information about the host genotypes. This 

will result in synergistic effects in the desired host stress adaptation in various environments. 

REFERENCES 

AI-Karaki G N and Al-Raddad A (1997). Drought stress and VA mycorrhizal fungi effects on 

growth and nutrient uptake of two wheat genotypes differing in drought resistance. 

Crop Res. (Hisar) 13, 245-257. 

517

Allen M F (1991). The ecology of mycorrhizae. Cambridge Studies in ecology; ISBN 0 521 

33531 0; pp184. 

Auge R M; Stodola A J W (1990). An apparent increase in symplastic water contributes to 

greater turgor in mycorrhizal roots of droughted Rosa plants. New Phytol. 115, 285- 

295. 

Azcon R, Ocampo JA (1981) Factors affecting the vesicular-arbuscular infection and 

mycorrhizal dependency of thirteen wheat cultivars. New Phytol 87:677-685 Barnes 

DK, Vance CP, Heichel GH, Peterson MA, Ellis WR (1988) Registration of a nonnodulation 

and three ineffective nodulation alfalfa germplasms. Crop Sci 28:721-722 

Azcon-Aguilar C; Barea J M (1996). Arbuscular mycorrhizas and biological control of soilborne 

plant pathogens. Mycorrhiza 6, 457-464. 

Azcon-Aguilar C; Jaizme-Vega M C; Calvet C (2002). The contribution of arbuscular 

mycorrhizal fungi to the control of soil-borne pathogens. In: S Gianinazzi, H Schuepp, J 

M Barea, K Haselwandter, eds. Mycorrhizal Technology and Agriculture. Birkhäuser- 

Verlag, Basel. 187-197. 

Balaji B, Poulin M J, Vierheilig H and Piche Y 1995 Responses of an arbuscular mycorrhizal 

fungus, Gigaspora margarita, to exudates and volatiles from the Ri T-DNAtransformed 

roots of nonmycorrhizal and mycorrhizal mutants of Pisum sativum L. 

Sparkle. Exp. Myco!. 19,275-283. 

Baon J B, Smith S E and Alston A M 1993 Mycorrhizal responses of barley cultivars differing 

in P effieiency. Plant Soil 157, 97-105. 

Barker S J, Stummer B, Gao L, Dispain I, O'Connor P J and Smith SE 1998 A mutant in 

Lyeopersicon esculentum Mil!. with highly reduced VA mycorrhizal colonization: 

isolation and preliminary characterisation. Plant J. 15,791-797. 

Bertheau Y, Gianinazzi-Pearson V, Gianinazzi S (1980) Developpement et expression de 

l'association endomycorhizienne chez le BIe 1. Mise en evidence d'un effet varietal. 

Ann Amelior Plant 30:67-78 

Bethlenfalvay G J (1992). Mycorrhizae and Crop Productivity. In: G J Bethlenfalvay, R G 

Linderman, eds Mycorrhizae in sustainable agriculture. ASA Publ. No. 54. Madison, 

USA. 1-28. 

Blair D A (1987) A comparative study of mycorrhizal associations between Glomus versiforme 

and roots of Lotus and Trifolium. MSc Thesis, University of Guelph, Guelph, Ontario 

Boyetchko S M and Tewari J P 1995 Susceptibility of barley cultivars to vesicular-arbuscular 

mycorrhizal fungi. Can. J. Plant Sei. 75, 269-275. 

Cheng Y, Dengsha B, Sun L, Feldmann F, Feng G (2008): Utilization of arbuscular 

mycorrhizal fungi during production of micropropagated potato Solanum tuberosum. 

In: Feldmann F, Kapulnik Y, Baar J:: Mycorrhiza Works, ISBN 978-3-941261-01-3; 

47-59. © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

Cordier C; Gianinazzi S; Gianinazzi-Pearson V (1996). Colonization patterns of root tissues by 

Phytophtora nicotianae var. parasitica related to reduced disease in mycorrhizal 

tomato. Plant Soil 185, 223-232. 

Cordovilla M P, Ligero F, Lluch C and Cordovilla M P 1995 Infiuence of hast genotypes on 

growth, symbiotic performance and nitrogen assimilation in faba bean (Vieia faba L.) 

under salt stress. Plant Soil 72, 289-297. 

Dag A, Ben-Gal A, Yermiyahu U, Zipori I, Wininger S, Kapulnik Y (2008): Response of olive 

trees under arid conditions to arbuscular mycorrhizal fungi. In: Feldmann F, Kapulnik 

518

Y, Baar J:: Mycorrhiza Works, ISBN 978-3-941261-01-3; 196-202. © Deutsche 


De la Bastide P Y, Krapp B R and Piche Y 1995 Population structure and mycelial phenotypic 

variability of the ectomycorrhizal basidiomycete Laccaria bieolor (Maire) Orlon. 


Dehne H -W (1982). Interaction between VAM-fungi and plant pathogens. Am. Phytopath. 

Soc. 72 (8), 1115-1119. 

Duc E, Trouvelot A, Gianinazzi-Pearson V, Gianinazzi S (1989) First report of nodnodulating 

plant mutants (myc-) obtained in pea (Pisum sativum L.) and Faba bean (Vicia faba L.). 

Plant Sci 60:215-222 

Estaun V, Calvet C, Hayman DS (1987) Influence of plant genotype on mycorrhizal infection: 

response of three pea cultivars. Plant SoiI103:295-298 

Estaún V, Pera J, Busquets M, Camprubí A, Parladé J, Calvet C (2008): Efficacy of arbuscular 

and ectomycorrhizal fungi in a rehabilitated limestone quarry in the Mediterranean area. 

In: Feldmann F, Kapulnik Y, Baar J:: Mycorrhiza Works, ISBN 978-3-941261-01-3; 

243-253. © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

Feldmann F (1997). Charakterisierung des Vorkommens von arbuskulären Mykorrhizasymbiosen 

in Pflanzennutzungssystemen des Braunschweiger Raumes. Braunschw. 

Naturkdl. Schr. 5 (2), 491-503. 

Feldmann F (1998a). Symbiontentechnologie in der Praxis: Arbuskuläre Mykorrhiza im 

Gartenbau. Thalacker-Medien, Braunschweig, ISBN 3-87815-109-8. 

Feldmann F (1998b). The Strain - Inherent Variability of Arbuscular Mycorrhizal 

Effectiveness: II. Effectiveness of single spores. Symbiosis. 25, 131-143. 

Feldmann F (2003). Weltweiter Handel mit Mykorrhizapilz-Inokulum – eine Risikoanalyse, 

Schriftenr. BMVEL Angew. Wissenschaft 498, 165-175 

Feldmann F (2008): Mycorrhiza for plant vitality: mycorrhizal fungi as factors of integrated 

plant protection in urban horticulture. In: Feldmann F, Kapulnik Y, Baar J: Mycorrhiza 

Works, ISBN 978-3-941261-01-3; 69-77. © Deutsche Phytomedizinische Gesellschaft, 


Feldmann F, Hallmann J, Wagner S, Long X-q, Yang R, Schneider C, Hutter I, Ceipek B, Feng 

G, Fan J, Zheng X, Wang C: (2008) Mycorrhizal fungi as biological components of the 

integrated cucumber production project BIOMYC – promising results for mycorrhizal 

technology transfer to horticultural practice. In: Feldmann F, Kapulnik Y, Baar J:: 

Mycorrhiza Works, ISBN 978-3-941261-01-3; 79-92. © Deutsche Phytomedizinische 


Feldmann F; Boyle C (1998). Concurrent development of arbuscular mycorrhizal colonization 

and powdery mildew infection on three Begonia hiemalis cultivars. Zeitschrift für 

Pflanzenkrankheiten und Pflanzenschutz. 105 (2), 121-129. 

Feldmann F; Grotkass C (2002). Directed inoculum production – shall we be able to design 

populations of AMF to achieve predictable symbiotic effectiveness? In: S Gianinazzi, H 

Schuepp, J M Barea, K Haselwandter, eds. Mycorrhizal Technology and Agriculture. 

Birkhäuser-Verlag, Basel. 261-280. 

Feldmann F, Schneider C (2008): How to produce arbuscular mycorrhizal inoculum with 

desired characteristics. In: Feldmann F, Kapulnik Y, Baar J: Mycorrhiza Works, ISBN 

978-3-941261-01-3; 292-310. © Deutsche Phytomedizinische Gesellschaft, 

Braunschweig, Germany; pp 292-311; 

519

Feldmann F; Hutter I; Niemann P; Weritz J; Grotkass C; Boyle C (1999). Einbindung der 

Mykorrhizatechnologie in die Heil- und Zierpflanzenproduktion sowie den Endverkauf. 

Mitteilungen der Biologischen Bundesanstalt. 363, 6-38. 

Feldmann F; Idczak E; Martins G; Nunes J; Gasparotto L; Preisinger H; Moraes V H F; 

Lieberei R (1995). Recultivation of degraded, fallow lying areas in central Amazonia 

with equilibrated polycultures: Response of useful plants to inoculation with VAmycorrhizal 

fungi. Angewandte Botanik. 69, 111-118. 

Feldmann F; Junqueira N T V; Lieberei R (1989). Utilization of vesicular-arbuscular 

mycorrhiza as a factor of integrated plant protection. Agriculture, Ecosystems and 

Environment. 29, 131-135. 

Forbes P J; Ellison C H; Hooker J E (1996). The impact of AMF and temperature on root 

system development. Agronomie, 16, 617-620. 

Garcia-Garrido J M; Ocampo J A (1988). Interaction between Glomus mosseae and Erwinia 

carotovora and its effects on the growth of tomato plants. New Phytol. 110, 551-555. 

Garcia-Garrido J M; Ocampo J A (1989). Effect of VA mycorrhizal infection to tomato on 

damage caused by Pseudomonas syringae. Soil Biol. Biochem. 21, 165-167. 

Gianinazzi-Pearson V 1996 Plant cell responses to arbuscular mycorrhizal fungi: getting to the 

roots of the symbiosis. Plant Cell 8, 1871-1883. 

Gianinazzi-Pearson V, Gollote A, Lherminier J, Tisserant B, Franken P, Dumas-Gaudot E, 

Lemoine M C, Van Tuinen D and Gianinazzi S 1995 Cellular and molecular 

approaches in the characterization of symbiotic events in functional arbuscular 

mycorrhizal associations. Can. J. Bot. 73, S526--S532. 

Graham JH, Eissenstat DM, Drouillard DL (1991) On the relationship between a plant's 

mycorrhizal dependency and rate of vesicular-arbuscular mycorrhizal colonization. 

Funct Ecol 5:773-779 

Hetrick BAD, Wilson GWT, Cox TS (1992) Mycorrhizal dependence of modern wheat 

varieties, landraces, and ancestors. Can J Bot 70:2032-2040 

Hooker J E; Jaizme-Vega M; Atkinson D (1994). Bio control of plant pathogens using 

arbuscular mycorrhizal fungi. In: S Gianinazzi, H Schuepp, eds. Impact of arbuscular 

mycorrhizas on sustainable agriculture and natural ecosystems. ALS, Birkhäuser- 

Verlag, Basel. 191-200. 

Kapulink Y, Kushnir U (1991) Growth dependeney of wild, primitive and modern cultivated 

wheat lines on vesicular-arbuscular mycorrhiza fungi. Euphytiea 56:27-36 

Karagiannidis N, Nikolaou N and Mattheou A 1995 Influence of three VA-mycorrhiza species 

on the growth and nu trient uptake of three grapevine roolstocks and one table grape 

cultivar. Vitis 34,85-89. 

Khan A G; Belik M (1995). Occurrence and ecological Significance of Mycorrhizal Symbiosis 

in Aquatic Plants. In: A Varma, B Hock, eds. 1995 Mycorrhiza. Springer Berlin. 627- 

668. 

Leyval C; Joner J; del Val C; Haselwandter K (2002). Potential of arbuscular mycorrhizal 

fungi for bioremidiation. In: S Gianinazzi, H Schuepp, J M Barea, K Haselwandter, eds. 

Mycorrhizal Technology and Agriculture. Birkhäuser-Verlag, Basel. 175-186. 

Linderman R G; Paulitz T C (1990). Mycorrhizal-rhizobacterial interactions. In: D Hornby, 

Long X, Cui W-D, Yong R, Feldmann F: (2008) Enhanced yield and disease tolerance of field 

cotton, field pepper and potted marigold following AMF inoculation. In: Feldmann F, 

520

Kapulnik Y, Baar J:: Mycorrhiza Works, ISBN 978-3-941261-01-3; 151-159. © 


MacMahon J A, Schimpf D J,Anderson D C, Smith K G, Bayn R L Jr (1981). An organismcentered 

approach to some community and ecosystem concepts. Journal of Theoretical 

Biology, 88, 287-307. 

Manske GGB (1989) Genetical analysis of the efficiency of VA mycorrhiza with spring wheat. 

Agric Ecosystems Environ 29:273-280 

Manske GGB (1990) Genetical analysis of the efficiency of VA mycorrhiza with spring wheat. 

1. Genotypical differences and a reciprocal cross between an efficient and non-efficient 

variety. In: Bassam EL et al. (ed) Genetical aspects of plant mineral nutrition. Kluwer, 

Dordrecht, pp 397-405 

Martin-Laurent F, Van Tuinen D, Dumas-Gaudot E, GianinazziPearson V, Gianinazzi S and 

Franken P 1997 Differential display analysis of RNA accumulation in arbuscular 

mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene. Mol. Gen. 

Genet. 256, 37-44. 

Mercy MA, Shivashankar G, Bagyaraj DJ (1990) Mycorrhizal colonization in cowpea is host 

dependent and heritable. Plant Soil121:292-294 

Nemec Sand Datnoff L 1993 Pepper and tomato cultivar responses to inoculation with Glomus 

intraradices. Adv. Hortic. Sci. 7, 161-164. 

Peterson R L, Bradbury S M (1995): Use of plant mutants, intraspecific variants, and non-hosts 

instudying mycorrhiza formation and function; In: Varma A, Hock B: Mycorrhiza. 

ISBN 3 540 58525 7; 157-180. 

Pinochet J; Calvet C; Camprubi A; Fernandez C (1996). Interactions between migratory 

endoparasitic nematodes and AMF in perennial crops: A review. Plant Soil. 185, 183- 

190. 

Pivonia S, Levita R, Cohen S Gamliel A, Wininger S, Ben-Gal A, Yermiyahu U, Kapulnik Y 

(2008):: Reducing the effects of biotic and abiotic stresses on pepper cultivated under 

arid conditions using arbuscular mycorrhizal (AM) technology. In: Feldmann F, 

Kapulnik Y, Baar J: Mycorrhiza Works, ISBN 978-3-941261-01-3; 203-215. © 


Pozo M J; Slezack-Deschaumes S; Dumas-Gaudot E; Ginaninazzi S; Azcon-Aguilar C (2002). 

Plant defense responses induced by arbuscular mycorrhizal fungi. In: S Gianinazzi, H 

Schuepp, J M Barea, K Haselwandter, eds. Mycorrhizal Technology and Agriculture. 

Birkhäuser-Verlag, Basel. 103-112. 

Rengel Z (2002). Breeding for better symbiosis. Plant and Soil 245, 147-162. 

Rosendahl C N; Rosendahl S (1991). Influence of VAMF (Glomus spp.) on the response of 

Cucumber (Cucumis sativus) to salt stress. Environ. Exp. Bot. 31, 313-318. 

Schmid T, Meyer J, Oehl F (2008):: Integration of Mycorrhizal Inoculum in High Alpine 

Revegetation. In: Feldmann F, Kapulnik Y, Baar J: Mycorrhiza Works, ISBN 978-3- 

941261-01-3; 265-275. © Deutsche Phytomedizinische Gesellschaft, Braunschweig, 

Germany 

Schneider C, Hutter I, Feldmann F (2008): Vitalizing symbionts in acclimatization of the 

medicinal plant Baptisia tinctoria (L.) Vent. – an established plant production factor for 

ten years. In: Feldmann F, Kapulnik Y, Baar J:: Mycorrhiza Works, ISBN 978-3- 

941261-01-3; 38-46. © Deutsche Phytomedizinische Gesellschaft, Braunschweig, 

Germany 

521

Schönbeck F (1987). Mykorrhiza und Pflanzengesundheit. Ein Beitrag zum biologischen 

Pflanzenschutz. Angew. Botanik. 61, 9-13. 

Schönbeck F; Dehne H -W (1979). Untersuchungen zum Einfluß der endotrophen Mykorrhiza 

auf Pflanzenkrankheiten. Z. Pflanzenkrankh. Pflanzenschutz. 86(2), 103-112. 

Slezack S; Dumas-Gaudot E; Paynot M; Gianinazzi S (2000). Is a fully established arbuscular 

mycorrhizal symbiosis required for bioprotection of Pisum sativum roots against 

Aphanomyces euteiches? Mol. Plant Microb. Interac. 13, 238-241. 

Stöppler H, Kölsch E, Vogtmann H (1990) Vesicular-arbuscular mycorrhiza in varieties of 

winter wheat in a low external input system. Biol Agric and Hortic 7:191-199 

Strasser R J (1988) A concept for stress and its application in remote sensing. In: Lichtenthaler 

H K (ed) Applications of chlorophyll fluorescence. Kluwer Academic Publishers pp 

333-337 

Takács T, Biró I., Németh T, Vörös I (2008):: Selection and application of infective and 

effective AMF strains for phytoremediation of metal contaminated soils. In: Feldmann 

F, Kapulnik Y, Baar J: Mycorrhiza Works, ISBN 978-3-941261-01-3; 254-264. © 


Toth R, Page T, Castleberry R (1984) Differences in mycorrhizal colonization of maize 

selections for high and low ear leaf phosphorus. Crop Sci 24:994-996 Toth R, Toth D, 

Starke D, Smith DR (1990) Vesicular-arbuscular mycorrhizal colonization in Zea mays 

affected by breeding for resistance to fungal pathogens. Can J Bot 68:1039-1044 

Trotta A; Varese G C; Gnavi E; Fusconi A; Sampo S; Berta G (1996). Interactions between the 

soil-borne root pathogen Phytophtora nicotianae var. parasitica and the arbuscular 

mycorrhizal fungus Glomus mosseae in tomato plants. Plant 185, 199-209. 

Tschirner E, Brandt C, Deininger D: Benefits of Mycorrhiza Inoculation of Trees and Bushes 

at Roadsides. In: Feldmann F, Kapulnik Y, Baar J: (2008): Mycorrhiza Works, ISBN 

978-3-941261-01-3; 282-290. © Deutsche Phytomedizinische Gesellschaft, 


Tsimilli-Michael and Strasser (2002). Mycorrhization as a stress adaptation procedure. In: S 

Gianinazzi, H Schuepp, J M Barea, K Haselwandter, eds. Mycorrhizal Technology and 

Agriculture. Birkhäuser-Verlag, Basel; 199-210. 

Tsimilli-Michael M, Eggenberg P, Biro B, Köves-Pechy K, Vörös I, Strasser R J (2000) 

Synergistic and antagonistic effects of arbuscular mycorrhizal fungi and Azospirillum 

and Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa, probed by the 

chlorophyll a polyphasic fluorescence transient O-J-I-P. Applied Soil Ecology 15:169- 

182 

Vierheilig H, Ocampo JA (1991a) Susceptibility and effectiveness of vesiculararbuscular 

mycorrhizae in wheat cultivars under different growing conditions. Biol Fertil Soils 

11:290-294 

Vierheilig H, Ocampo JA (1991b) Receptivity of various wheat cultivars to infection by V Amycorrhizal 

fungi as influenced by inoculum potential and the relation of V AMeffectiveness 

to succinic dehydrogenase activity of the mycelium in the roots. Plant 

Soil133:291-296 

Xavier L J C and Germida J J 1998 Response of spring wheat cultivars to Glomus clarum NT4 

in a P-deficient soil containing arbuscu1ar mycorrhizal fungi. Can. J. SoH Sei. 78, 481- 

484. 

522

Matyjaszczyk E: Registration of Plant Protection Products in Poland and the Problem of Resistence. In: Feldmann 

F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 523-529; ISBN 978-3- 


12-1 Registration of Plant Protection Products in Poland and the Problem of 

Resistence 

Matyjaszczyk E 

Plant Protection Institute - National Research Institute, ul. W. Węgorka 20, 60-318 Poznań, 

Poland 

Email: E.Matyjaszczyk@ior.poznan.pl 

ABSTRACT 

To prevent the development of resistance, the rotation of active substances (ASs) is 

necessary. At present, however, the number of ASs permitted for use in plant 

protection in the European Union is being continually reduced. In Poland, this is 

accompanied by the reductions of approved uses of plant protection products 

(PPPs). The farmers associations are not strong or determined enough to order the 

studies and apply for the widening of the scope of use of PPPs on their own. As a 

result, for most minor crops there is a very limited number of PPPs available, and 

the rotation of ASs is sometimes impossible. This creates a very serious potential 

for resistance development. 

INTRODUCTION 

Due to accession to the European Union (01.05.2004), Poland implemented a number of EU 

law regulations concerning among others, agriculture and plant protection. The Directive 

91/414 regarding placing plant protection products on the market has had the biggest impact on 

the registration of plant protection products in Poland has. The result of implementing EU rules 

regarding plant protection, which seems to be the most noticeable for Polish farmers so far, is 

the reduction of the number of PPPs placed on the Polish market, as well as the reduction of 

the available ASs of PPPs. The reality of numerous ASs and PPPs being withdrawn from the 

market can be observed in all EU member states. The reason for this lies in the review carried 

out in the EU to ensure that the ASs of PPPs used are safe for human, animals and the 

environment. Withdrawal of the ASs which are not safe enough is obviously beneficial and it 

was the intended effect of the review. Lack of safety however is not the only reason of 

523

withdrawal. AS can be withdrawn from use in plant protection in the territory of the EU due to 

two reasons: 

• the producer is not able to prove that it is safe for the environment 

• the producer has not supported the AS through the review process 

Producers often do not support ASs through the review process because of the high costs of the 

review. This means that part of the ASs is being withdrawn from use in EU, purely from 

financial reasons. Excessive withdrawals of ASs are not indifferent for environment because 

such can influence the increase of probability of resistance development. 


The PPPs as well as ASs placed on the Polish market and withdrawn from use in Poland in the 

period from EU accession to the end of 2008 (01.05.2004-31.12.2008) were analysed. The 

sources of data were information of the Polish Ministry of Agriculture and Rural Development. 

The possibilities of further changes were shown on the basis of current and prepared legal acts. 

DISCUSSION 

In January 2009 there were 798 plant protection products placed on the Polish market (Ministry 

2009). The most numerous groups of plant protection products in Poland are herbicides and 

fungicides. From the date of accession, the products withdrawn significantly outnumbered the 

new registered PPPs (see Table 1). The decline of PPPs placed on the market is noticeable in 

all groups of plant protection products, especially in case of herbicides – since Poland’s 

membership in the EU, the number of herbicides placed on the Polish market decreased by 59. 

In the case of zoocides (e.g. insecticides, nematicides, molluscicides, rodenticides and 

acaricides) there was decline of 44 PPPs. The number of fungicides and other PPPs decreased 

by 7 and 4 respectively. In the Table 1 the PPP containing a number of AS for which the 

derogation for use in Poland was granted are not included (among them 15 PPP qualified for 

use in ecological farming in Poland). PPP with derogation will be finally withdrawn from the 

Polish market in 2010. The decrease of PPP placed on the Polish market is a problem for 

farmers. A matter of concern is also the fact that the products withdrawn had, in most cases, 

been present on the Polish market for many years and the farmers knew them well, given that 

many of them were produced in Poland. Following the withdrawals, farmers need advice on 

what can be used in their place. In the case of major crops, there are usually effective products 

available which can substitute for the ones withdrawn. The problem is that they are often 

considerably more expensive. 

Reduced number of PPP available is accompanied by the reductions of approved uses. In 

Poland (as in most member states) the PPP are registered for 10 years. After this period the 

producer must apply for the re-registration. The re-registration is granted after documentation 

assessment according to current requirements. As the requirements change to re-register the 

PPP for their former uses, the producer is very often called upon to supply new study results. 

524

The lack of this study results translates into the withdrawal of the particular uses from the 

label. It can be estimated that about 70% of PPP re-registered in Poland have fewer approved 

uses than previously. Numerous minor uses are excluded from the labels because the producers 

of PPPs have no financial interest to finance additional study results which are required 

according to new registration procedure. The farmers associations are, on the other hand, not 

influential or determined enough to comission the studies and apply for the widening of the 

scope of use of PPPs on their own. As a result, for most minor crops there is a very limited 

number of PPPs available, and the rotation of ASs is sometimes impossible. This creates a very 

serious potential for resistance development. 

Table 1. Changes in number of plant protection products placed on the Polish market 

Products Fungicides Herbicides Insecticides Others Total 

New registrations 62 57 18 27 164 

Re-registrations 33 30 14 18 95 

Withdrawals 69 116 62 31 278 

Source: Personal elaboration of data from Polish Ministry of Agriculture and Rural Development 

Active 

Substances 

withdrawn 

Active 

Substances 

registered 

Table 2. The active substances totally withdrawn from use in plant protection in Poland 

and placed for the first time on the Polish market since EU accession 

(01.05.2004-31.12.2008) 

Herbicides Fungicides Insesticides Others 

alachlor 

benomyl azinphos-methyl lecithin 

atrazine 

dichlofluanid benfuracarb 

cycloate 

fentin acetate carbofuran 

dichlorprop triadimefon carbosulfan 

dimethipin tridemorph cyhexatin 

fluoroglycofen triforine diazinon 

haloksyfop-R ofurace fenitrothion 

imazapyr oxadixyl malathion 

imazethapyr oxine-copper methomyl 

naptalam 

oxydemeton- 

prometryn 

methyl 

sethoxydim 

tebufenozide 

simazine 

thiodicarb 

terbacil 

triazamate 

trifluralin 

trichlorfon 

bifenox benthiavalicarb clothianidin 1-methylcyclo 

triticonazole spirodiclofen 

cyfluthrin 

propene 

Source: Personal elaboration of data from Polish Ministry of Agriculture and Rural Development 

525

When discussing the impact of available PPP on possibility of resistance development, we 

should, however, not only consider the number of products placed on the market. The number 

of available ASs is probably more relevant. Table 2 presents the ASs totally withdrawn from 

the Polish market during the analyzed period as well as the ASs placed on the market for the 

first time. It should be stressed that the AS is listed as withdrawn only if: 

− at least one PPP containing this AS was registered in Poland 

− the withdrawal is complete, it means for all PPP containing this AS the decision about 

withdrawal was given. 

The reason of withdrawal of all ASs listed in Table 2 was lack of inclusion to the Annex 1 of 

the Directive 91/414. However the AS to be withdrawn with valid derogation for use in Poland 

are not listed. 

The AS is listed as registered in Table 2 if: 

− a PPP containing this AS was placed on the Polish market during analyzed period 

− no other PPP containing this AS had been earlier registered in Poland. 

Upon analyzing Table 2 we can observe that during discussed period, ASs withdrawn from use 

in plant protection in Poland (39) very significantly outnumbered the newly registered (7). The 

biggest gap we can observe in case of herbicides where 15 ASs had been totally withdrawn 

from use in Poland while only a single new one had been registered. In the analyzed period, 14 

ASs used in zoocides had been totally withdrawn while 3 were newly registered, while in case 

of fungicides 9 were withdrawn and 2 registered. “Other” plant protection products are the only 

group where the number of ASs withdrawn and registered was equal. It must be mentioned, 

however, that several ASs from this group (like garlic extract or grapefruit extract) are in the 

period of derogation and will be withdrawn from use in Poland in the year 2010. It should be 

stressed that Table 2 does not demonstrate the complete results of the EU review for two 

reasons: 

− the review is not yet completed (it will probably conclude in the end of 2009) 

− due to the time consuming legislation procedure not all results of withdrawal decisions 

given by EU Comission in 2008 were visible in Poland in December 2008. 

This suggests that in subsequent years further reduction of number of ASs available for Polish 

farmers are expected. Reduction of accessible AS decrease possibility of their rotation and thus 

increase probability of resistance development. 

The possibility of AS rotation is so far sufficient in most major crops in Poland. In minor crops 

however sometimes there is no possibility of rotation. It can be a serious problem for Polish 

agriculture, because Poland is a country with a big number of minor crops (vegetables, herbs, 

fruits and some minor agricultural crops). Minor crops are often grown by small farmers, so 

that lack of protection in this case is likely to also create some social problems. The detailed 

comparative assessment of possibilities of protection of three crops: winter wheat, carrot and 

mint in Poland in years 2002 and 2008 was performed (Matyjaszczyk 2009). The number of 

PPP registered to control each group of significant harmful organisms in selected crops was 

526

analysed. It was found that in the mentioned period the possibility of protection of all crops 

was significantly reduced (with the exception of disease control in winter wheat). In the season 

2009 the sufficient (considering the resistance preventing strategy) rotation of ASs on carrot 

plantations will be not possible in Poland (Dobrzański 2008). Carrots are grown in Poland on 

over 30,000 ha of land.. 

As was previously mentioned numerous minor crops are withdrawn from labels of PPPs during 

the re-registration process. It means that they can be not legally used in the protection of this 

crops in spite of the fact that they are remain being placed on the market. Farmers of minor 

crops use the available AS without regard to resistance preventing strategy because if they 

would like to rotate the AS they would break the law. It is a very serious potential reason of 

resistance development. In such a case it is no question of if but rather of when the resistance 

will develop. It is only a matter of time. 

It should be also stressed that the weeds and very often also pests or diseases can be harmful to 

numerous crops. It means that the harmful organism which has developed resistance on the 

minor crop plantation will propagate and negatively influence other crops. Since the harmful 

organism is resistant to certain AS the number of ASs available to control this particular 

organism is reduced. In turn, the possibility of AS rotation on the area where resistant harmful 

organism occurs is reduced. Such circumstances increase the probability of further resistance 

development – it means development of resistance against more than one group of ASs (thus 

creating super resistant species) as well as development of resistance of the other organisms 

occurring in this area. This two phenomena (developing super resistant species and resistance 

development among susceptible species) can take place in parallel. 

Public opinion in the European Union has a reluctant attitude towards PPPs and demands 

higher standards of protection for humans, as well as for the environment. To fulfill this 

demand, the Thematic Strategy for Sustainable use of Pesticides is to be implemented in all EU 

Member States. The new law aims to advance harmonization rules regarding PPPs in Member 

States, providing improved protection of humans and the environment against the negative 

influence of PPPs. It is worth emphasizing that in the new law, as well as in the Directive 

91/414, the objective of protecting human or animal health and the environment has a priority 

over the objective of improving plant production. New rules regarding the registration of plant 

protection products will probably contribute to further withdrawals of AS. 

The British authority responsible for registration of plant protection products has prepared the 

assessment of the impact of proposed Regulation of European Parliament and the Council 

concerning the placing of plant protection products on the market on crop protection in UK 

(Assessment 2008) - it can be estimated that the numbers for Poland will be similar. According 

to the assessment and considering the latest version of the proposal (European 2009) the total 

reductions of available ASs will be 5 to 15% and taking into consideration the groups of ASs: 

− Insecticides 6-10% 

− Fungicides 8-32% 

− Herbicides 4-10% 

527

This signals further withdrawals of ASs in subsequent years. As a result the resistance of 

harmful organisms against PPPs will probably become increasingly important problem in EU 

agriculture. 

How can we diminish the risk of resistance development? Faced with the reality of a 

decreasing number of accessible ASs, the task is not simple. There are however activities 

undoubtedly both beneficial and practicable. The problem with sufficient minor crops 

protection seems to be an important potential source of resistance in Poland. The scope of use 

of a number of products already registered could be widened following studies regarding safety 

and efficacy of PPPs in minor crops. As producers of PPPs are not interested in performing 

these studies, for financial reasons it seems that perhaps some kind of governmental 

intervention (like decreasing of registration fees for the PPP with minor crops in labels or 

founds for efficacy studies of PPP for minor crops) would be favourable. Another crucial issue 

is the reliable, up-to-date and accessible information about resistance development. The 

appropriate international websites do exist, but the data is usually uploaded on a voluntary 

basis, so the information is often not complete. In the light of growing risk of resistance 

development the measures should be found to ensure full information. The training for the 

advisors and farmers regarding resistance preventing strategy should be also ensured, 

especially on the areas where resistance has been confirmed. 

Changes following the requirements of the Directive 91/414 and the new Regulation 

concerning the placing of plant protection products on the market contribute to improvement of 

the environment. Not only farmers, but also all EU residents will benefit from this. On the 

other hand, because of the review of ASs, a significant number of ASs and PPPs is being 

withdrawn from use, some of them purely for financial reasons. The list of PPPs available in 

Poland, especially for minor crops has been significantly reduced, and further reductions are 

expected. The reductions will be continued following the provisions of the new Regulation. 

This can contribute to the development of resistance. Initiatives to prevent the resistance are 

necessary. Some of them require governmental support. The available (although probably not 

sufficient) methods are among others: appropriate trainings, ensuring full information 

regarding resistance and widening the palette of products for protection of minor crops. 

REFERENCES 

Assessment of the impact on crop protection in the UK of the “cut-off criteria” and substitution 

provisions in the proposed Regulation of the European Parliament and of the Council 

concerning the placing of plant protection products on the market. Pesticides Safety 

Directorate, May 2008, 46 pp 

European Parliament legislative resolution of 13 January 2009 on the Council common 

position for adopting a regulation of the European Parliament and of the Council on the 

placing of plant protection products on the market and repealing Council Directives 

79/117/EEC and 91/414/EEC. Text adopted P6_TA-PROV(2009)0011 

http://www.europarl.europa.eu/sides/getDoc.do?pubRef=-//EP//TEXT+TA+P6-TA- 

2009-0011+0+DOC+XML+V0//EN (date of access 29.01.2009) 

528

Dobrzański A (2008). Problemy z ochroną marchwi przed chwastami. Owoce, Warzywa, 

Kwiaty 23, 26-28. 

Matyjaszczyk E (2009). The consequences of changes on the list of plant protection products 

placed on the Polish market for selected crops. Progress in Plant Protection Book of 

Abstracts, 81-82. 

Ministry of Agriculture (2009) List of plant protection products placed on the Polish market 

http://www.bip.minrol.gov.pl/DesktopDefault.aspx?TabOrgId=647&LangId=0 (date of 

access 15.01.2009) 

529

Consmüller N, Beckmann V, Petrick M: The Adoption of Bt-Maize in Germany: An Econometric Analysis. In: 



12-2 The Adoption of Bt-Maize in Germany: An Econometric Analysis 

Consmüller N 1 , Beckmann V 1 , Petrick M 2 

1 

Humboldt Universität zu Berlin, Department of Agricultural Economics and Social Sciences, 

Philippstr. 13, 10099 Berlin, Germany 

2 

IAMO, Department of Agricultural Policy, Theodor-Lieser-Str. 2, 06120 Halle, Germany 

Email: nicola.consmueller@agrar.hu-berlin.de 

530 

Abstract 

In this study, we theoretically and empirically investigate the determinants of 

Bt-maize adoption in German regions. Specifically, we ask how the regulatory 

framework, the farm structures as well as the socio-political environment of GM 

expansion in Germany have influenced regional adoption rates. Following a 

description of the relevant legal and economic framework in Germany, we develop 

theoretical hypotheses concerning regional variation in Bt-maize adoption and test 

them econometrically with unique data at the Federal States (Länder) and County 

(Landkreis) level. The study provides evidence that the adoption of Bt-maize in 

different regions is positively affected by the amount of maize grown per farm and 

by the European Corn Borer (ECB) infestation rates. There is also some evidence 

that the Bt-maize adoption is negatively affected by the activities of the anti-GMO 

movement and the establishment of GMO-free zones. 

INTRODUCTION 

Since 2005 Bt-maize resistant to ECB (European Corn Borer, Ostrinia nubilalis HÜBNER) has 

been allowed for commercial cultivation in Germany. Subsequently, adoption has been picking 

up in the East German Federal States, notably in Brandenburg, Mecklenburg-Western 

Pomerania, Saxony, and Saxony-Anhalt. These are dominated by large farm structures and are 

among the least densely populated areas of Germany. Although ECB infestation is reported to 

be a serious problem in the southern parts of Germany as well (e.g. Bavaria and Baden- 

Württemberg) (Beckmann & Schleyer, 2006), Bt-maize adoption rates have been much lower 

there, rarely exceeding 10 ha per State (BVL, 2008). At the same time, public controversy 

concerning the principal desirability of genetically modified (GM) crops in German agriculture

has gained new momentum, including partially violent destruction of fields sown with Btmaize 

by members of anti-GM movements. These opponents argue that GM crop production 

may pose unpredictable risks to human health and the environment, and that the technology 

may favour undesirable farming structures and practices (www.gentechnikfreie-regionen.de). 

Based on two regional panel data sets, we analyse the determinants of varying adoption rates in 

Germany. Given the paucity of rigorous analysis of Bt-maize adoption in Europe, it is the first 

systematic study that analyses the influence of structural and political determinants of adoption 

in a multiannual setting. Following a description of the relevant legal and economic framework 

in Germany in the second section, we develop theoretical hypotheses concerning regional 

variation in Bt-maize adoption in the third section. The econometric methodology to test them 

with unique data at the State (Länder) and County (Landkreis) level is developed in the fourth 

section. The fifth section presents the results and the final section concludes. 

LEGAL FRAMEWORK FOR GROWING BT-MAIZE IN GERMANY 

Following the EU legislation (2001/18/EC, 1829/2003, 1830/2003 and 2003/556/EC), 

Germany incorporated rules of ex-ante regulation such as a general code of Best Management 

Practice (BMP) as well as the creation of a public site register and ex-post liability rules (joint 

and several liability) into the German Genetic Engineering Act (GenTG) in 2004, coming into 

force in January 2005. During the first three years of commercial cultivation (2005 until 2007), 

the German Genetic Engineering Act (GenTG) combined rather flexible ex-ante regulations 

with strict liability rules because concrete and scientifically based safety measures to keep 

cross-pollination of maize below the labelling threshold of 0.9% were not agreed upon yet 

(GenTG 2006). This legal gap was initially filled by recommendations of the seed industry 

which suggested the installation of 20 m conventional hybrid maize buffer zones around Btmaize 

fields. However, during the first years little experience existed regarding the possible 

risk of outcrossing, the risk of economic damages and finally the risk of being held liable. 

Thus, the fist years were characterised by high uncertainty and little practical experience. 

In 2008, isolation distances for GM maize of 150 m and 300 m respectively were defined by 

the new regulation on GM crop production (Gentechnik-Pflanzenerzeugungsverordnung, 

GenTPflEV), which are, however, not relevant for our data analysis. However, as a matter of 

flexibility, the new GenTG allows farmer to enter into private arrangements to reduce the 

minimum distance requirements. All additional costs of ex-ante regulations and ex-post 

liability which emerge from the GenTG have to be carried by the GM farmer exclusively. This 

includes field registration in a national cadastre, compliance with security measures, and 

liability in case of damage (Consmüller et al., 2008). Only the costs of testing for GM presence 

have to be borne by the non-GM farmer. 

531

DETERMINANTS OF BT-MAIZE CULTIVATION 

Against the regulatory background for GM crop cultivation in Germany and the significance of 

the anti-GM movement as well as from literature review we hypothesise that a number of 

factors affect the benefits and costs of Bt-maize adoption. These include: ECB infestation rates, 

the maize area cultivated per farm, the ownership rights in land, the importance of organic 

farms in a region, the share of GM-free regions and the strength of the anti-GM activists and 

finally time (Beckmann & Wesseler 2007; Beckmann et al. 2006). While some have been 

discussed in the literature, several others have not been considered in adoption research so far: 

1 ECB infestation rates 

From a farm management perspective, potential infestation with ECB should be the prime 

reason for the adoption of Bt-maize. Resistance against this pest is the single benefit of this 

maize variety and the profitability of Bt-maize adoption is crucially determined by the 

opportunity costs of doing so. High adoption rates are therefore to be expected in those regions 

where ECB has been a recurrent problem. Literature on the adoption of Bt-maize in the U.S. 

reveals that the cultivation is confined to those areas with heavy infestation rates of the ECB. 

We assume that this also applies to Germany where high pest incidence is reported from the 

Oderbruch region in Brandenburg (Schröder et al. 2007) and parts of Baden-Württemberg and 

Bavaria (Degenhardt et al. 2003). To test the effect of ECB infestation rates, meaningful data 

on economically relevant infestation rates are required. One plausible measure is the frequency 

of infestation because it depicts the heaviness of infestation in terms of the percentage of 

infested plants and thus the need for the farmer to take action according to the economic 

threshold. Unfortunately, corresponding data for Germany are unavailable at the Federal States 

level. We therefore had to confine our analysis to counties in Brandenburg, for which data 

were collected for the years 2005 to 2007 and published by the LVLF (Landesamt für 

Verbraucherschutz, Landwirtschaft und Flurneuordnung). 

2 Maize acreage per farm 

Assuming that ECB infestation is a recurring problem, the second important factor affecting 

the economic benefits of adopting Bt-maize is the amount of maize planted on a farm. Since 

Bt-maize is an embodied technology, viz. incorporated in the new product, the economic 

benefits of Bt-maize increase with the extent of maize cultivation. The incremental benefits of 

growing Bt-maize compared with the untreated control are estimated up to 93 € per ha 

(Degenhardt et al. 2003) 1 . Thus, without considering the costs of the regulatory environment 

and assuming a constant infestation, the benefits would increase linearly with the area of maize 

cultivated on the farm. In this case, the Bt-technology would be scale neutral, but the overall 

1 Brooks (2007) has reviewed the gross margin for several European countries. In Spain, gross margin benefits of 

growing Bt-maize were estimated between 67 and 330 € per ha; in France between 98 and 120 € per ha. 

532

incentives to adopt Bt-maize would increase with the size of maize acreage per farm. However, 

ex-ante regulations and ex-post liability of coexistence introduce additional costs, some of 

which may have a fixed cost character (Beckmann & Wesseler 2007). Messean et al. (2006) 

report additional on-farm costs for creating buffer zones of between 60 and 78 € per ha 

depending on the size of the Bt-maize field, the width of the buffer zone and the adoption rate 

of Bt-maize in the region. The authors further note that the smaller the GM fields the higher the 

on-farm costs per ha caused by the establishment of buffer zones. Until the recent amendment 

of the German Genetic Engineering Act in 2008, best management practices for the cultivation 

of Bt-maize were defined as ‘all measures to reduce the probability of cross-pollination’ (e.g. 

buffer zones, safety distances etc., GenTG 2006) and buffer zones of 20 m were the most 

common measure to facilitate coexistence. However, the installation of buffer zones or safety 

distances requires a certain amount of cultivation area depending on the required width. In the 

case of a 20 m buffer zone, this theoretically means that for planting only 1 ha of Bt-maize, a 

field of nearly 2 ha in total will be needed (see Figure 1). 

Figure 1. Requirements for total field size according to different safety distances 

Recent legal restrictions (the GenTPflEV 2008) have tightened these requirements even more. 

Minimum distance is set at 150 m to conventionally and 300 m to organically cultivated 

adjacent fields. For planting 1 ha of Bt-maize the minimum necessary field size will hence 

increase up to 16 and 49 ha, respectively. Bt-maize adoption is thus strongly dependent on the 

possibility to create large maize fields. While regulations before 2008 were less strict, this 

factor will gain importance in the future. Besides the buffer zones and the minimum distance 

requirements, the ex-ante regulation may also include fixed costs, such as the registration of 

Bt-plantation in the public site register and informing neighbours. 

Summing up, the ex-ante regulation and ex-post liability rules introduced in Germany turn a 

size neutral technology into a size dependent one, leading to the hypothesis that larger farms or 

more precisely farms that plant more maize are more likely to adopt, given that maize is 

533

subjected to ECB infestation. The influence of the farm size on the adoption of GM crops has 

been discussed intensively by Fernandez-Cornejo & McBride, 2002, Gómez-Barbero et al., 

2008 and some of these authors also report a significant influence of the actual farm size on the 

adoption of Bt-maize. 

3 Ownership rights 

Farmers interested in Bt-maize adoption face another potential obstacle if they are not the 

owner of their land. There are recent attempts of landlords to prohibit the cultivation of 

Bt-maize, because they fear liability claims in case of cross-pollination or a long term negative 

side effects on their property. Beyond this, many municipalities have already banned the 

cultivation of GM crops from their land and the same holds true for the Protestant Church in 

Germany (Evangelische Kirche in Deutschland, EKD) (e.g. http://www.epv.de/node/3371). 

Taking this development into account, we suppose that the adoption of Bt-maize is 

significantly influenced by land ownership rights, favouring farms with more land in individual 

ownership. 

4 Importance of organic farms in a region 

Organic production is obliged to refrain from any use of genetic engineering and is legally 

protected against negative side effects of GM crop cultivation by larger distance requirements 

since 2008. However, the significance of organic farming may also affect other conventional 

farmers in the neighbourhood in their adoption decision. There are mainly two reasons why a 

farmer might not adopt Bt-maize if his neighbours are organic farms: 1) higher likelihood to 

face economic losses due to liability claims because organic produce receives a premium price 

in Germany and 2) the need to create large maize stands (at least 49 ha for planting 1 ha Btmaize) 

to keep the prescribed distance of 300 m to his neighbour(s). Although the larger 

distance to organic farming was not required from 2005 to 2008, in practice farmer kept larger 

distances to organic farmers (Consmüller et al., 2008). Therefore we would expect that a 

higher share of organic farming leads to a lower adaptation rate. 

5 Number and size of GMO-free zones 

An interesting phenomenon of resistance to Bt-maize in Germany and Europe is the 

establishment of GMO-free zones (Gentechnikfreie Regionen), which has been observed since 

2003. GMO-free zones are cooperative arrangements among farmers, land owners or 

downstream enterprises. This initiative has been supported by the German Association for 

Environmental Protection and Nature Conservation (BUND) in order to prohibit GM crops on 

German fields. To become a member of a GMO-free zone, the farmer must contractually 

refrain from planting GM varieties on his farm. In those regions where significant initiatives 

for GM free zones are emerging, the social pressure on farms intending to plant Bt-maize 

might be high. Thus a region with a large share of GMO-free zones may have a negative 

534

influence on the adoption of Bt-maize. At the same time, it is possible that the establishment of 

GMO-free zones is itself driven by the expansion of Bt-maize in a given region. Hence, it is an 

empirical question whether Bt-maize expansion and the establishment of GMO-free zones 

reinforce or drive out each other. 

6 Significance of anti-GMO activists 

Many environmental groups (e.g., BUND 2 , Greenpeace) are actively involved in the anti-GM 

movement and support the establishment of GMO-free zones. Since farmers have to report GM 

field location and size three months before seeding to the competent authority, Greenpeace and 

other groups provide detailed information on the location of fields or organise campaigns in 

order to exert pressure on the GM farmers. In past years, destructions of GM fields have often 

taken place by members of the German anti-GM movement. A high density of activists in 

nature groups could therefore be an indicator for GM-opposition in a region and is expected to 

affect the Bt-maize adoption negatively. 

7 Time 

As for other technologies, adoption of Bt-maize is affected by the time dimension. The benefits 

and costs of Bt-maize adoption are subject to high uncertainty. On the one hand, the ECB 

infestation rates may vary from year to year; on the other hand the risk for farmers being held 

liable for economic damages due to outcrossing is very difficult to estimate. The experiences 

gained over time may reduce the uncertainty and lead to increasing adoption in the following 

period. 

Summing up, the ECB infestation rates and the maize grown per farm are the two factors 

generating the benefits of Bt-maize adoption, while the regulatory and social environment 

impose costs that have partly a fixed cost character. In regions with a high share of rented land, 

organic agriculture, GM-free regions and many anti-GMO activists we expect the adoption rate 

to be lower. 

ECONOMETRIC ANALYSIS 

In order to test the previous hypotheses, we utilize panel datasets at the Federal States and 

County level. These datasets include regionally aggregated information about GMO adoption 

and various structural and socioeconomic variables on an annual basis between 2005 and 2007. 

They cover the early history of commercial Bt-maize cultivation in Germany. Data was 

obtained from the Federal Statistic Office in Germany, the BVL 3 , the statistical service of the 

2 

Friends of the Earth Germany 

3 

Bundesamt für Verbraucherschutz und Lebensmittelsicherheit 

535

churches and from the webpage of the GMO-free regions. The following analysis takes only 

into account the years in which Bt-maize cropping was legally possible and subject to the first 

regulatory environment, that is from 2005 until 2007. As outlined above, the legal environment 

changed significantly in 2008. 

Our data allows principally straightforward testing of the previous hypotheses, by using a 

linear regression model: 

y = x 

′ 

β + ε , i = 1, �, N, t = 1, � , T. 

, (1) 

it it it 

where y it is hectares under Bt-maize cultivation for given regions and years, x it is a vector of 

determinants, β the vector of coefficients that is to be estimated, and ε it a conventional, 

identically and independently distributed error term. Estimated confidence intervals for β 

allow to statistically test the above hypotheses. N is the number of regions and T the number of 

years. As two modifications of the general model in (1) we estimate a pooled OLS with period 

effects (equation 2) and a fixed effects model (equation 3) either with or without period effects. 

y = α + λ + x 

′ 

β + ε (2) 

it t it it 

y = α + λ + x 

′ 

β + ε (3) 

it i t it it 

The dependent variable y it indicates the Bt-maize cultivation in May of the respective year. 

Although farmers are required by law to register the sowing area of Bt-maize early in the year, 

normally not later than end of January, they often adjust their plans until sowing in end of April 

or beginning of May. In the last years, usually more than 30% of the initially announced Btmaize 

area was withdrawn. This may have different reasons, among others that neighbours 

adjust their cultivation plans or that GM farmers yield to the pressure of anti-GMO activists. 

Thus, from a decision making point of view the opportunities and constraints of the current 

year must be taken into account. For this reason, the explanatory variables x it originate mainly 

from the same year. Data from the Agriculture Structure Survey are gathered usually in 

March/April. Data from the GM-free zones are usually summarised in June. 

Among the explanatory variables, the ECB infestation rate and the maize area per farm are the 

most important factors determining the private benefits of Bt-maize cultivation. Unfortunately, 

systematic and complete annual data on ECB infestation rates is missing. At the Federal States 

level, the Federal Government of Germany provided information on infestation rates only for 

the year 2005. The indicator used displays the maize area in ha, where at least 10% of the 

plants are infested by the ECB. In contrast, the Federal State of Brandenburg provides annual 

information on the frequency of ECB infestation for the Counties (data source LVLF). This 

indicator describes the percentage of plants infested by ECB but does not provide exact 

information on the infested area. In the analysis we make use of both indicators. 

Because of the regional aggregation of the data, the maize acreage per farm can only be 

calculated as a regional average, i.e. the maize area divided by the number of farms. Although 

not all farms cultivate maize the indicator provides information on possible farm-level 

536

profitability to plant Bt-maize. It is important to note that the aggregate data on Bt-maize 

adoption is the effect of individual decision making. Form an individual point of view, the 

infested maize area on the farm counts and not the total area in the region. If the total infested 

area within a region is high, but the individual infested area small, no Bt-maize will be planted, 

as private benefits do not outweigh the costs. The Bt-maize acreage in a given region may 

grow if Bt-maize growing farms extend their cultivation or if new farms start growing Btmaize. 

Unfortunately, annual data on maize cultivation is only available for the Federal States 

level. For the County level in the State of Brandenburg information on maize plantation exists 

only for 2007. 

As it was argued, the Bt-maize cultivation may be negatively affected by the significance of 

organic farming, amount of rented land, GMO-free zones and the anti-GM movement. The 

significance of organic farming is indicated by the share of organic farming in the Utilisable 

Agricultural Area (UAA). For the ownership in land, we used the share of owned land in the 

UAA, and for the GMO-free zones the share of declared GM-free land in total UAA. Finally as 

an indicator for the strength of the anti-GM movement we used share of BUND members in the 

total population. The data availability differs between the Federal States and the County level. 

The share of rented land and the number of environmental activists are not available for 

Brandenburg Counties. We therefore estimate different models for the two aggregation levels. 

There are two methodological problems in estimating consistent parameters in (1). First, as 

[Bt-maize area in the Federal States] shows, the various States differ by orders of magnitude in 

their cultivation levels of Bt-maize. 4 One likely reason is the principal differences in farm 

structures between East and West Germany. Furthermore, there may be important latent 

variables having an impact on y it , such as climatic and soil conditions, or unobserved abilities 

and preferences of farmers and consumers. Second, several variables in x it may not be 

independent of the Bt-maize cultivation decisions of farmers. Notably, this could be the case 

for the maize area planted per farm and for the establishment of GMO-free zones which were 

probably be set up in response to impending or actual Bt-maize cultivation in a given region. 

Both problems will make ε it no longer independently distributed, so that estimates of β are 

inconsistent. 

We address the first of these concerns by including regional fixed effects in the regression 

model. As a consequence, β will capture only the effect of relative changes in x it on y it , 

independent of the absolute level of Bt-maize cultivation. To the extent that they are time 

invariant, also the effects of all latent determinants of y it will in this way be eliminated. In 

order to filter out the effects of changes in the overall environment that are identical for all 

farms, such as annual price variation, we also include year dummies in the model. 

4 

There are five observations with zero Bt-maize in the dataset. While this indicates slight censoring of the 

dependent variable, we ignore this problem in the following. 

537

The second concern is addressed by estimating an instrumental variable regression (2SLS) for 

the Federal States level. The idea is to first estimate for maize area per farm and GMO-free 

zones which endogenise these variables. It uses predictions from a first stage instrumental 

variable equation to estimate the equations of the system in the second stage. The results of this 

model are presented in addition to a more conventional single equation pooled OLS model. As 

data on maize cultivation and environmental activists is missing for Brandenburg Counties, we 

present single equation results for this model only. 

RESULTS 

Estimation results for German Federal States are displayed in Fehler! Verweisquelle konnte 

nicht gefunden werden.. Model A presents the results from a pooled ordinary least squares 

(OLS) model with time effects, whereas model B shows an instrumental variable (IV) model 

where the maize area per farm is instrumented with the average farm size per region. This 

model accounts for the possible endogeneity of the maize area per farm. Model C presents a 

fixed-effects model that also takes into account possible regional and time effects. 

538 

Table 1 Regression estimates for Bt-maize cultivation in the German Federal States 

Explanatory variables Pooled OLS 

period effects 

(A) 

Bt-maize a( ECB-infested area 

(ha in 2005) 

Coefficient pvalue 

Pooled IV 


(B) 


Fixed Effects and 


(C) 


Mean 

values 

-0.002 0.327 -0.001 0.564 - - - 28707 

Maize area per farm (ha) 25.06 *** 0.001 18.10 ** 0.018 184.61 *** 0.003 7.67 

Land in cultivators’ 

ownership (% of UAA) 

-2.098 0.603 -2.623 0.524 54.59 0.268 31.67 

Organic farming area 

(% of UAA) 

GMO-free zones 

(% of UAA) 

BUND members 

(% of population) 

13.33 0.464 19.20 0.305 54.59 0.561 5.36 

2.91 0.790 0.030 0.998 45.39 0.245 5.05 

347.06 0.233 256.41 0.387 -5055.3 * 0.075 0.36 

Year 2006 (dummy) 40.16 0.613 42.55 0.599 -94.45 0.205 0.33 

Year 2007 (dummy) 149.30 * 0.070 158.15 * 0.060 -116.36 0.260 0.33 

Constant -234.78 0.182 -178.21 0.319 -1716.7 0.365 

Adjusted R² 0.391 0.369 0.811 

Notes: Source: Authors’ calculations. a Dependent variable is Bt-maize per region in the same year (ha). Model 

(B) uses farm size in ha as an instrument for maize cultivation. ** (***): significant at 5% (1%) level. N=39 for 

all regressions.

The results show that the main factor affecting the plantation of Bt-maize is the average maize 

area grown on the farm. This result is robust over the whole range of models calculated. 

Surprisingly, the ECB infested area does not have a significant impact. There may be several 

reasons for this: First, the information of the ECB infestation originates from 2005 and is not 

updated for 2006 and 2007. Thus, the dynamics of the infestation rates could not be taken into 

account. Second, in Federal States where the infested area is large in total, but small per farm, 

farmers are unlikely to adopt Bt-maize because of the fixed regulatory (and social) costs. This 

seems to be the case in particular for Bavaria and Baden-Württemberg where in the ECB 

infested area is estimated with 180,000 and 60,000 ha, but the adoption of Bt-maize is only 5.8 

and 7.2 ha (2007) respectively. The farm size and the maize cultivation per farm are among the 

smallest in Germany. Land ownership, organic farming, GMO-free regions and BUND 

members have no effect in models A and B. In model C, the increase over time in the number 

BUND members has a significant negative impact on the adoption of Bt-maize. This suggests 

that the anti-GM groups have a negative impact on Bt-maize adoption. 

Table 2. Regression estimates for Bt-maize area in Brandenburg Counties 

Explanatory variables Pooled OLS with 


(A) 


Fixed Effects 

(B) 

Fixed effects 

with period effects 

(C) 


Mean 

values 


ECB Infestation (frequency) 6.072 *** 0.000 3.505 * 0.074 2.378 0.247 20.48 

Organic farming area 

(% of UAA) 

-1.019 0.467 11.432 0.627 5.341 0.822 10.88 

GMO-free zones 

(% of UAA) 

-3.228 0.924 -8.664 * 0.052 -9.155 ** 0.043 0.98 

Year 2006 37.600 - 58.452 * 0.097 0.33 

Year 2007 24.373 - 39.726 0.279 0.33 

Const. -55.214 -112.21 0.664 -55.066 0.832 

Adjusted R² 0.416 0.529 0.340 

Notes: Dependent variable is Bt-maize area in subsequent year (ha). Model (B) includes 13 county dummies, 

model (C) 13 county and two year dummies. *, **, ***: significant at 10, 5 and 1% level. N=42 for all 

models. 

Source: Authors’ calculations. 

The results for Brandenburg Counties are shown in Fehler! Verweisquelle konnte nicht 

gefunden werden.. Certain variables were not available, such as maize per farm (which was 

only available for 2007), land ownership and the members of BUND. However, the 

information on the ECB infestation goes more into the details as they provide yearly indicators 

for the frequency of infestation. The main interest here is whether infestation with ECB affects 

Bt-maize adoption in the following year. The pooled OLS model (A) as well as the fixed 

539

effects model (B) demonstrates a positive effect, as expected, which is significantly different 

from zero at least at the 1 and 10 percent level respectively. However, the effect vanishes once 

year dummies are included in the fixed effects model. It follows from a closer inspection of the 

data (not shown in the table) that relative changes of adoption rates in Brandenburg Counties 

follow previous year infestation rates with ECB rather well. Even so, as both infestation and 

adoption rates are uniformly low in the first year of our sample, the model cannot statistically 

discriminate between a general macro effect and an effect of ECB infestation if year dummies 

are included. Interestingly, the increasing size of GMO-free zones has a statistically negative 

effect in model B and C on the Bt-maize adoption rates in Brandenburg. 

CONCLUSIONS 

Our analysis shows that the regional differences in Bt-maize adoption are affected by 

agricultural structures and the activities of the anti-GMO movement. The regulatory 

environment in Germany introduces additional fixed and variable cost to adopters of Bt-maize. 

Although Bt-maize is a scale neutral technology controlling for damages caused by the 

European Corn Borer (ECB) the additional fixed and variable costs transform the technology 

into a scale dependent one. As the empirical analysis of panel data at the Federal States level 

show, the maize area grown per farm is the single most important factor explaining regional 

and temporal variance in Bt-maize adoption. At the Federal States level no relationship could 

be identified between the ECB infestation rates and the Bt-maize adoption. One main reason 

seems to be that farms with little maize acreage resign completely from Bt-maize adoption 

even if they face high ECB infestation rates. In contrast, at the Brandenburg County level the 

ECB infestation frequency turns out to be an important factor explaining the adoption of Btmaize. 

Brandenburg, however, is characterised by large-scale maize farming, where the size of 

maize strands are unlikely to constrain Bt-maize adoption. 

Surprisingly, other factors such as land ownership and organic agriculture do not explain the 

regional and temporal variation of Bt-maize adoption on the Federal State level. However, 

there is some indication that anti-GMO activists and GMO-free zones have a negative impact 

on Bt-maize adoption. Whereas at the level of the Brandenburg Counties the increasing size of 

GMO-free zones constrains the adoption of Bt-maize, this could not be confirmed for the level 

of the Federal States. 

REFERENCES 

2001/18/EC: Directive 2001/18/EC of the European Parliament and of the Council of 12 March 

2001 on the deliberate release into the environment of genetically modified organisms 

and repealing Council Directive 90/220/EEC. 

1829/2003: Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 

22 September 2003 on genetically modified food and feed. 

1830/2003: Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 

22 September 2003 concerning the traceability and labelling of genetically modified 

540

organisms and the traceability of food and feed products produced from genetically 

modified organisms and amending Directive 2001/18/EC. 

2003/556/EC: Commission Recommendation of 23 July 2003 on guidelines for the 

development of national strategies and best practices to ensure the coexistence of 

genetically modified crops with conventional and organic farming. 

GenTG: Gentechnikgesetz in der Fassung der Bekanntmachung vom 16. Dezember 1993 

(BGBl. I S. 2066), zuletzt geändert durch Artikel 1 des Gesetzes vom 1. April 2008 

(BGBl. I S. 499) 

GenTPflEV: Gentechnik-Pflanzenerzeugungsverordnung vom 7. April 2008 (BGBl. I S. 655) 

Beckmann V; Schleyer C (2006). Neue Formen der Kooperation von Landwirten bei der 

Befürwortung und Ablehnung der Agro-Gentechnik. Agro-Gentechnik im ländlichen 

Raum: Potenziale, Konflikte und Perspektiven. Forum für interdisziplinäre Forschung. 

Berlin-Brandenburgische Akademie der Wissenschaften. Dettelbach, J.H. Röll. 

Beckmann V; Soregaroli C; Wesseler J (2006). Coexistence Rules and Regulations in the 

European Union. Amer. J. Agr. Econ. 88 (5), pp. 1193-1199. 

Brookes G (2007). The benefits of adopting genetically modified, insect resistant (Bt) maize in 

the European Union: first results from 1998-2006 plantings. PG Economics Ltd. 

Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL), Standortregister: 

http://194.95.226.237/stareg_web/showflaechen.do (date of access: 15.09.2008) 

Consmüller N; Beckmann V; Schleyer C (2008). Koordination und Kooperation beim Bt- 

Maisanbau in Brandenburg – eine explorative Untersuchung betrieblicher Strategien der 

Koexistenz. Berichte über Landwirtschaft 86 (2), pp. 242-261. 

Degenhardt H; Horstmann F; Mülleder N (2003) Bt-Mais Anbau in Deutschland: Erfahrungen 

mit dem Praxisanbau von 1998 bis 2002. Mais 2, pp. 75-77. 

Fernandez-Cornejo J; McBride W D (2002). Adoption of Bioengineered Crops. Agricultural 

Economic Report No. 810. 

Gentechnikfreie Regionen: www.gentechnikfreie-regionen.de (date of access: 15.09.2008). 

Gómez-Barbero M; Berbel J; Rodríguez-Cerezo E (2008). Adoption and performance of the 

first GM crop introduced in EU agriculture: Bt maize in Spain. JRC Scientific an 

Technical Reports. 

LVLF (2007). Bericht des Landesamtes für Verbraucherschutz, Landwirtschaft und 

Flurneuordnung zur Überwachung des Anbaus von Bt-Mais MON810 im Jahr 2007. 

www.mluv.brandenburg.de/cms/media.php/2335/btmais07.pdf (date of access: 

15.09.2008) 

Messean A; Angevin F; Gómez-Barbero M; Menrad K; Rodríguez-Cerezo E (2006). New case 

studies on the coexistence of GM and non-GM crops in european agriculture. Joint 

Research Centre; Europäische Kommission; Institute for Prospective Technological 

Studies. 

Schröder G; Goetzke G; Kuntzke D (2006). Perspektiven der Kontrolle des Maiszünslers 

(Ostrinia nubilalis Hbn.) mit Insektiziden – Versuchsergebnisse aus dem Oderbruch. 

Gesunde Pflanzen 58, pp. 143-151. 

541

Schiemann J, Devos Y, Lheureux K: Environmental Risk Assessment of Transgenic Plants Resistant to Biotic and 

Abiotic Factors. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 


12-3 Environmental Risk Assessment of Transgenic Plants Resistant to 

Biotic and Abiotic Factors 

Schiemann J 1 , Devos Y 2 , Lheureux K 2 

1 Julius Kühn Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for 

Biosafety of Genetically Modified Plants, Erwin-Baur-Str. 27, D-06484 Quedlinburg 

2 European Food Safety Authority (EFSA), GMO Unit, Parma, Italy 

email: joachim.schiemann@jki.bund.de 

INTRODUCTION 

Before genetically modified (GM) plants resistant to biotic and abiotic factors are tested in 

experimental field releases or placed on the market they have to undergo a rigorous 

environmental risk assessment (ERA). For experimental field releases, ERA is performed by 

national authorities, befor placing on the market by the European Food Safety Authority 

(EFSA) in consultation with EU Member States. EFSA is the keystone of EU risk assessment 

regarding food and feed safety. In close collaboration with national authorities and in open 

consultation with its stakeholders, EFSA provides independent scientific advice and clear 

communication on existing and emerging risks. The EFSA Panel on genetically modified 

organisms (GMOs) provides independent scientific advice on the safety of (i) GMOs such as 

plants, animals and micro-organisms, on the basis of Directive 2001/18/EC on the deliberate 

release into the environment of GMOs, and (ii) GM food and feed, on the basis of Regulation 

(EC) No 1829/2003 on GM food and feed. The GMO Panel carries out risk assessments in 

order to produce scientific opinions and advice for risk managers. Its ERA work is based on 

reviewing scientific information and data in order to evaluate the safety of a given GMO. This 

helps to provide a sound foundation for European policies and legislation and supports risk 

managers in taking effective and timely decisions. 

REGULATORY VERSIGHT OF GM PLANTS AND THEIR DERIVED FOOD AND 

FEED PRODUCTS 

Process-based versus product-based approach 

In Europe, a process-based system was put in place for the regulation of GMOs as the breeding 

techniques used for their production were considered new and raised specific safety concerns. 

542

A GMO is thus mainly characterised by the breeding techniques used to produce it and is 

defined as an organism in which the genetic material has been altered in a way that does not 

occur naturally by crossing and/or natural recombination (EC 2001). Breeding techniques 

falling under the EU GMO definition are (1) recombinant nucleic acid breeding techniques 

(involving the formation of new combinations of genetic material by the insertion of nucleic 

acid molecules produced by whatever means outside an organism, into any virus, bacterial 

plasmid or other vector system and their incorporation into a host organism in which they do 

not naturally occur but in which they are capable of continued propagation); (2) breeding 

techniques involving the direct introduction into an organism of heritable material prepared 

outside the organism (including micro-injection, macro-injection and micro-encapsulation); 

and (3) cell fusion (including protoplast fusion) or hybridisation techniques where live cells 

with new combinations of heritable genetic material are formed through the fusion of two or 

more cells by means of methods that do not occur naturally. In-vitro fertilisation, natural 

transformation processes (such as: conjugation, transduction, transformation), and polyploidy 

induction are currently excluded from the GMO definition. 

In the United States (US) and Canada, a product-based approach is followed for the regulation 

of GMOs (Macdonald & Yarrow 2003; McHughen & Smyth 2008; Smyth & McHughen 

2008). Legislations focus on risks of products, and not the breeding techniques of production, 

as genetic engineering per se is not considered inherently risky. Because the focus is on novel 

traits or attributes introduced into a plant, rather than the method of production, plants and their 

derived food and feed products are regulated under the existing regulatory system. 

Regulatory framework for GMOs in the EU 

In the early 1990s, two European Directives for the use of GMOs were adopted to ensure the 

protection of human and animal health and the environment, and to guarantee consumers’ 

freedom of choice without misleading consumers/users. Directive 90/219/EEC, which has been 

amended by Directive 98/81/EEC, regulated the contained use of GM (micro)organisms, whilst 

Directive 90/220/EEC regulated the deliberate release of GMOs into the environment, covering 

both the release for research purposes (part B) and for commercial use as or in products (part 

C). This triad reflects the stepwise process GM plants go through, beginning with experiments 

under contained use (e.g. laboratory, greenhouse) through experimental release, up to the 

placing on the market. According to the step-by-step principle, the containment of GMOs can 

be reduced and the scale of release increased gradually, if assessment of earlier steps indicates 

that the next step can be taken. 

Since 15 May 1997, Regulation No (EC) 258/97 – the so-called Novel Food Regulation – 

removed food products derived from GM plants from the Deliberate Release Directive’s scope. 

This Regulation covered risk assessment procedures, marketing and labelling of all types of 

novel food products including those produced by new plant breeding techniques such as 

genetic engineering, as well as food without a history of safe use in the EU. 

543

On 17 October 2002, Directive 2001/18/EC replaced (the older) Directive 90/220/EEC. With 

it, the precautionary principle was explicitly adopted as a guide, risk assessment criteria were 

broadened to include direct, indirect, immediate, delayed and cumulative long-term adverse 

effects, post-market environmental monitoring became obligatory, the need for a common 

methodology for the environmental risk assessment was established, an additional rigorous risk 

assessment of antibiotic resistance marker genes was introduced, the existing labelling 

provisions applying to GM food were extended to all marketed products containing GMOs, the 

general concept of traceability at all stages of commercialisation was introduced, the 

transparency in the decision-making process was increased, the consultation of the public 

became mandatory in the authorisation procedure, the possible consultation of an ethics 

committee was confirmed, and the implementation of national cultivation registers that record 

the locations where GM plants have been grown was required. 

Adding on to Directive 2001/18/EC, Regulation No (EC) 178/2002 laid down general 

principles of food law and procedures in food and feed safety. With this Regulation, the 

application of the precautionary principle is further extended to risk analysis of all food and 

feed products in the EU, whether or not of GM-origin. In response to a multiple wave of food 

crises that caused considerable concerns in European publics about food safety and the ability 

of regulatory authorities to fully protect consumers, the European Food Safety Authority 

(EFSA) was created as a European-wide risk assessment body. By providing ‘independent, 

objective and transparent’ science-based advice, EFSA aims to ensure a high level of consumer 

protection and to restore and maintain confidence in the EU food supply. 

Since 18 April 2004, Regulation No (EC) 1829/2003 on GM food and feed covers the 

commercialisation and risk assessment of GM food and feed such as food/feed containing or 

consisting of, food/feed produced from, and food/feed containing ingredients produced from 

GMOs, as well as seed-propagating material. Prior to this date, approvals for human food use 

were required under the Novel Food Regulation, whereas feed use was assessed under 

Directive 2001/18/EC and its predecessor. The amended approval procedure is centralised 

around EFSA and based on a ‘one door–one key’ approach whereby all commercial uses can 

be covered in a same GM crop market dossier. Moreover, it also introduces the need for a GM 

crop market dossier to cover both food and feed uses, as it avoids market approval for a single 

use in case a product is likely to be used both for food and feed uses. Regulation No (EC) 

1830/2003 complements, clarifies and makes operational some of the labelling and traceability 

objectives of previous legislations. 

RISK ASSESSMENT PRINCIPLES 

Interplay of risk assessment, risk management and risk communication 

GMOs and their derived food and feed products are generally subjected to a risk analysis 

before they can be commercialised (Craig et al. 2008; Paoletti et al. 2008). In the EU, the risk 

analysis consists of risk assessment, risk management and risk communication. In risk 

544

assessment, potential adverse impacts associated with a specific activity are scientifically 

characterised on a case-by-case basis, whilst in risk management, policy alternatives to accept, 

minimise or reduce the characterised risks are weighed and, if needed, appropriate prevention 

and control options are selected. Because risk managers and regulators rely on risk assessments 

to make an informed decision on whether or not to approve a certain use of a GM plant, it 

should explain clearly what assumptions have been made during the risk assessment, and what 

is the nature and magnitude of uncertainties associated with the characterised risks. The 

decision whether a certain risk is acceptable and/or tolerable under a particular set of 

conditions is not part of the risk assessment itself, as this choice is not only based on scientific 

criteria, but also involves political, social, cultural and economic considerations. Theoretically, 

there is a functional and temporal separation between risk assessment and risk management in 

order to reduce any conflict of interest and to protect the scientific integrity of risk assessment 

(Johnson et al. 2007). Risk communication is defined as an interactive exchange of information 

and opinions on risk throughout risk analysis, running between risk assessors, risk managers 

and other interested parties. It includes the explanation of risk assessment findings and of the 

basis on which risk management decisions are made (EFSA 2006). 

Even though there are considerable differences between countries in regulatory requirements 

for GM plants, environmental priorities (including the preservation of biodiversity) as well as 

risk terminology, most risk assessments of GM plants follow a science-based assessment 

process that estimates the level of risk through comparison with a non-GM counterpart (Hill 

2005; Paoletti et al. 2008). In addition, regulatory requirements involve consideration of a 

range of issues relevant to the overall risk assessment in order to determine the impact of the 

GM plant on human/animal health and the environment relative to the non-GM plant, and thus 

its relative safety (Conner et al. 2003; Craig et al. 2008). Some of these elements are discussed 

in the next section. 

Risk assessment methodology and terminology 

Despite the considerable variation among risk assessment frameworks for GM plants regarding 

risk assessment steps, risk assessment generally comprises several sequential steps: (1) 

problem formulation as beginning; (2) hazard assessment that examines potential hazards and 

their magnitude; (3) exposure assessment that covers levels and likelihood of exposure; and (4) 

integrative risk characterisation in which the magnitude of consequences and the likelihood of 

occurrence are integrated (EFSA 2006). In the EU, the consideration of mitigation options such 

as post-market monitoring is not included as a fifth step in the risk assessment framework, as 

risk assessment is kept separated from risk management (Hill 2005). The terms hazard and risk 

are often interchangeably used in the EU (see e.g. Johnson et al. 2007), but have different 

meanings. The term hazard is associated with the potential of an agent or situation to cause 

adverse effects. It refers to an inherent property of that agent or situation. Risk is recognised as 

a function of the probability and severity of an adverse effect occurring to human and animal 

health or the environment following exposure to a hazard, under defined conditions. 

545

Problem formulation 

In order to identify the areas of greatest concern or uncertainty relating to risks, each risk 

assessment begins with the identification and formulation of the problem, usually in the context 

of regulatory decision-making (Hill & Sendashonga 2003; Wolt et al. 2009). The problem 

formulation phase involves defining environmental assessment endpoints, which are explicit 

and unambiguous targets for protection, and developing a methodology that will help to direct 

the risk characterisation and to produce information that will be relevant for regulatory 

decision-making. According to the US Environmental Protection Agency, this is generally 

done on the basis of a conceptual model and an analysis plan (US EPA 1998). The information 

that is considered during problem formulation takes many forms, including published scientific 

literature, expert opinion, stakeholder deliberation, and data developed by the applicant and 

submitted to the regulatory authority as part of a market registration dossier (Romeis et 

al.,2008). As such, existing knowledge of the system (plant–stressor–environment–hazard– 

exposure) is summarised during the problem formulation. If the level and quality of the 

available information is high, the risk assessment can build on existing knowledge, in turn 

reducing the number of risk hypotheses that will need to be tested for characterising the risk. In 

the following, we focus on environmental risk assessment of GM plants. 

Assessment endpoints 

Assessment endpoints are operationally defined by an ecological entity (i.e. arthropod natural 

enemies) and attributes of that entity (i.e. regulation of arthropod pest populations) that could 

potentially be impacted by the GM plant or its associated farm management practice (stressor) 

and that require protection from harm (Suter 2000). It is not an abstract goal such as ecosystem 

health or sustainability, but a real, operationally definable property of a component of the 

environment that reflects management or protection goals set by public policy. A typical 

assessment endpoint is the abundance and species richness of certain groups of organisms, in 

case protection of biodiversity is a management goal (Romeis et al. 2008). Because arthropod 

natural enemies fulfil relevant ecological functions by contributing to the natural regulation of 

arthropod pest populations within crop fields in agricultural landscapes, they can be identified 

as the entity to be preserved with the biological control functions they perform as attribute 

(Sanvido et al. 2008). 

Once assessment endpoints have been set, the environmental quality to be preserved needs to 

be defined. To allow regulatory decision-making, assessment endpoints should be defined as 

far as possible using measurable criteria, so that change in these endpoints can be identified. 

This also includes defining the magnitude and both the spatial and the temporal scales relevant 

for the entity and the attribute to be preserved. The magnitude describes to what extent the 

environmental quality should be preserved (or above what threshold a change would be 

considered a disturbance in environmental quality). The spatial and temporal scales are the 

habitats in which the environmental quality and the period during which the environmental 

quality should be preserved, respectively (Sanvido et al. 2008; Storkey et al. 2008). 

546

Conceptual model 

The conceptual model describes the consequential exposure scenario that links GM plant 

deployment to the assessment endpoint (valued entity). Thereby, key relationships are 

described between the GM plant, the valued entity, pathways of exposure through which the 

GM plant may affect the valued entity either directly or indirectly (= exposure profile), and 

potential impact of the GM plant to the environment. The conceptual model includes the 

available information on the nature of the stressor, its proposed use (including the intended 

scale of cultivation), reasonable exposure profiles, and potential responses of the assessment 

endpoint as a result of exposure. A well-structured conceptual model in which the components 

of the system are detailed will allow the identification and formulation of relevant risk 

hypotheses that are necessary to make assumptions and predictions about how a stressor could 

affect an assessment endpoint (Raybould 2006; Nickson 2008). It is important to bear in mind 

that risk hypotheses are not null hypotheses, but rather proposed answers to reasonable 

questions about how the assessment endpoint(s) will respond to the stressor(s) (Raybould 

2007; Nickson 2008; Storkey et al. 2008). Conceptual models can take an array of forms going 

from simple statements towards complex flowcharts and diagrams. 

Analysis plan 

The last step of the problem formulation comprises an analysis plan, in which data needed and 

the approach to be taken for data acquisition and synthesis are delineated for testing the risk 

hypotheses. Hence, scenarios defined in the conceptual model are placed in the context of an 

analysis plan. Two important aspects included in the analysis plan are the selection of measures 

to be used in the risk assessment (measurement endpoints) and the prioritization of the data 

needed. These measurement endpoints cover properties of the GM plant, its transgenic 

proteins, or both, and usually constitute estimates of hazard or exposure (Raybould 2006). A 

measurement endpoint defines the indicator of change in the assessment endpoint that will 

actually be recorded as part of comparative study of the environmental impact of a GM plant or 

its associated management practice (Storkey et al. 2008). 

The prioritizing of testing enables to allocate human and financial resources in a proper way 

(Qi et al. 2008), so that only essential data for characterizing the risk are collected (Raybould 

2006). It is during the planning phase that decisions are made about the most appropriate ways 

to measure the response of each assessment endpoint to the GM plant. It is, for instance, 

important to realize that for practical reasons not all potentially exposed terrestrial arthropods 

can be considered for regulatory testing (Romeis et al. 2008). Therefore, it is necessary to 

select appropriate species that can be tested effectively under laboratory conditions or that are 

available in sufficient numbers in the field to give statistically meaningful results (Gathmann et 

al. 2006; Todd et al. 2008). This selection of species is based on several criteria: ecological 

relevance, susceptibility to known or potential stressors (sensitivity and exposure), 

anthropocentric value that is usually defined in public policy through management goals, and 

testability (Todd et al. 2008). Hence, the risk assessment may consider species with special 

547

aesthetic or cultural values or species of conservational importance and that are classified as 

threatened or endangered. The number and type of species that are to be tested will depend 

upon the risk hypotheses generated during the problem formulation (Romeis et al. 2008). Once 

specific measurements are chosen and given a priority, appropriate methods of measurement 

are selected and noted in the analysis plan (US EPA 1998). 

The information from the problem formulation and the processes described above is the crucial 

starting point for risk assessments, as it enables detecting effects that indicate a potential risk in 

a structured and logic way. Having a properly constructed analysis plan based on a conceptual 

model that is clearly linked to assessment endpoints helps to guide the collection of relevant 

data useful for a risk assessor to evaluate hazard and exposure and ultimately estimate and 

characterize risk. Moreover, it helps to make the risk assessment process transparent and 

comprehensive and thus to allow regulatory decision-making. In contrast, poor problem 

formulation in risk assessments may fail to identify the most important questions to be solved 

and can lead to the collection of data that might be irrelevant for demonstrating the safety of a 

GM plant (Raybould 2006). 

Risk assessment principles and concepts 

Several principles and concepts are to be considered during the risk assessment of GM plants. 

Risk assessment of GM plants should (1) be science-based where quantitative information is 

available and use qualitative information in the form of expert judgment; (2) use a comparative 

approach; (3) be case-specific; (4) be iterative and examine conclusions already made based on 

new information; and (5) follow a tiered approach. 

Comparative risk assessment and familiarity concept 

According to the comparative risk assessment concept, the importance of risks posed by a GM 

plant is placed in the context of risks posed by current non-GM comparators (e.g. non-GM 

recipient or parental organism). As such, differences between the GM plant and comparator are 

established. The underlying assumption of this comparative assessment approach for GM 

plants is that traditionally-bred plants have a history of safe use for the consumer or animals 

and the environment, and familiarity for the consumer. The concept of familiarity is based on 

the fact that most GM plants are developed from crop plants, the biology of which is wellknown. 

The knowledge about the non-GM plant, gained through experience over time, can 

therefore be used in a risk assessment to establish differences associated with the genetic 

modification and the subsequent management of the GM plant. According to the Organisation 

for Economic Co-operation and Development, familiarity will derive from the knowledge and 

experience available from conducting a risk analysis prior to scale-up of any new plant line or 

crop plant variety in a particular environment, and from previous applications for similar 

constructs and traits in similar or different crop plants (OECD 1993). However, it is important 

to bear in mind that familiarity is not an endpoint in risk assessment and does not necessarily 

548

mean safety. If differences between the GM plant and comparator have been identified, it needs 

to be defined whether these differences have any significance for the assessment endpoints 

(Raybould 2007). 

Case-by-case principle 

According to the case-by-case principle, the source and target environments, biological and 

ecological characteristics of a GM plant, the scale and frequency of deliberate release, and the 

interactions among these elements should be considered when performing an environmental 

risk assessment (Andow & Zwahlen 2006; Garcia-Alonso et al. 2006). 

Iterative and adaptive 

It is recognised that an environmental risk assessment is framed within the scientific 

knowledge available at the time it is conducted, and that regulatory decisions must be made 

acknowledging that these shortcomings may not be resolved. Therefore, under current EU 

legislation, it is recommended to describe these scientific uncertainties, which generally relate 

to possible cumulative and long-term risks due to the large-scale exposure of different 

environments to GM plants when grown at a larger scale over long periods (EFSA 2008). In 

this respect, post-market environmental monitoring (PMEM) of GM plants, which became 

mandatory under current EU legislation, allows for the collection of additional data during the 

commercialisation phase of a GM plant. The scientific knowledge derived from the monitoring 

of GM plants, experiences gained from their cultivation, and any other new knowledge 

(generated through, for instance, biosafety research) will provide valuable information for risk 

assessors who will use this information for continually updating environmental risk 

assessments and reducing remaining uncertainties. 

PMEM of GM plants is mandatory in all applications for deliberate release submitted under 

Directive 2001/18/EC and Regulation (EC) No 1829/2003, and aims at (1) studying any 

possible adverse effects of the GM plant identified in the formal pre-market risk assessment 

procedure, and (2) to identify the occurrence of adverse effects of the GM plant or its use 

which were not anticipated in the environmental risk assessment (Sanvido et al. 2005; EFSA 

2006). 

Risk assessments are always iterative in the sense that regulatory decisions are temporary, 

reversible and adaptable in the light of new information that becomes available. Under 

Directive 2001/18/EC, the duty of re-examination has been strengthened by limiting the 

duration of market consent to a maximum period of ten years. 

Tiered approach 

An environmental risk assessment is generally conducted in a tiered manner, where 

information collected in lower tiers directs the extent and nature of the experimentation 

549

conducted in higher tiers. Thereby, both hazards and exposure are evaluated within different 

tiers that progress from worst-case scenario conditions framed in highly controlled laboratory 

environments to more realistic conditions in the field (Dutton et al. 2003; Wilkinson et al. 

2003; Andow & Zwahlen 2006; EFSA 2006; Garcia-Alonso et al. 2006; Bartsch et al. 2008; 

Nickson 2008; Romeis et al. 2008). In general, tiers 1 and 2 aim to identify potential hazards, 

whilst tier 3 identifies the likely exposure levels. The conclusion regarding potential risks 

drawn at each tier will lead to a regulatory decision after the residual uncertainty of the 

assessment has been defined or to additional investigations (Romeis et al. 2008). If a risk is 

identified, decision-making can consider whether risk management should be implemented to 

reduce risk. It is important that throughout the assessment, the problem being addressed 

remains appropriate, and is revised if necessary. 

Lower-tier tests serve to identify and test potential hazards under worst-case scenario 

conditions and thus involve conservative assumptions. By exposing target and non-target biota 

likely to be directly exposed to the GM plant or its products to high levels of the GM plant or 

its products, the likelihood of detecting potential adverse effects on these organisms increases. 

These studies are conducted under controlled laboratory or growth room conditions in order to 

quantify effects in relation to known exposure levels, to provide high levels of replication and 

control, and to increase the statistical power for testing the established hypotheses. Indirect 

effects of the GM plant on organisms not directly exposed to the GM plant, but are one or two 

steps behind in the food chain (e.g. predators and parasites of primary phytophagous or plant 

pathogenic organisms) are generally assessed in the second tier. Second tier studies are also 

generally conducted under controlled laboratory, growth room or glasshouse conditions in 

order to measure effects in relation to known exposure levels (EFSA 2006). If no hazards are 

identified and the GM plant is not different from the comparator, the tested product is regarded 

as safe. 

However, in case potential hazards are detected in early-tier tests or if unacceptable 

uncertainties about possible hazards remain, additional information is required to confirm 

whether the observed effect might still be detected at more realistic rates and routes of 

exposure (EFSA 2006; Garcia-Alonso et al. 2006; Bartsch et al.. 2008; Nickson 2008; Romeis 

et al. 2008). Progression to larger-scale experiments in higher tiers aims to provide 

increasingly refined estimates of exposure. Field trials are then established in which the 

cultivation of the GM plant is conducted with greater environmental realism. As such, actual 

levels of exposure of different biota can be quantified. In comparison with the comparator plant 

and its management, likely ecological adverse effects due to the GM plant and its management 

can be determined. While higher-tier studies offer greater environmental realism, they may 

have lower statistical power due to the higher variability of environmental conditions (e.g. 

climate) that can mask effects generated by the GM plant or its product (Bartsch et al. 2008). 

In exceptional cases, higher-tier studies may be conducted at the initial stage when early-tier 

tests are not possible or meaningful. As such, many risk assessments are conducted in tiered 

manner, meaning that risk assessment studies increase in complexity depending upon the 

findings at each level of assessment (Hill & Sendashonga 2003). In cases where uncertainty 

550

about the risk remains after higher-tier studies, one can always return to lower tiers to conduct 

additional studies (Romeis et al. 2008). 

The tiered approach is consistent with the iterative or adaptive nature of risk assessment where 

conclusions are reviewed when new information is obtained. As such, the uncertainty in risk 

assessment is reduced because each tier is guided by results obtained in the previous tier, and 

specific, testable, and relevant hypotheses are formulated based on these data (Andow & 

Zwahlen 2006; EFSA 2006; Garcia-Alonso et al. 2006; Bartsch et al. 2008; Nickson 2008; 

Romeis et al. 2008). 

REFERENCES 

Andow D A; Zwahlen C (2006). Assessing environmental risks of transgenic plants. Ecology 

Letters, 9, 196-214. 

Bartsch D; Gathmann A; Saeglitz C; Sinha A (2008). Field testing of transgenic plants. In: 

Plant biotechnology and genetic principles, techniques, and applications, ed C N 

Stewart, Jr, pp. 311-323. John Wiley & Sons, Inc. 

Batista R; Saibo N; Lourenço T; Oliveira M M (2008) Microarray analyses reveal that plant 

mutagenesis may induce more transcriptomic changes than transgene insertion. 

Proceedings of the National Academy of Sciences of the United States of America, 105, 

3640-3645. 

Conner A J; Glare T R; Nap J-P (2003). The release of genetically modified crops into the 

environment. Part II: overview of ecological risk assessment. The Plant Journal, 33, 

19-46. 

Craig W; Tepfer M; Degrassi G; Ripandelli D (2008). An overview of general features of risk 

assessment of genetically modified crops. Euphytica, 164, 853-880. 

Dutton A; Romeis J; Bigler F (2003) Assessing the risks of insect resistant transgenic plants on 

entomophagous arthropods: Bt-maize expressing Cry1Ab as a case study. BioControl, 

48, 611-636. 

EC (2001). Directive 2001/18/EC of the European Parliament and of the Council of 12 March 

2001 on the deliberate release into the environment of genetically modified organisms 

and repealing Council Directive 90/220/EEC. Official Journal of the European 

Communities, L106, 1-39. 

EFSA (2006). Guidance document of the Scientific Panel on Genetically Modified Organisms 

for the risk assessment of genetically modified plants and derived food and feed. EFSA 

Journal, 99, 1-100. 

EFSA (2008) Environmental Risk Assessment of Genetically Modified Plants - Challenges and 

Approaches. Scientific Colloquium Series of the European Food Safety Authority N° 8 

June 2007, http://www.efsa.europa.eu/cs/BlobServer/Event_Meeting/sci_coll_8_ 

summary_report.pdf 

Garcia-Alonso M; Jacobs E; Raybould A; Nickson T E; Sowig P; Willekens H; Kouwe P v d; 

Layton R; Amijee F; Fuentes AM; Tencalla F (2006). A tiered system for assessing the 

risk of genetically modified plants to non-target organisms. Environmental Biosafety 

Research, 5, 57-65. 

551

Gathmann A; Wirooks L; Hothhorn L A; Bartsch D; Schuphan I (2006). Impact of Bt-maize 

pollen (MON810) on lepidopteran larvae living on accompanying weeds. Molecular 

Ecology, 15, 2677-2685. 

Hill R A; Sendashonga C (2003). General principles for risk assessment of living modified 

organisms: lessons from chemical risk assessment. Environmental Biosafety Research, 

2, 81-88. 

Hill R A (2005). Conceptualizing risk assessment methodology for genetically modified 

organisms. Environmental Biosafety Research, 4, 67-70. 

Johnson K L; Raybould A F; Hudson M D; Poppy G M (2007). How does scientific risk 

assessment of GM crops fit within the wider risk analysis? Trends in Plant Science, 12, 

1-5. 

Macdonald P; Yarrow S (2003). Regulation of Bt crops in Canada. Journal of Invertebrate 

Pathology, 83, 93-99. 

McHughen A; Smyth S (2008). US regulatory system for genetically modified [genetically 

modified organisms (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnology 

Journal, 6, 2-12. 

Nickson T E (2008). Planning environmental risk assessment for genetically modified crops: 

problem formulation for stress-tolerant crops. Plant Physiology, 147, 494-502. 

OECD (1993). Safety Evaluation of Foods Derived by Modern Biotechnology, Concepts and 

Principles. Paris: Organization for Economic Co-operation and Development. ISBN: 

92-64-13859-5. 

Paoletti C; Flamm E; Yan W; Meek S; Renckens S; Fellous M; Kuiper H (2008). GMO risk 

assessment around the world: some examples. Trends in Food Science & Technology, 

19, S66-S74. 

Qi A; Perry J N; Pidgeon J D; Haylock L A; Brooks D R (2008). Cost-efficacy in measuring 

farmland biodiversity – lessons from the Farm Scale Evaluations of genetically 

modified herbicide-tolerant crops. Annals of Applied Biology, 152, 93-101. 

Raybould A (2006). Problem formulation and hypothesis testing for environmental risk 

assessments of genetically modified crops. Environmental Biosafety Research, 5, 

119-125 

Raybould A (2007). Ecological versus ecotoxicological methods for assessing the 

environmental risks of transgenic crops. Plant Science, 173, 589-602. 

Romeis J; Bartsch D; Bigler F; Candolfi M P; Gielkens M M C; Hartley S E; Hellmich R L; 

Huesing J E; Jepson P C; Layton R; Quemada H; Raybould A; Rose R I; Schiemann J; 

Sears M K; Shelton A M; Sweet J; Vaituzis Z; Wolt J D (2008). Assessment of risk of 

insect-resistant transgenic crops to nontarget arthropods. Nature Biotechnology, 26, 

203-208. 

Sanvido O; Widmer F; Winzeler M; Bigler F (2005). A conceptual framework for the design of 

environmental post-market monitoring of genetically modified plants. Environmental 

Biosafety Research, 4, 13-27. 

Sanvido O; Romeis J; Bigler F (2008). An approach for post-market monitoring of potential 

environmental effects of Bt-maize expressing Cry1Ab on natural enemies. Journal of 

Applied Entomology, DOI: 10.1111/j.1439-0418.2008.01367.x. 

Smyh S; McHughen A (2008). Regulating innovative crops technologies in Canada: the case of 

regulation genetically modified crops. Plant Biotechnology Journal, 6, 213-225. 

552

Storkey J; Bohan D A; Haughton A J; Champion G T; Perry J N; Poppy G M; Woiwod I P 

(2008). Providing the evidence base for environmental risk assessments of novel farm 

management practices. Environmental Science & Policy, 11, 579-587. 

Suter G W (2000). Generic assessment endpoints are needed for ecological risk assessment. 

Risk Analysis, 20, 173-178. 

Todd J H; Ramankutty P; Barraclough E I; Malone L A (2008). A screening method for 

prioritizing non-target invertebrates for improved biosafety testing of transgenic crops. 

Environmental Biosafety Research, 7, 35-56. 

US EPA (1998). Guidelines for Ecological Risk Assessment, EPA/630/R095/002F. U.S. 

Environmental Protection Agency, Risk Assessment Forum, 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=12460 

Wilkinson M J; Sweet J; Poppy G M (2003). Risk assessment of GM plants: avoiding 

gridlock? Trends in Plant Science, 8, 208-212. 

Wolt J D; Keese P; Raybould A; Fitzpatrick J W; Burachik M; Gray A; Olin S S; Schiemann J; 

Sears M; Wu F (2009). Principles of environmental risk assessment for genetically 

modified plants: problem formulation. Transgenic Research (submitted). 

553

Latxague E, Barzman M, Bui S, Abrassart C, Ricci P: ENDURE Foresight Study: A Tool for Exploring Crop 

Protection in Europe in 2030 and Its Implications for Research. In: Feldmann F, Alford D V, Furk C: Crop Plant 



12-4 ENDURE Foresight Study: A Tool for Exploring Crop Protection in 

Europe in 2030 and Its Implications for Research 

Latxague E 1 , Barzman M 2 , Bui S 2 , Abrassart C 1 , Ricci P 2 

1 

INRA, UAR 1241 Prospective, 147 rue de l’Université, 75338 Paris cedex 07 

2 INRA, UR1284 Institut Sophia – AGROBIOTECH, 400 route des Chappes, BP 167, 06903 

Sophia-Antipolis 

554 

ABSTRACT 

The ENDURE Network Of Excellence is carrying a foresight study, which aims at 

defining long-term research priorities for European crop protection in 2030. 

Five scenarios on the future of crop protection in Europe at the 2030 horizon were 

built. They are based on expert statements and on a literature review, considering a 

broad scope of factors. These range from global context (food and energy demand 

and supply), political context in Europe (regulations, objectives ascribed to 

agriculture), the food and non-food system (retailers, consumers, farmers), all the 

way to innovation & research capabilities and crop protection engineering. 

The overall approach of this foresight study is not predictive but rather explorative, 

and aimed at building contrasted scenarios which are considered as tools to help to 

address a number of questions the European research community. The scenarios’ 

ability to raise interesting questions to research is a priority as they intent to be used 

in interactive meetings open to all ENDURE partners and crop protection 

stakeholders. 

INTRODUCTION 

A turning point for crop protection in Europe 

Advances in plant protection have contributed considerably to increasing yields and ensuring 

regular production. Easy to obtain and apply, and rather inexpensive, chemical control products 

have proved to be extremely efficient and reliable in a very large number of cases. European 

farming has developed production systems based on using these products and is currently 

highly dependent on pesticides. Today, their systematic use is being called into question 

(Aubertot et al. 2005), with the increasing awareness of their negative impacts, the

demonstration of undesirable adverse effects on ecosystems, on non-targeted useful or 

domestic species and on human health. 

Revealing this turn in crop protection, ambitious legislations on placing of plant protection 

products on the market and the sustainable use of pesticides have been implemented, both at 

the European and national levels. This regulative framework for change has been followed by a 

general mobilisation of all stakeholders: agricultural services, farmers’ organisations, 

agrochemical industries, NGOs and research institutes. 

ENDURE: a research network for diversifying crop protection approaches 

ENDURE, the European Network for the DURable Exploitation of crop protection strategies, 

is a network of excellence funded by the European Union under the Framework 6 program. It 

brings together more than 10 countries and 300 researchers in the fields of agronomy, biology, 

ecology, economics and the social sciences. 

A European network of expertise and knowledge is being developed, and progressively 

enhanced by teams from other Member States and countries outside Europe. The network will 

establish itself as a world leader for the development and implementation of diverse and 

sustainable crop protection strategies, aiming at reducing our dependency to pesticides. The 

ENDURE network is not a decision-making body but is providing tools and knowledge to 

stakeholders who make decisions about the optimisation and reduction of pesticide use 

(ENDURE, 2007). 

Within this framework, a foresight reflexion – one of the first on this topic at the European 

scale – had been engaged in 2007 and enabling to imagine different worlds for crop protection, 

new potential roles and responsibilities for all stakeholders. 

OBJECTIVES AND METHODOLOGY 

Foresight as a reflexion tool 

ENDURE foresight study intends to better understand the interrelations that model the 

evolution of crop protection and aims at identifying long-term research priorities for European 

crop protection in 2030. This project is a foundation to elicit a debate on the relationships 

between the EU policy on pesticides and crop protection issues, the consequences on 

agriculture in Europe, and on the research priorities to be identified at EU and national levels. 

The overall approach of this study is not a predictive one – extrapolation of quantitative trends, 

forecasting the future configurations of crop protection – but rather an explorative one. It aims 

at building contrasted scenarios which are considered as tools (or "though experiments") to 

help to address a number of questions the European research community. Thus, the scenarios 

are diverse and “provocative” enough to create a mechanism for discussion and collective 

learning, and to trigger interesting questions for the research agenda. 

555

Conventional methodology completed by a participative approach 

We followed a classical foresight methodology, identifying key-drivers for the systems, 

developing and combining assumptions on these drivers and finally building scenarios 

choosing coherent options among these combinations. Resulting scenarios are considering a 

broad scope of factors ranging from global context (food and energy demand and supply), 

political context in Europe (regulations, objectives ascribed to agriculture), the food and nonfood 

system (retailers, consumers, farmers), all the way to innovation & research capabilities 

and crop protection engineering. 

In addition to this conventional building phase, because of the participative dimension of being 

part of a network, we launched an early debate phase aimed at successively enriching the 

scenarios. Various stakeholders including agrochemical companies, NGOs and researchers 

were interviewed on the basis of provisional documents, bringing specific elements to the 

debate, deepening typical points and allowing a continuous emergence of the scenarios. 

A QUICK GLIMPSE AT FIVE SCENARIOS FOR CROP PROTECTION IN 2030 

The five scenarios built are illustrating five worlds, starting with different contexts, having 

different rules within the EU and resulting in different types of agriculture. Crop protection has 

specific features in each of these worlds. However, they can be gathered in three groups: 

Agricultural free market scenarios 

The first couple of scenarios show the EU as a major player in a free globalised market for 

agricultural goods. In “The Commodity-market Player”, priority is on competitiveness for 

basic commodities. The use of pesticide is the first solution, although accountability of 

stakeholders is enhanced. In “The Specialised High-tech Grower”, the EU is taking advantage 

of strengthened regulation on plant protection products, turning to competitive specialty 

products. In this scenario, crop protection is mainly ensured through high-tech methods. 

Scenarios answering crisis situations 

The second couple of scenarios present crisis situations that led to extreme political choices in 

favour of strong European governance. In “The Sustainable Food Provider”, food and feed 

self-sufficiency is the main goal ascribed to European agriculture, and producers’ first priority 

is to minimise pest problems with robust agricultural systems. In “The Energy-saving 

Producer”, European landscape is deeply modified by high energy prices. Food is produced 

locally, within very dense cities or empty countryside, and farmers are demanded to reduce 

their energy consumption, even for protecting their crops. 

Local breakthrough scenario 

In the last scenario, “The Community-conscious Farmer”, the EU hands over to territoires 

much of the responsibility for their own development. Thus, they are competing for residents, 

visitors and investors and need to promote their assets to make themselves as attractive as 

556

possible. Agriculture is understood as essential and its productive function accompany other 

functions contributing to the value of territoires (land management, environment and 

biodiversity preservation, tourism). Crop protection is participative and designed according to 

local needs and conditions. 

CONCLUSION: IMPLICATIONS FOR RESEARCH 

This foresight study underlines questions addressed to research and striking gaps: objectives of 

varietal selection, regulation for biocontrol agents, etc. Going through the scenarios, several 

challenges and opportunities for research come to mind. As an example we can cite “The 

Community-conscious Farmer” who will have to use ecological knowledge (including 

landscape ecology) to redesign systems to maintain pests at acceptable levels, although 

research on this topic is just starting. 

In fact, the scenarios’ ability to raise interesting questions to research is a priority as they intent 

to be used in interactive meetings open to all ENDURE partners and crop protection 

stakeholders. During such meetings, participants exchange about the scenarios and discuss the 

consequences for European research (such as priorities, competencies, organisation, financing 

and partnerships), for all scientific disciplines, following or marking a break with current 

research programs, being underlying or innovative. Innovative practices, ongoing changes, 

weak signals, potential breakthroughs are example of items that are identified during this new 

participative phase. 

All the scenarios illustrate that along with the new social demand for a more sustainable 

agriculture, crop protection stakeholders have the possibility to play new roles. In particular, 

public research may become a key-actor in the innovation process going from expressing 

diagnostics towards designing new crop protection solutions. 


This work has been supported by the European Commission in the framework of the ENDURE 

Network of Excellence. We thank C Jez, O Mora and S Paillard for their methodological 

contribution to this foresight study. 

REFERENCES 

Aubertot J N; Barbier J M; Carpentier A; Gril J J; Guichard L; Lucas P; Savary S; Savini I; 

Voltz M (2005). Pesticides, agriculture and the environment. Reducing the use of 

pesticides and limiting their environmental impact. Collective Scientific Expert Report, 

INRA and Cemagref (France). 

ENDURE (2007). Diversifying crop protection. www.endure-network.eu (13.02.2009) 

557

558 

IUFRO MEETING UNIT 7.02.04: VIRUSES IN FOREST AND URBAN TREES

Vogel S, Tantau H, Mielke N, Sarker R H, Khan S, Mühlbach H-P: Double strand RNA patterns indicate plant 

viruses associated with dieback affected Dalbergia sissoo trees in Bangladesh. In: Feldmann F, Alford D V, Furk 

C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 559; ISBN 978-3-941261-05-1; © Deutsche 


IUFRO-1 Double strand RNA patterns indicate plant viruses associated 

with dieback affected Dalbergia sissoo trees in Bangladesh 

Vogel S 1 , Tantau H 1 , Mielke N 1 , Sarker R H 2 , Khan S 3 , Mühlbach H-P 1 

1 

University of Hamburg, Biocentre Klein Flottbek, Ohnhorststr. 18, D-22609 Hamburg, 

Germany 

2 

Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh 

3 

BCSIR laboratories, Dhaka-1205, Bangladesh 

Email: muehlbach@botanik.uni-hamburg.de 

Abstract 

The dieback of sissoo is a devastating disease occurring in Bangladesh as well as in 

India, Nepal, Pakistan and Afghanistan. Fungi, bacteria, and insects were reported 

to be associated with the dieback syndrome, but the causal agent(s) were not yet 

identified unequivocally. Our studies are focused on the molecular detection and 

characterization of putative pathogens from dieback affected sissoo including 

viruses, viroids, phytoplasms, bacteria and fungi. Electron microscopic inspection 

of leaf homogenates revealed the presence of virus-like particles 60-130 nm in 

diameter. Preparation and gel electrophoretic analysis of double stranded RNA 

(dsRNA) from affected leaf material allowed the detection of dsRNA patterns, 

which may indicate the presence of plant viruses. Cloning and sequencing of cDNA 

fragments should help to clarify the question whether plant viruses play a role in the 

dieback disease of Dalbergia sissoo. 

559

Kallinen A, Lindberg I, Valkonen J: Detection and distribution of European Mountain Ash Ringspot-Associated 

Virus (EMARAV) in Finland. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 


Germany 

IUFRO-2 Detection and distribution of European Mountain Ash Ringspot- 

Associated Virus (EMARAV) in Finland 

Kallinen A, Lindberg I, Valkonen J 

Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland 

Email: jari.valkonen@helsinki.fi 

560 

Abstract 

A virus was recently characterized from mountain ash (rowan) (Sorbus aucuparia 

L.) displaying ringspot symptoms in Germany and designated as European 

mountain ash ringspot-associated virus (EMARAV) (Mielke & Muehlbach, J. Gen. 

Virol. 88: 1337-1346, 2007). Similar symptoms are common in mountain ash in 

Finland and have been documented by A.E. Jamalainen already in 1957. In this 

study, reverse transcription polymerase chain reaction (RT-PCR) and dotblot 

hybridization using digoxigenin-labeled RNA probes were used to test 73 mountain 

ash trees, including 16 trees with no virus-like symptoms. EMARAV was detected 

in all tested trees from 16 districts of Finland and Viipuri, Russia. Hence, 

EMARAV is associated with the ringspot disease and can cause latent infections in 

mountain ash. The putative nucleocapsid (NP) gene sequence of EMARAV was 97- 

99 % identical among the 17 isolates characterized indicating strong purifying 

selection. Amino acid substitutions were detected in only two positions at the Nterminus 

and one position at the C-terminus of NP. The 3' untranslated region was 

more variable (94-99 %).

Schlatermund N, Mielke N, Mühlbach H-P: European mountain ash ringspot-associated virus (EMARAV) and 

relationship to other ss(-) RNA viruses: Protein characterisation, RNA localisation and quantification. In: 



IUFRO-3 European mountain ash ringspot-associated virus (EMARAV) 

and relationship to other ss(-) RNA viruses: Protein 

characterisation, RNA localisation and quantification 

Schlatermund N, Mielke N, Mühlbach H-P 

Biozentrum Kl. Flottbek, Ohnhorststr. 18, 22609 Hamburg, Germany 

Email: nanette.schlatermund@botanik.uni-hamburg.de 

Abstract 

European mountain ash ringspot-associated virus (EMARAV) is a novel, still 

unclassified plant RNA virus, which was found to be associated with chlorotic 

ringspots and mottling symptoms on leaves of European mountain ash trees (Sorbus 

aucuparia L.) in many parts of Europe. EMARAV has a multipartite genome of four 

ss(-) RNAs, each of them carrying a single ORF. We could identify a RNAdependent 

RNA-polymerase (P1), showing sequence similarities to the replicases of 

the virus family Bunyaviridae and the phytopathogenic genus Tenuivirus, a putative 

glycoprotein precursor (P2) and a putative nucleocapsid protein (P3) so far. New 

sequence analyses of the proteins presume close relation between EMARAV and a 

novel, still unassigned virus associated with Fig mosaic. The fourth protein P4, 

encoded by the smallest RNA, is still of unknown function. Recent analyses in 

Drosophila S2 cells and in Nicotiana benthamiana 16C line indicated that this 

protein might be a suppressor of post transcriptional gene silencing (PTGS). 

Further analyses concentrate on the function of the putative glycoproteins. Transient 

transfection of the putative precursor P2 in Nicotiana benthamiana protoplasts 

indicates its localisation nearby the Golgi apparatus. Unlike it is described for 

Tospoviruses (Bunyaviridae), coexpression of P3 seems to have no influence on the 

P2 stability. 

All four genomic RNAs are detectable by in situ RT-PCRs and in situ hybridisation 

with RNA probes in European mountain ash tissues. In further investigations, 

EMARAV specific RNAs were quantified by real time RT-PCR (Sybr Green) 

during the year in leaves and bark. All genomic (-) RNAs were present in higher 

concentrations than (+) RNAs as it is typical for ss(-) RNA viruses.Significant 

amounts of vRNAs were detectable all year long. Thus, virus diagnostic using real 

time RT-PCR is possible even in wintertime. 

561

Novikova L, Ikogho B, Ludenberg I, Mielke N, Muehlbach H-P: Co-expression of viral proteins P2 and P3 of 

European mountain ash ringspot-associated virus (EMARAV) in plant protoplasts. In: Feldmann F, Alford D V, 



IUFRO-4 Co-expression of viral proteins P2 and P3 of European mountain 

ash ringspot-associated virus (EMARAV) in plant protoplasts 

Novikova L, Ikogho B, Ludenberg I, Mielke N, Muehlbach H-P 

University of Hamburg, Biocentre Klein Flottbek, Ohnhorststr. 18, 22609 Hamburg, Germany 

Email: LenaNovikova@web.de 

562 

Abstract 

The European mountain ash ringspot-associated virus (EMARAV) is a novel and 

yet not classified plant virus. It is associated with characteristic disease symptoms 

of European mountain ash (Sorbus aucuparia L.). EMARAV has a single-stranded 

(ss), segmented RNA genome of negative orientation. Each of the viral RNAs 

encodes one protein: a RdRp (P1), a putative glycoprotein precursor (P2), a putative 

N-Protein (P3) and a protein of unknown function (P4). 

In order to characterize the putative glycoprotein precursor P2 and the putative 

nucleocapsid protein P3, both proteins were expressed in mesophyll protoplasts of 

Nicotiana rustica. Western-Blot analysis support the theory of a processing of P2 

into two separate glycoproteins G2 (N-terminal; 22,7 kDa) and G1 (C-terminal; 

51,6 kDa). Due to the smaller size of the detected G2-specific protein (~ 19 kDa), 

the N-terminal signal peptide is probably cut off. No evidence for a stabilizing 

influence of the nucleocapsid protein P3 on the expression of P2 was observed, 

though the P3 co-transfected protoplasts showed an additional G2-specific protein 

band of about 50 - 54 kDa, that cannot be attributed to any EMARAV protein due 

to its dimension. 

Furthermore, the presence of transient P2-wtGFP fusion protein in P2-alone or P3cotransfected 

protoplasts could be demonstrated in comprehensive fluorescent 

microscope studies with green fluorescence detection. The green fluorescence of the 

P2-wtGFP fusion protein localized in globular structures within the cell. By using a 

G2-specific and a Golgi antibody, the co-localization of these structures was 

assigned to the Golgi apparatus. This indicates that the morphogenesis of 

EMARAV could take place in this cell compartment, similar to that of the 

bunyaviruses. Thus, it confirms the phylogenetic relation of the EMARAV to this 

virus family. Again no stabilizing effect on the expression of P2 or its localization 

in the cell by co-expression with the P3 N-protein, could be observed.

Nóbrega F 1 , Vidal R 2 , Serrano R: Investigation on virus-like particles associated to decline of Quercus suber. In: 



IUFRO-5 Investigation on virus-like particles associated to decline of 

Quercus suber 

Nóbrega F 1 , Vidal R 2 , Serrano R 2 

1 

Instituto Nacional dos Recursos Biológicos, Quinta do Marquês, 2780-159 Oeiras, Portugal; 

2 

Faculdade de Farmácia da Universidade de Lisboa, Avenida das Forças Armadas, 1600-083 

Lisboa, Portugal; Email: mariafilomenanobrega@gmail.com 

Abstract 

Over the last few decades, ecophysiological disturbances of the cork oak (Quercus 

suber) forest have been observed. In affected areas, the decline symptons of cork 

trees evolved by the deterioration of the crown which, starting with leaf necrosis 

and vigour loss, led some trees to sudden death. Factors grouped as follows: 

predisposing factors (social system, physical complex and production system), 

trigger factors (abiotic factors such severe drought spells and human intervention 

factors) and acceleration factors (pests and diseases and human action-wounds of 

cork stripping, stripping intensity and dead trees maintenance) were reported to be 

associated with the cork oak decline. However, the result obtained up to now have 

not satisfactorily explained the exact nature and specific causes of the phenomena. 

Scanning (SEM) and Transmission electron microscopy (TEM) of symptomatic 

leaves homogenates revealed the presence of isometric virus-like particles with 20- 

30 nm in diameter and the presence of 5-6 giant, unusual rod-shaped virus particles 

about 2-3 µm in length, with an end round and the other one flattened, emerging 

from a broken, putative proteinaceous occlusion body. 

Further studies, focused on the extraction and analysis by electrophoresis of double 

stranded RNA (dsRNA) from leaf material showed the presence of a pattern of 

multiple dsRNA bands. A degenerate oligonucleotide primer PCR (DOP-PCR) for 

the non-specific amplification of virus as well as general PCR primer sets for 

different virus genera were used. Cloning and sequencing of some cDNA fragments 

allowed the obtention of sequences without similarity with virus sequences. 

Consequently, the goal of our studies is to improve the knowledge about the 

detection and molecular characterization of virus-like particles associated to decline 

of Quercus suber. 

563

Bargen S von, Langer J, Rumbou A, Gentkow J, Büttner C: Cherry leaf roll virus (CLRV) - genome organisation 

of the RNA1. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 


IUFRO-6 Cherry leaf roll virus (CLRV) - genome organisation of the 

RNA1 

Bargen S von 1 , Langer J 1 , Rumbou A 1 , Gentkow J 2 , Büttner C 1 

1 

Faculty of Agriculture and Horticulture, Section Phytomedicine, Humboldt-Universität zu 

Berlin, Lentzeallee 55/57, D-14195 Berlin, Germany 

2 

Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany 

Email: susanne.von.bargen@agrar.hu-berlin.de 

564 

Abstract 

The complete organisation of the Cherry leaf roll virus genome, a virus which 

affects many fruit trees and other woody hosts, has not been determined to date. 

However, partial sequence information of the bipartite virus which is available of 

the 3´ proximal portion including the complete 3´ non-coding region (NCR) of the 

genomic RNA1 and RNA2 has led to the classification as a subgroup c nepovirus. 

Sequences of the RNA1 of two CLRV isolates from different host plants (CLRV- 

E395 originating from Rheum rhabarbarum and CLRV-E326 from Juglans regia) 

were obtained and compared with other nepoviruses. The genomic structure of the 

CLRV-RNA1 coding for a polyprotein corresponds with other established subgroup 

c nepoviruses like Tomato ringspot virus (ToRSV), Blackcurrant reversion virus 

(BRV) and Peach rosette mosaic virus (PRMV). The polyprotein of the rhubarb 

isolate (ORF12-6350 nt; 2112 amino acids) contains a N-terminal protease cofactor 

(PCo), adjacent is a nucleotide-binding protein-domain (NTB), followed by the 

sequences coding for the genome-linked viral protein (VPg), a protease (Pro) and 

the viral replicase (RdRp). Putative protein functions were predicted by 

identification of characteristic sequence motifs (Argos 1988; Gorbalenya et al. 

1989a and 1989b; Rott et al. 1995, Wang et al. 1999). The region coding for the 

putative CLRV-VPg protein was identified with the computer programs 

NetPicoRNA V1.0 and. NetCorona V1.0., and exhibited highest similarities to the 

corresponding ToRSV-VPg. Predicted specific protease recognition sequences in 

the CLRV isolates (Q1121/S1122 and Q1150/S1151) also corresponded to ToRSV.

REFERENCES 

Argos, P. (1988). A sequence motif in many polymerases. Nucleic Acids Res 16, 9909–9919. 

Gorbalenya, A.E., Blinov, V.M., Donchenko, A.P., Koonin, E.V. (1998a). A NTP-binding 

motif is the most conserved sequence in a highly diverged monophyletic group of 

proteins involved in positive strand RNA viral replication. Journal of Molecular 

Evolution 28, 256–268. 

Gorbalenya, A.E., Donchenko, A.P., Blinov V.M., Koonin E.V. (1989b). Cysteine proteases of 

positive strand RNA viruses and chymotrypsin-like serine proteases. FEBS Letters 243, 

103–114. 

Rott, M.E., Gilchrist, A., Lee, L., Rochon D.M. (1995). Nucleotide sequence of tomato 

ringspot virus RNA1. Journal of General Virology 76, 465–473. 

Wang, A., Carrier, K., Chisholm, J., Wieczorek, A., Huguenot, C., Sanfaçon, H. (1999). 

Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: 

definition of the domains for the VPg, protease and putative RNA-dependent RNA 

polymerase. Journal of General Virology 80, 799-809. 

565

Rumbou A, Bargen S von, Büttner C: Study on transmission modes of Cherry leaf roll virus: genetic basis of seed 

transmissibility based on the model system CLRV/A. thaliana and investigation of possible vectors. In: Feldmann 

F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 566; ISBN 978-3-941261-05- 

1; © Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany 

IUFRO-7 Study on transmission modes of Cherry leaf roll virus: genetic 

basis of seed transmissibility based on the model system 

CLRV/A. thaliana and investigation of possible vectors 

Rumbou A, Bargen S von, Büttner C 



Email: amirumbou@yahoo.gr 

566 

Abstract 

Cherry leaf roll virus constitutes a worldwide dispersed Nepovirus that naturally 

infects a wide range of woody hosts. Although the virus is only reported to be 

transmitted in nature vertically - through seed and pollen -, the underlying 

mechanisms of seed infection are not specified. After transmission experiments of 

CLRV on birch and Arabidopsis thaliana seedlings, vertical spread of the virus is 

believed to be achieved due to virus presence in the embryo rather than in the seed 

coat. To confirm the speculated indirect embryo invasion, we intent to use the 

model system CLRV/A. thaliana to investigate the protein-protein interactions 

during seed embryo infection. In this way we expect to confirm the virus invasion 

of the floral meristem, to localize the virus in the gametes and gametophytes and 

intify host and viral determinants involved in seed transmission. Concerning farther 

epidemiological studies on CLRV we intent to investigate possible transmission 

through vectors on birch forests and elderberry plantations in Germany and Finland. 

Transmission of CLRV through aphids and mites is speculated; this may constitute 

one factor potentially responsible for the recent broad CLRV epidemics in north 

European birch forests.

Langer J, Bargen S von, Büttner C: Molecular properties of Cherry leaf roll virus. In: Feldmann F, Alford D V, 



IUFRO-8 Molecular properties of Cherry leaf roll virus 

Langer J, Bargen S von, Büttner C 



Email: langerj@rz.hu-berlin.de 

Abstract 

The Cherry leaf roll virus (CLRV) is a globally distributed pathogen occurring 

primarily on deciduous, fruit and ornamental trees from at least 17 genera, including 

many economically important trees like walnut, cherry and birches. CLRV is a 

nepovirus of the Comoviridae within the Picornavirus superfamily with a bipartite 

genome organisation and protein expression strategy resembling other members of 

the genus. Nepoviral RNAs exhibit 3´ non-coding regions (3´ NCR) with extensive 

sequence identities (80-100 %), exclusively illustrated by the members of the 

nepovirus subgroup c, including the CLRV, with very large 3´ NCRs of over 1500 

nt. Sequence comparisons between the RNA1 and RNA2 specific 3´ NCRs of six 

different CLRV isolates from different host plant species and phylogenetic groups 

displayed almost identical 3´ NCRs (97.5-99.5 %) for five CLRV isolates. A 

raspberry isolate exhibits 3´ NCRs with only 73.8 % sequence identity, raising the 

question about the prerequisite of sequence identity within the 3´ NCRs of a RNA 

population of an individual CLRV strain. So far, the question for the benefit of the 

long 3´ NCRs in any replication or translation mechanism is still unanswered, but 

the selective 3´ NCR sequence conservation of almost all previously analyzed 

nepovirus isolates, confirmed a strict necessity of identity for maintaining 

functional sequences within this region. It is commonly considered that 

homologous recombination is responsible for the 3´terminal sequence identity. But 

this is only one of several efficient mechanisms to ensure viability of RNA 

populations, at least for the CLRV since a raspberry isolate with non-homologous 

3´ NCRs was found in this study. 

Furthermore, a stable secondary hairpin structure was predicted within the analyzed 

3´ NCRs of all six different CLRV isolates. This is located in a region with high 

sequence variability of up to 34 % and the conservation of this secondary structure 

suggests that it represents an important functional domain within the 3´terminus of 

CLRV-RNAs. 

567

Langer J, Bargen S von, Büttner C: Epidemiological investigations on Cherry leaf roll virus. In: Feldmann F, 



IUFRO-9 Epidemiological investigations on Cherry leaf roll virus 

Langer J, Bargen S von, Büttner C 



Email: langerj@rz.hu-berlin.de 

568 

Abstract 

Cherry leaf roll virus (CLRV) is globally distributed in woody and herbaceous plant 

species including 17 genera. The wide host range and geographical distribution of 

CLRV indicate a fast adaptability to different hosts and therefore a genetic 

heterogeneicity among CLRV-isolates of different origins. This was confirmed by 

molecular and serological analyses, which also revealed that phylogenetic 

affiliations are strongly correlated with the host plant species. This reflects the 

natural mode of transmission by pollen and seeds which require a high degree of 

host specificity of CLRV-isolates. However, transmission barriers are not absolute, 

as some CLRV isolates were found in phylogenetic groups not accordingly to their 

host plant species. Conclusively, further efficient modes of transmission must be 

relevant for CLRV distribution in natural habitats. In order to prove whether 

molecular properties reflect biological characteristics, the mechanical 

transmissibility of genetically diverse Cherry leaf roll virus (CLRV) isolates to 

different woody host plant species was tested. In an outdoor study three CLRV 

isolates from elderberry, walnut and sweet cherry were inoculated by stem slashing 

on Sambucus nigra, Juglans regia, Prunus avium, Sorbus aucuparia, Betula 

pendula. CLRV infected trees were detected by IC-RT-PCR, but molecular analysis 

could not identify the inoculated CLRV isolates as the causal agents. Thus 

indications towards varying capabilities of genetically diverse CLRV isolates to 

adapt to different hosts were not gained. As uninoculated control trees were found 

to be infected, we conclude that CLRV infection of trees was due to transmission 

from natural sources. This is supported by our findings of CLRV contaminated 

aphids sampled from elderberry (Sambucus nigra) seedlings of the experimental 

plot. It is shown that CLRV is a pathogen that may easily be disseminated by 

natural ways of transmission to healthy woody hosts.

Czesnick H, Langer J, Bargen S von, Büttner C: Analysis of the 3´ non-coding region of Cherry leaf roll virus, a 

nepovirus of subgroup c. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic 


Germany 

IUFRO-10 Analysis of the 3´ non-coding region of Cherry leaf roll virus, a 

nepovirus of subgroup c 

Czesnick H, Langer J, Bargen S von, Büttner C 



Email: phytomedizin@agrar.hu-berlin.de 

Abstract 

Cherry leaf roll virus (CLRV) is a member of the family Comoviridae, genus 

Nepovirus. The bipartite genome consists of RNA1 (7918 nucleotides, nt) and 

RNA2 approx. 6800 nt in length. Each RNA comprises a single open reading frame 

encoding one polyprotein being cleaved proteolytically into functional proteins. 

Both genomic RNAs contain a VPg at the 5´ terminus as well as a 3´ terminal 

poly(A) tail. The genome organisation of CLRV is according to other members of 

the genus; especially the long 3´ non.coding region (3´ NCR) of about 1600 nt of 

both RNAs is a typical feature of the Nepovirus subgroup c. 

Sequence comparisons of coding regions of the RNA1 (a 523 nt fragment of the 

RNA dependent-RNA polymerase, RdRp), RNA2 (coat protein, CP, 1539-1542 nt) 

and the complete 3´ NCR (1557-1602 nt) revealed that parts of the untranslated 

region exhibited higher sequence conservation than protein-coding parts of the 

genome. CLRV isolates analysed originated from different locations and host 

plants, thus representing various phylogenetic clusters as proposed by Rebenstorf et 

al. (2006). Protein encoding sequences exhibited a maximal nucleotide diversity of 

23 %, whereas it was found that the 5´ proximal part of 3´ NCR directly adjacent to 

the stopcodon of the polyprotein showed higher variability (33 %). The middle part 

of the 3´ NCR revealed 25 % nucleotide variability while the 3´ terminal region was 

highly conserved (17 %). These findings of sequence conservation support the role 

of the 3´ NCR involved in translational regulation (Dreher and Miller 2006). 

Furthermore, 3´ NCRs of RNA1 and RNA2 originating from individual CLRV 

isolates are not identical, although they exhibit only moderate sequence variability 

of max. 2 %. This is in accordance with the 3´ NCR sequences of other nepoviruses 

exhibiting isolate specific diversity of 3´ NCRs of 1 % (Tomato ringspot virus) and 

7 % (Blackcurrant reversion virus, BRV). 

569

REFERENCES 

Dreher, T.W., Miller, A. (2006). Translational control in positive strand RNA plant viruses. 

Virology 344, 185 – 197. 

Rebenstorf, K., Candresse, T., Dulucq M.J., Büttner, C., Obermeier, C. (2006). Host Species 

dependent Population Structure of a Pollen-Borne Plant Virus, Cherry leaf roll virus. 

Journal of Virology, 80, 2453 – 2462. 

570

Grubits E, Bargen S von, Langer J, Jalkanen R, Büttner C: Cherry leaf roll virus: a threat to Finnish Betula spp.. 

In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 571-572; ISBN 


IUFRO-11 Cherry leaf roll virus: a threat to Finnish Betula spp. 

Grubits E 1 , Bargen S von 1 , Langer J 1 , Jalkanen R 2 , Büttner C 1 

1 



2 

Metla, Finnish Forest Research Institute, Rovaniemi, Finland 


Abstract 

Cherry leaf roll virus, CLRV, was detected in Finland in several Betula pubescens 

ssp. pubescens (downy birch) trees exhibiting symptoms of a viral disease (Jalkanen 

et al. 2007); the virus could also be confirmed in B. pendula (silver birch), both are 

dominating deciduous tree species in the country. CLRV was found in B. nana 

(dwarf birch), B. pubescens ssp. czerepanovii (mountain birch) as well as B. 

pubescens ssp. appressa (Kiilopää birch) comprising key components of the arctic 

ecosystem. A single B. pendula var. carelica (curly birch) an ornamental tree 

variety used as expensive veneer wood was also found to be CLRV infected. 

Fragments of the 3´non-coding region (3´NCR) were amplified by application of 

CLRV specific IC-RT-PCR. 

Testing symptomatic birch trees confirmed CLRV infected birches including 6 

different species or subspecies respectively over the country. 

CLRV specific fragments from 3 downy birches from Rovaniemi, 2 silver birch 

trees (Lieksa, Vaasa) and one mountain birch (Inari) were sequenced. Genetic 

relationships were investigated by PCR-RFLP as well as sequence comparison with 

CLRV isolates characterised previously by Rebenstorf et al. (2006), who 

established 5 different phylogenetic groups (A-E) depending on the host plant. Nine 

individual CLRV clones obtained from 6 different Betula trees revealed two 

different fragment sizes, 404 bp and 412 bp, which were in accordance with 

grouping of Finnish CLRV isolates by PCR-RFLP (Buchhop et al. 2009). Unlike 

clustering of CLRV strains from birches growing in the UK and Germany 

exclusively within group A, Finnish CLRV isolates exhibited highest sequence 

identities to isolates clustered in phylogenetic group B, D or E. Furthermore, from 

two trees more than one sequence variant of CLRV was detected indicating a higher 

sequence variability of the virus not only in the Finnish birch population, but also in 

individual trees. 

571

REFERENCES 

Jalkanen R, C Büttner, S von Bargen (2007). Cherry leaf roll virus, CLRV, abundant on Betula 

pubescens in Finland. Silva Fennica 41, 755-762. 

Buchhop J, S von Bargen, C Büttner. Differentiation of Cherry leaf roll virus isolates from 

various host plants by immunocapture-reverse transcription-polymerase chain reactionrestriction 

fragment length polymorphism according to phylogenetic relations. Journal 

of Virological Methods published online (2009), doi: 10.1016/j.jviromet.2008.12.010, 

in press. 

Rebenstorf K, T Candresse, M J Dulucq, C Büttner, C Obermeier (2006). Host Species 

dependent Population Structure of a Pollen-Borne Plant Virus, Cherry leaf roll virus. 

Journal of Virology 80, 2453–2462 

572

Arndt N, , Bargen S von, Grubits E, Jalkanen R, Büttner C: Occurrence of EMARAV and CLRV in tree species 

native to Finland. In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors 


Germany 

IUFRO-12 Occurrence of EMARAV and CLRV in tree species native to 

Finland 

Arndt N 1 , Bargen S von 1 , Grubits E 1 , Jalkanen R 2 , Büttner C 1 

1 



2 

Metla, Finnish Forest Research Institute, Rovaniemi, Finland 


Abstract 

Since the first report of accumulation of virus-like symptoms in downy birch 

(Betula pubescens) and silver birch (B. pendula) in Fennoscandia in 2007 by 

Jalkanen et al., Cherry leaf roll virus (CLRV) could be associated with the disease 

symptoms. Samples from symptomatic birch species showing leaf roll and 

proliferation, chlorosis, vein banding and mottling of leaves were collected in the 

following years from different regions in the country and assessed for a CLRV 

infection by RT-PCR. Furthermore, mountain ash trees (Sorbus aucuparia) with 

ringspot and mottling symptoms characteristic for an infection with the European 

mountain ash ringspot-associated virus (EMARAV; Mielke and Mühlbach, 2007) 

were included in the study as well as singular trees of other woody host species 

native to Finland. It was found that red elderberry (Sambucus racemosa) as well as 

six different Betula species which are typical deciduous tree species of the boreal 

forests were infected by CLRV; besides many virus affected silver and downy 

birches from locations all over the country, an individual sampled curly birch (B. 

pendula var. carelica) as well as several dwarf (B. nana), mountain (B. pubescens 

ssp. czerepanovii), and Kiilopää birches (B. pubescens ssp. appressa) growing in 

the northern part of the country up to the alpine tree line were CLRV affected. 

As expected symptomatic S. aucuparia trees were found to be infected by 

EMARAV; however an infection with CLRV of mountain ash could also be 

confirmed in two sampled trees revealing a mixed infection with the two viruses in 

a single case. 

573

574 

Sequence analysis of CLRV samples originating from birches in Finland based on 

short fragments of the coat protein (112 bp) and 3´ non-coding region (375 bp) 

revealed unique phylogenetic relationships of the virus isolates. 

REFERENCE 

Jalkanen, R., Büttner, C., von Bargen, S. (2007). Cherry leaf roll virus, CLRV, abundant on 

Betula pubescens in Finland. Silva Fennica 41, 755-762. 

Mielke, N., Mühlbach, H.P. (2007). A novel, multipartite, negative-strand RNA virus is 

associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.). 

Journal of General Virology 88, 1337-1346.

Bandte M, Essing M, Büttner C: Investigations on virus-diseased elm trees (Ulmus laevis L.) in eastern Germany. 

In: Feldmann F, Alford D V, Furk C: Crop Plant Resistance to Biotic and Abiotic Factors (2009), 575; ISBN 978- 


IUFRO-13 Investigations on virus-diseased elm trees (Ulmus laevis L.) in 

eastern Germany 

Bandte M, Essing M, Büttner C 




Abstract 

On elm trees in a public park planted in 1830 virus-like leaf symptoms and dieback 

were observed. Investigations focused on the identification of the casual agent. An 

infection with Cherry leaf roll virus (CLRV), Elm mottle virus (EMV), Arabis 

mosaic virus (ArMV) and Tobacco ringspot virus (TRSV), well known viruses to 

infect elm trees, could be excluded by bioassays and serological tests. 

Flexible particles of approximately 750 nm were isolated repeatedly from diseased 

elms. These particles are transmissible in plant sap of diseased elm leaves to 

herbaceous indicator plants such as Chenopodium species. The virus disqualifies as 

a member of the Potyviridae family based on an ELISA and an RT-PCR assay using 

a potyvirus genus-specific broad-spectrum polyclonal antibody and family-specific 

primers, respectively. Also no potyvirus-like pinwheel inclusions were found in leaf 

cells of infected indicator plants in electron microscopic studies. 

575

Bandte M, Fabich S, Bargen S von, Büttner C: Studies on Quercus robur - a perspective. In: Feldmann F, Alford 



IUFRO-14 Studies on Quercus robur - a perspective 

Bandte M 1 , Fabich S 2 , Bargen S von 1 , Büttner C 1 

1 



2 

DLR Rheinhessen-Nahe-Hunsrück, Rüdesheimer Str. 60–68, 55545 Bad Kreuznach 


576 

Abstract 

Virus-like symptoms such as distinct chlorotic lesions, ringspots and chlorotic 

mottle are often observed on leaves of oak trees and seedlings (Quercus robur L) 

growing at several forest stands and nurseries in the northern part of Germany. 

The same symptoms were induced on young oak seedlings after grafting. So far, the 

causal agent was not transmissible by mechanical inoculation of plant sap to 

indicator plants. Investigations by serological means demonstrated that the agent of 

virus-like symptoms of oak is not related to viruses widely spread in the forest 

ecosystem such as Tobacco mosaic virus, Tobacco necrosis virus, Brome mosaic 

virus, Cherry leaf roll virus and European mountain ash ringspot-associated virus. 

Different completely base paired double-stranded RNA (dsRNA) fragments 

indicated at 1.5 to 2.0 kbp were isolated from oak. Three types of dsRNA banding 

patterns occurred in the investigated oak leaf tissue independent of a symptom 

development due to a virus infection. The fragments were partially characterized 

physically and molecular. The number of the conformational transition and the 

denaturation profile of the two dsRNA structures each in type 1 and 2 are analogous 

with those of the four dsRNA structures of type 3. The denaturation profile of the 

individual dsRNA structures is very characteristic and allows the classification to 

one of the types by visual evaluation. Sequence analysis strongly indicates towards 

the presence of RdRp coding dsRNAs which are associated with the Partitivirus 

family, comprising two plant pathogenic cryptovirus genera not causing symptoms 

in their hosts. The characteristics of isolated dsRNA exclude them from being 

intermediate products of the causal agent of the disease.

Crop Plant Resistance to Biotic and Abiotic Factors ... - Die DPG

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