1 · Web view07.30 Breakfast 08.15 Coaches to Central Science Laboratory Session I Biology,...

Papers from these proceedings should be cited as for the example below:

Hoyte SM, Carbone I, Kohn LM, 2001. Isolate variability amongst Sclerotinia sclerotiorum from New Zealand kiwifruit orchards. In: Young CS, Hughes KJD, eds. Proceedings of Sclerotinia 2001 – The XI International Sclerotinia Workshop, York 8th-12th July 2001, York, England: Central Science Laboratory, York, England. 97-98.

The proceedings can also be viewed on the BSPP Website at:

http://www.bspp.org.uk

The Workshop is being held jointly under the auspices of the British Society for Plant Pathology and the Sclerotinia Committee of the International Society for Plant Pathology.

Donations in support of this workshop are gratefully acknowledged from:

British Society for Plant PathologyInternational Society for Plant Pathology

SyngentaBlackwell Science Ltd

Papers edited by

Caroline Young & Kelvin Hughes

Organising Committee:

Dr N V Hardwick (CSL, York)Dr M J Hocart (SAC, Edinburgh)

Prof. J R Steadman (University of Nebraska, USA)Prof. J M Whipps (HRI, Wellesbourne)Dr C S Young (ADAS, Wolverhampton)

Mr K J D Hughes (CSL, York)

Many thanks to Maggi Churchouse, Marketing Officer and Sue Sainty for secretarial assistance

BSPP web pages at: http://www.bspp.org.ukISPP web pages at:

http://www.isppweb.org/subcom.htm

Sclerotinia 2001 1

Welcome

A very warm welcome to delegates to the 11th International Sclerotinia Workshop. It is both a pleasure and a privilege to be your hosts for this the first meeting outside North America. We trust you will enjoy your stay in Yorkshire. You may wonder why we have decided on a split campus arrangement, with accommodation at the University of York and the talks and posters at the Central Science Laboratory (CSL). This will enable you to have the benefit of being near York, and the opportunities to experience a city with over 2000 years of history, and that of a modern laboratory with excellent conference facilities and active research on Sclerotinia and many other plant pathogens.

It seems a long time ago when, at the 7th International Congress of Plant Pathology held in Edinburgh in 1998, it was proposed that the next Workshop should be held in Europe. Since then the Sclerotinia Group has made a successful application to the International Society for Plant Pathology to become one of its special interest committees. This Workshop is therefore the first to be held under the banner of the ISPP. We will be holding the first meeting of the Committee in the last formal session of the Workshop to which everyone is of course invited.

The emphasis of the Workshop is on participation and informality. The programme is your programme, compiled from your submissions. We have provided plenty of opportunities for discussion during the formal sessions and contact over meal breaks, transport to and from CSL and during the tour. We hope you will take every opportunity to participate to the full by exchanging information on all aspects of this fascinating pathogen.

We would like to take this opportunity of thanking our donors, the British Society for Plant Pathology, the International Society for Plant Pathology, Blackwell Science Ltd and Syngenta for their generosity and support. Also we would like to thank the staff of the University of York and CSL who have enabled us to stage the Workshop.

Nigel HardwickFor and on behalf of the Organising Committee

Sclerotinia 2001 2

British SocietyFor Plant Pathology

President:Prof Chris Gilligan

International Society for Plant Pathology

President: Dr Peter Scott

The XI th International Sclerotinia Workshop

Central Science Laboratory,York, UK

Programme

Keynote speaker:

Prof. Linda Kohn, University of Toronto

Sclerotinia 2001 3

Programme

Sunday 8 July

16.00-19.00 Registration University of York19.00 Welcome reception19.30 Dinner

Monday 9 July

07.30 Breakfast08.15 Coaches to Central Science Laboratory

Session I Biology, taxonomy and molecular biology Page 15Chair: Nigel Hardwick, Central Science Laboratory, UK

09.00 Nigel Hardwick (Central Science Laboratory, UK) Welcome and introduction to CSL.

09.15 Keynote Address Prof L Kohn, University of Toronto, Canada

Integrating our genotypic diversity data towards a global picture of population subdivision and fine-scale structure in Sclerotinia - and implications for disease management.

Page 17

10.00 N Aytkhozhina & N Kolokolova, Institute of Microbiology and Virology, Kazakhstan

Characteristics of Kazakhstani isolates of Sclerotinia sclerotiorum.

Page 21

10.20 LS Kull, WL Pedersen & GL Hartman, University of Illinois, USAClonality and aggressiveness of Sclerotinia sclerotiorum.

Page 23

10.40 Discussion

11.00 TEA/COFFEE

Session II Biology, taxonomy and molecular biology (continued) Page 25Chair: Linda Kohn, University of Toronto, Canada

11.30 SB Durman, AB Menendez & AM Godeas, Ciudad Universitaria, ArgentinaMycelial compatibility groups in Sclerotinia sclerotiorum from agricultural fields in Argentina.

Page 27

11.50 ME Matheron & M Porchas, University of Arizona, USAImpact of soil moisture and temperature on viability of sclerotia of Sclerotinia minor and S. sclerotiorum.

Page 29

12.10 CR Lane & PA Beales, Central Science Laboratory, York, UKCamellia flower blight – first European record of Ciborinia camelliae an EU quarantine listed member of the family Sclerotinaceae.

Page 31

12.30

12.50 Discussion

13.00 LUNCH

14.00 Tour of Central Science Laboratory, Nigel Hardwick and colleagues

Sclerotinia 2001 4

Session III Posters I Page 87Chair: Jim Steadman, University of Nebraska, USA - 192

14.45 First session presenters to be by their posters.

15.45 COFFEE (to be taken during the poster sessions)

Session IV Posters II

Page 87 - 192

15.45 Second session presenters to be by their posters.

16.45 General discussion on posters led by Jim Steadman.

17.45 Depart for University

19.30 DINNER

Tuesday 10th

07.30 Breakfast

08.15 Coaches to CSL

Session V ControlChair: Alison Stewart, Lincoln University, New Zealand Page 33

09.00 JM Whipps, Horticulture Research International, UKDevelopments in the use of Coniothyrium minitans for the biocontrol of Sclerotinia sclerotiorum.

Page 35

09.20 P Lüth, PROPHYTA GmbH, Malchow, GermanyThe control of Sclerotinia spp. and Sclerotium cepivorum with the biological fungicide Contans®WG - experiences from field trials and commercial use

Page 37

09.40 BD Nelson, T Christianson & P McClean, North Dakota State University, USAEffects of bacteria on sclerotia of Sclerotinia sclerotiorum.

Page 39

10.00 PAG Elmer, SM Hoyte, T Reglinsky, R Marsden, F Parry, Horticultural and Food Research Institute, Hamilton, New Zealand Epicoccum nigrum: a biological control agent for the control of Sclerotinia

sclerotiorum in New Zealand Kiwifruit (Actinidia deliciosa)

Page 41

10.20 C Martin, Agriphyto, Perpignan, FranceIntegrated soil disease management on sclerotinia drop of salad by solarisation and green manure sequence in southern France (Roussillon).

Page 43

10.40 Discussion

11.00 COFFEE

Sclerotinia 2001 5

Session VI Control (continued) Page 45Chair: Alison Stewart, Lincoln University, New Zealand

11.30 JM Krupinsky, DL Tanaka, SD Merrill & RE Ries, USDA, Mandan, North Dakota, USA

Sclerotinia disease on canola, crambe, safflower, and sunflower as influenced by previous crops.

Page 47

11.50 MP McQuilken, Scottish Agricultural College, UK

Integrated control of sclerotinia disease in field-grown lettuce in Scotland.Page 49

12.10 RF Vieira, CMF Pinto & TJ de Paula Júnior, EPAMIG, BrazilFumigation with benomyl and fluazinam and their fungicidal effects in soil for white mould control (Sclerotinia sclerotiorum) control on dry beans.

Page 51

12.30 O Carisse, Agriculture and Agri-Food Canada, HRDC, Canada.Effect of Microsphaeropsis ochracea, a new biological control agent, on germination of sclerotia of Sclerotinia sclerotiorum.

Page 53

12.50 Discussion

13.00 LUNCH

Session VII TourLeader: Nigel Hardwick

14.00 to 18.30 Coach tour of North Yorkshire.

19.30 DINNER (York)

Wednesday 11th

07.30 Breakfast08.15 Coaches to CSL

Session VIII Resistance Chair: Jim Steadman, University of Nebraska, USA Page 55

09.00 V Hahn, Z Micic, AE Melchinger &E Bauer, University of Hohenheim, GermanyQTL-analysis of Sclerotinia sclerotiorum resistance in sunflower

Page 57

09.20 P Miklas, R Riley, K Grafton & P Gepts, USDA, USAQuantitative trait loci (QTL) conditioning resistance to white mould in common bean.

Page 59

09.40 DH Simmonds, PI Donaldson, T Anderson, S Hubbard, A Davidson, S Rioux, I Rajcan & P Gober, Agriculture & Agri-Food, Canada

Development of white mould resistant soybean.

Page 61

10.00 J Steadman, J Kolkman & KM Eskridge, University of Nebraska, Lincoln, USASearch for resistance to Sclerotinia sclerotiorum in common bean – screening and sources.

Page 63

10.20 HA Melouk & KD Chenault, Oklahoma State University, USASclerotinia blight resistance research in peanut.

Page 65

10.40 Discussion

11.00 COFFEE

Sclerotinia 2001 6

Session IX Pathology and epidemiology Page 67Chair: Berlin Nelson, North Dakota State University, USA

11.30 E Twengström, J Yuen & R Sigvald, University of Agricultural Sciences, Sweden

Apothecium development from sclerotia of Sclerotinia sclerotiorum in relation to rain and crop density.

Page 69

11.50 SM Hoyte, R Beresford & PG Long, Horticultural and Food Research Institute, Hamilton, New Zealand

Effects of temperature and relative humidity on colonisation of kiwifruit (Actinidia deliciosa) petals by Sclerotinia sclerotiorum ascospores.

Page 71

12.10 HA McCartney, A Heran, J Freeman & Q Li, IACR-Rothamsted, UKAscospores, petals and infection of oilseed rape by Sclerotinia sclerotiorum (see

abstracts P5.5 and P5.6)

Page 73

12.30 P Gladders, C Young, J Smith, M Watling, L Hiron, ADAS, UKFactors contributing to the adhesion of petals to oilseed rape leaves and risk of

sclerotinia stem rot.

Page 75

12.50 Discussion

13.00 LUNCH

Session X Epidemiology Page 77Chair: John Whipps, Horticulture Research International, UK

14.00 JP Clarkson, JM Whipps & CS Young, Horticulture Research International, UKEpidemiology of Sclerotinia sclerotiorum on lettuce

Page 79

14.20 J Freeman, C Calderon, E Ward & HA McCartney, IACR-Rothamsted, UKPolymerase chain reaction (PCR)-based assays for the detection of inoculum of Sclerotinia spp.

1

14.40 C Kora, MR McDonald & GJ Boland, University of Guelph, CanadaRelationships among environmental, pathogen and crop variables and their influence on sclerotinia rot of carrot [Sclerotinia sclerotiorum (Lib.) de Bary].

Page 83

15.00 GW Bourdôt, GA Hurrell & MD De Jong, AgResearch Limited, New ZealandRisk analysis of Sclerotinia sclerotiorum as a mycoherbicide for pasture weed control in New Zealand.

Page 85

15.20 Discussion

15.30 COFFEE

Session XI ISPP Sclerotinia CommitteeChair: Jim Steadman, University of Nebraska, USA

16.00 ISPP Sclerotinia Committee: business meeting (open to all)

17.45 Depart for University

19.30 DINNER

Thursday 12th

07.30 Breakfast08.15 Departure

Sclerotinia 2001 7

Posters Page 87

1. Biology, Taxonomy and Molecular Biology Page 89

1.1 Biochemical changes in abnormal sclerotia of Sclerotinia sclerotiorum 1HC Huang1 & JMYeung2,

1Agriculture and Agri-Food Canada, Research Centre, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1; 2National Food Processors Association, 1350 I Street NW, Washington, DC 20005, USA

1.2 Breeding, genetics, and mapping of QTL for architectural avoidance and physiological Page 93resistance to white mould in common beanDP Coyne1, SO Park1, JR Steadman1 & PW Skroch2

1 University of Nebraska, Lincoln, NE USA 68583; 2 8274 Andy Road, Waterloo, IL USA 62298

1.3 Introns can differentiate Sclerotinia sclerotiorum and S. trifoliorum Page 95KS Powers, JR Steadman, BS Higgins & TO PowersDepartment of Plant Pathology, University of Nebraska , Lincoln, Nebraska 68583-0722

1.4 Isolate variability amongst Sclerotinia sclerotiorum from New Zealand kiwifruit orchards Page 97

SM Hoyte1, I Carbone2 & LM Kohn2

1The Horticultural and Food Research Institute of New Zealand, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand; 2Erindale College, University of Toronto, Mississauga, Ontario L5L 1C6, Canada

1.5 Diagnostic assays for monilinia brown rot species; members of the family Sclerotiniaceae. Page 99

KJD Hughes, CR Lane & JN Banks Central Science Laboratory, MAFF, Sand Hutton, York, YO41 1LZ, U.K.

1.6 Regulation of lytic enzymes production upon plant infection by Sclerotinia sclerotiorum Page 101C Bruel, P Cotton, N Girard, R Letoublon, N Poussereau, G Billon-Grand, M-B Martel, C Rascle & M FèvreLaboratoire de Biologie Fongique – ERS 2009. Université Claude Bernard. Bat. 405 RdC. 43 Bd du 11 nov 1918. 69622 Villeurbanne.

1.7 Sclerotinia stem infection in flax in Western CanadaPage 103

KY Rashid Agriculture and Agri-Food Canada, Cereal Research Centre, Morden Research Station, Morden, Manitoba, Canada R6M 1Y5

2. Chemical Control Page 105

2.1 Management of sclerotinia blight, Sclerotinia minor, in Texas, USA peanut fields Page 107TA Lee, Jr. Department of Plant Pathology & Microbiology, Texas A&M University, Research & Extension Centre, Stephenville, TX 76401, USA.

2.2 Plant densities and fungicide effects on the intensity of white mould (Sclerotinia sclerotioru) Page 109of dry beansRF Vieira, CMF Pinto & ESG MizubutiEPAMIG, C.P. 216, Viçosa, MG 36571-000, Brazil.

2.3 Investigation of the mechanisms by which the post-harvest modification of the internal gas Page 111atmosphere of top fruit, helps sustain the innate resistance of host tissues to attack by fungal pathogens O BeletNatural Resources Institute, Food Systems Department, Medway University Campus, Chatham Maritime, ME4 4TB, UK

Sclerotinia 2001 8

2.4 Seed treatment for the control sclerotinia basal-stalk rot/wilt in sunflower Page 113KY Rashid & J SwansonAgriculture and Agri-Food Canada, Cereal Research Centre, Morden Research Station, Morden, Manitoba, Canada R6M 1Y5

2.5 Chemical control of Sclerotinia sclerotiorum with acetylsalicylic acid Page 115A Moret, M Nadal, N Canti & S SánchezDept. de Biologia Vegetal, Fac. De Biologia, 645, E-08028 Barcelona

2.6 Benzimidazole resistance of Sclerotinia sclerotiorum in French oilseed rape crops Page 117A Penaud1, B Huguet2, V Wilson2 & P Leroux3

1CETIOM, Centre de Grignon, B.P. 4, 78850 Thiverval-Grignon, France; 2SRPV Lle de France, 10 Rue de Séminaire, 94516 Rungis cedex, FR; 3INRA, Unité de Phytopharmacie er Médateurs Chimiques, Route de St Cyr, 78026 Versailles cedex, FR.

2.7 Effect of cycloheximide on curing of the hypovirulence of Sclerotinia sclerotiorum strain Page 119Ep-1PNG Li,1 D Jiang1, D Wang1, B Zhu1 & SR Rimmer2

1Dept of Plant Protection, Huazhong Agricultural University. Wuhan, 430070; 2Saskatoon Research Centre, Agricultural and Agri-Food Canada, Saskatoon, SK, S7N OX2, Canada.

3. Biological Control Page 121

3.1 Study of the fungal flora of sunflower varieties with different response to Page 123Sclerotinia sclerotiorumMA Rodríguez, N Venedikian & AM GodeasDepartamento de Biología, F.C.E.y N., Universidad de Buenos Aires, Cindad Universitaria, pab. II, 1428 Buenos Aires, Argentina

3.2 Comparative study of fungal antagonists against Sclerotinia sclerotiorum Page 125MA Rodriguez & AM GodeasDepartamento de Biología, F.C.E.y N., Universidad de Buenos Aires, Cindad Universitaria, pab. II, 1428 Buenos Aires, Argentina.

3.3 The biological fungicide Contans WG - a preparation on the bases of the fungus Page 127Coniothyrium minitansP LüthPROPHYTA Biologischer Pflanzenschutz GmbH, Inselstrasse 12, 23999 Malchow, Germany

3.4 Control of Sclerotinia within carrot crops in North East Scotland: the effect of irrigation Page 129and compost application on sclerotinia germination. G Couper, A Litterick & C LeifertAberdeen University Centre for Organic Agriculture

3.5 Biocontrol of Sclerotinia sclerotiorum by film-coating Coniothyrium minitans onto seed Page 131and sclerotiaMP McQuilken1 & JM Whipps2

1Department of Plant Biology, The Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK; 2Plant Pathology and Microbiology Department, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK

3.6 Prolonged control of Sclerotinia sclerotiorum with Sporidesmium sclerotivorum Page 133CA Martinson1 & LE del Rio2

1Department of Plant Pathology, Iowa State University, Ames, IA 50011-1020, USA; 2Department of Plant Pathology, North Dakota State University, Fargo, ND 58105-5012, USA

3.7 Solarization in the management of lettuce drop (Sclerotinia spp.) Page 135V Gepp, E Silvera, S Casanova & Dy Tricot.Facultad de Agronomma, Avda. Garzsn 780, 12900 Montevideo, Uruguay,

Sclerotinia 2001 9

3.8 Wounding of weeds enhances Sclerotinia sclerotiorum as a mycoherbicide Page 137GA Hurrell & GW BourdôtAgResearch Ltd, Gerald Street, P O Box 60, Lincoln, New Zealand.

3.9 Management of sclerotinia rot in Indian mustard – An integrated approach Page 139S SinghNational Centre for Integrated Pest Management, Pusa Campus, New Delhi, India - 110 012

3.10 Comparative antagonistic activity of Trichoderma harzianum, T. viride and Epicoccum Page 141purpurescens against Sclerotinia sclerotiorumRS Singh & J KaurDepartment of Plant Pathology, Punjab Agricultural University, Ludhiana-141001, India.

3.11 Evaluation of selected Trichoderma isolates against Sclerotinia sclerotiorum causing white Page 143rot of Brassica napus L.A Srinivasan, IS Kang, RS Singh & J KaurDepartment of Plant Pathology, Punjab Agricultural University, Ludhiana-141001, India.

3.12 Biological control of sclerotinia diseases of vegetables using Coniothyrium minitans A69 Page 145A Stewart, N Rabeendran, IJ Porter, TM Launonen & J Hunt, Soil, Plant & Ecological Sciences Division, P.O. Box 84, Lincoln University, New Zealand.

3.13 Control of sclerotinia stem rot of canola by aerial application of Coniothyrium minitans Page 147G Li,1 S Wei1, D Wang1, D Jiang1 & HC Huang2

1Dept of Plant Protection, Huazhong Agricultural University. Wuhan, 430070; 2Lethbridge Research Centre, Agricultural and Agri-Food Canada, Lethbridge, Alberta, TIJ 4B1 Canada.

3.14 Suppression of apothecial formation in Sclerotinia sclerotiorum by bacteria Page 149X Feng & C ThaningPlant Pathology & Biocontrol Unit, SLU, PO Box 7035, SE 750 07 Uppsala, Sweden.

4. Resistance Page 151

4.1 Potential new sources of resistance to white mould in the Phaseolus core collections Page 153KF Grafton1, JB Rasmussen2, JR Steadman3 & C. Donohue2 1Plant Sciences Dep.; 2Plant Pathology Dep., North Dakota State University, Fargo, ND 58105;3Plant Pathology Dep., University of Nebraska, Lincoln, NE 68583.

4.2 Light sensitivity of quantitative resistance in soybean caused by Sclerotinia sclerotiorum Page 155BW Pennypacker Department of Agronomy, Penn State, University Park, PA. USA

4.3 Sclerotinia minor from peanut: isolate aggressiveness and host resistance on detached leaves Page 157BB Shew & JE HollowellDepartment of Plant Pathology, North Carolina State University, Box 7616, Raleigh NC USA 27695-7616.

4.4 Defence gene expression in Sclerotinia infected susceptible and resistant soybean lines Page 159A Dorrance, M Graham & T Graham Department of Plant Pathology, The Ohio State University, Wooster, OH 44691, USA

4.5 Lactofen induces multiple defense mechanisms in soybean Page 161T Graham, A Dorrance, S Landini & M Graham Department of Plant Pathology, The Ohio State University, Columbus, dH 43210, USA

4.6 Evaluation and breeding soybeans for resistance to sclerotinia stem rot Page 163TD Vuong1, LSG Kull1, D Hoffman, BW Diers1, WL Pederson1, L Aydogdu, RL Nelson2 & GL Hartman2

1Department of Crop Sciences, University of Illinois, 1101 Peabody Drive, Urbana, IL 61801, USA, email: 2USDA-ARS and Department of Crop Sciences, University of Illinois, 1101 Peabody Drive, Urbana, IL 61801, USA

Sclerotinia 2001 10

4.7 Tracing the origin of QTLs for resistance to Sclerotinia sclerotiorum in soybean ancestral Page 165lines V Arahana1, G Graef1, J Specht1 & JR Steadman2 1Department of Agronomy and Horticulture and 2Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0915

4.8 Pyramiding transgenes with QTLs to enhance resistance to Sclerotinia sclerotiorum in Page 167soybeanV Arahana1, G Graef1, T Clemente1, JR Steadman2, J Specht1, A Mitra2 & T Buhr1Department of Agronomy and Horticulture and 2Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0915

4.9 Use of induced resistance to control of soybean sclerotinia stem rot Page 169XB Yang & P Lundeen Department of Plant Pathology, Iowa State University, Ames 50011, USA

4.10 Inheritance and mechanisms of resistance to Sclerotinia sclerotiorum and in vitro Page 171mutagenesis via microspore culture in Brassica napus S-Y Liu, ZY Xu, JK Zhang, HZ Wang, YJ Huang & LY HeOil Crops Research Institute of CAAS, Wuhan 430062

4.11 QTL Analysis of Resistance to White Mould in Common Bean Page 173J. D. Kelly a and J.M. Kolkmanb aDepartment of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA; & bDepartment of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

5. Pathology and Epidemiology Page 175

5.1 Sclerotinia sclerotiorum - a new threat to mustard cultivation in Rajasthan, India Page 177A Shivpuri, AK Bhargava & HP ChhipaPlant Pathology Department, Agriculture Research Station, Durgapura, Jaipur, Rajasthan, India

5.2 Adaptation and importance of Sclerotinia sclerotiorum in North Dakota Page 179BD Nelson

Dept. Plant Pathology, North Dakota State University, Fargo, 58105, North Dakota, USA.

5.3 Environmental factors influencing apothecial production and lettuce infection by Page 181Sclerotinia sclerotiorum in field conditionsCS Young1, P Gladders1, JA Smith1, M Watling1, JM Whipps2 & JP Clarkson2

1ADAS 'Woodthorne', Wergs Road, Wolverhampton WV6 8TQ, UK; 2HRI, Wellesbourne, Warwick, CV35 9EF, UK

5.4 Infection of oilseed rape (Brassica napus) by petals containing ascospores of Page 183Sclerotinia sclerotiorumHA McCartney, A Heran & Q LiPlantPathogen Interactions, IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ,UK

5.5 Petal fall, petal retention and petal duration in oilseed rape crops Page 185HA McCartney, A Heran, Q Li & J FreemanPlant Pathogen Interactions, IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ,UK

5.6 Temporal and spatial distribution of ascospores of Sclerotinia sclerotiorum around Page 187tobacco greenhouses in North CarolinaHM Harzog, TA Melton & HD ShewDepartment of Plant Pathology, NC State University, Raleigh, NC 27695-7616, USA

5.7 Susceptibility of spring oilseed rape to sclerotinia stem rot in Poland and the Czech RepublicPage 189

1M Jedrycza, 1S Dakowska & 2E Plachka1Institute of Plant Genetics, Polish Academy of Sciences, Strzesznska 34, 60-479, Poland; 2OSEVA PRO, Institute of Oil Crops, Purkynova 6, 746-01 OPAVA, The Czech Republic.

Sclerotinia 2001 11

5.8 Factors affecting the production of ascospores of Sclerotinia sclerotiorum(Lib) de bary Page 191and their role in infection of sunflowerR Singh & NN TripathiDepartment of Plant Pathology, CCS HAY-Hisar-125004-India.

Delegate list Page 193

Sclerotinia 2001 12







Talks

Sclerotinia 2001 13

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Sclerotinia 2001 14

SESSION I

BIOLOGY, TAXONOMY AND MOLECULAR BIOLOGY

Chair:

Nigel Hardwick, Central Science Laboratory, UK

Sclerotinia 2001 15


Sclerotinia 2001 16

S1.1Keynote address

Integrating our genotypic diversity data: towards a global picture of population subdivision and fine-scale structure in Sclerotinia, with implications for disease management

L. M. Kohn

Department of Botany, University of Toronto, Mississauga, Ontario, Canada L5L 1C6

AbstractFour tenets of Sclerotinia population structure are presented. Genotyping tools (including new, easy-to-use microsatellite primers) and analyses that capture both ancient and contemporary population history at large and small geographical scales are reviewed. Finally, I propose integrated, cooperative research for global benefit.

IntroductionMy objective here is to convince you that pathogen variability impacts cultivar durability and disease management, that genotyping technology is now within reach of most of our labs, and that synergy will come from coordinated, cooperative study of Sclerotinia populations worldwide.

Four tenets of Sclerotinia population structure. All field samples of S. sclerotiorum made to date have been genetically heterogeneous. The world population of S. sclerotiorum is composed of subpopulations, some of which are older or isolated and endemic, others of which are younger or highly dispersed. From crop samples, there is more recombination in subpopulations in subtropical climates than in those in temperate zones. A few clonal genotypes of S. sclerotiorum predominate in any sample; preliminary data suggest that this extends to S. minor, but not to S. trifoliorum.

It makes sense to factor pathogen diversity into disease management. Inconsistent correlations between field and greenhouse disease evaluations (Kim et al., 2000) may stem from incorrect assumptions of genetic uniformity in pathogen populations. While differences in pathogenicity or aggressiveness among S. sclerotiorum genotypes have been difficult to demonstrate, we do know that some clones produce more apothecia per unit sclerotia than others and that some of these highly fecund clones are also sampled more frequently in the field. Fit clonal genotypes are epidemiologically significant. It makes sense to use them in screening for partial resistance in new varieties. It also makes sense to monitor pathogen genotypic diversity in performance trials; new crop varieties with partial resistance can be screened for durability against both established and emerging pathogen genotypes. Similarly, screening against frequently sampled pathogen genotypes makes sense in biocontrol (Carpenter et al., 1999). MethodsEfficient systems for characterizing and comparing genetic variability in S. sclerotiorum are already in hand and genotyping technology is now within reach of most of our labs. Until recently, population studies of S. sclerotiorum were designed to characterize contemporary field populations – as are most new marker systems. These studies made use of

Sclerotinia 2001 17

S1.1Keynote address

two, independent genotyping systems: mycelial compatibility groups (MCGs), and DNA fingerprints with a dispersed, repeated element, pLK44.20 (Carbone & Kohn, 1999; Carpenter et al., 1999; Cubeta et al., 1997; Errampalli & Kohn, 1995; Kohn et al., 1991; Kohli & Kohn, 1998; Kohli et al., 1995; Kull et al., 2000; Manandhar et al., 1998; for primer development see Carbone & Kohn, 1999; Carbone et al., 1999). When paired in culture, all members of an MCG fuse to form one confluent colony with no reaction line; they also share the same unique, complex DNA fingerprint (occasionally with 1-2 variant hybridizing bands among isolates). Members of different MCGs are incompatible, forming a barrage line where they meet; they also have DNA fingerprints that differ by relatively many bands (Carbone et al., 1999; Kohn et al., 1991). A group of isolates sharing the same DNA fingerprint and MCG is interpreted as a clone (note that the offspring are not “carbon copies” but rather a lineage in which mutations will accrue). Fifty-three fragments in each fingerprint are scored as present or absent; the Kohn laboratory maintains an archive of fingerprints for over 3,000 isolates from many hosts, from North America, New Zealand and Europe, to which fingerprints from new isolates can be compared by means of neighbor joining analysis. In order to determine how these clones are related (the clone genealogy or family tree), 385 of these isolates were haplotyped based on DNA sequence polymorphisms at 7 loci (300-4 000 bp at each locus; Carbone & Kohn, 2001; Carbone et al., 1999). Individuals of the same “haplotype” have the same nucleotides at variable positions as well as at invariant positions in one or more loci (genomic regions). “Genotype” is a more general term for individuals of S. sclerotiorum distinguished by a DNA fingerprint, karyotype (Errampalli & Kohn, 1996; Fraissinet-Tachet et al., 1996) or haplotype, or belonging to a mycelial compatibility group (MCG). Phylogenetic, coalescent and conventional chi square permutation and ANOVA statistical approaches not only trace genealogy but also detect associations of haplotype with geographical area, host, symptom type or genome size (Carbone & Kohn, 2001) as well as DNA fingerprint and MCG. Our lab has also developed 25 microsatellite markers, 14 of which also amplify S. minor, and 12 of which amplify S. trifoliorum (Sirjusingh & Kohn, 2001). Polymorphisms are detected at some microsatellite loci among isolates with the same fingerprint, so it will be possible to identify the most recently evolved genotypes (< 100 years). DNA fingerprint and microsatellite reference archives will be cross-referenced. To efficiently answer a research question, scale is a consideration when markers and sampling strategy are matched, e.g. continent vs. field, or thousands vs. hundreds of years of evolution.Sequence data, fingerprints and MCG designations from the Kohn laboratory can be accessed at http://www.erin.utoronto.ca/~kohn/

ResultsPractical questions can be answered. 1) Canola, grown in the SE US for only the past 20-30 years, is infected by both indigenous and mobile, dispersed genotype populations (Carbone & Kohn, 2001). Genotypes from these two kinds of populations show significant associations with symptom and host variety (unpublished). 2) Of 180 soybean and 21 edible bean isolates from Ontario, Quebec and Pennsylvania, only 15% of the genotypes were new, clones previously identified from Ontario canola in 1989 made up > 50% of the sample (unpublished). Soybeans from this geographical region are likely infected by inoculum residual from other crops.

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S1.1Keynote address

ConclusionsCooperative research will yield global benefits. People and agricultural produce are mobile as never before. I believe that our objective is an understanding of genetic diversity in Sclerotinia subpopulations worldwide. To achieve this, we should use the same genetic markers, expand and cross reference data bases, use the same positive controls, standards and voucher isolates, and provide genotyped and tested isolates of epidemiological significance for research on pathogenesis, control and resistance.

ReferencesCarbone I, Kohn LM, 2001. A microbial population-species interface: nested cladistic and

coalescent inference with multilocus data. Molec Ecol 10, 947-967.Carbone I, Kohn LM, 1999. A method for designing primer sets for speciation studies in

filamentous ascomycetes Mycologia 91, 553-556.Carbone I, Anderson JB, Kohn LM, 1999. Patterns of descent in clonal lineages and their

multilocus fingerprints are resolved with combined gene genealogies. Evolution 53, 11-21.

Carpenter MA, Frampton C, Stewart A, 1999. Genetic variation in New Zealand populations of the plant pathogen Sclerotinia sclerotiorum. New Zealand J Crop Hort Sci 27, 13-21.

Cubeta MA, Cody BR, Kohli Y, Kohn L,1997. Clonality in Sclerotinia sclerotiorum on infected cabbage in Eastern North Carolina. Phytopathology 87, 1000-1004.

Errampalli D, Kohn LM, 1995. Comparison of pectic zymograms produced by different clones of Sclerotinia sclerotiorum in culture. Phytopathology 85, 292-298.

Errampalli D, Kohn LM, 1996. Electrophoretic karyotypes of Sclerotinia sclerotiorum. Appl Env Microbiol 62, 4247-4251.

Fraissinet-Tachet L, Reymond-Cotton P, Fevre M, 1996. Molecular karyotype of the phytopathogenic fungus Sclerotinia sclerotiorum. Curr Genet 29, 496-501.

Kim HS et al., 2000. Reaction of soybean cultivars to Sclerotinia stem rot in field, greenhouse, and laboratory evaluations. Crop Sci 40, 665-669.

Kohn LM, Stasovski E, Carbone I, Royer J, Anderson JB, 1991. Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum. Phytopathology 81, 480-485.

Kohli Y, Kohn LM, 1998. Detection of random association among loci in clonal populations of Sclerotinia sclerotiorum. Fungal Genet and Biol 23, 139-149.

Kohli Y, Brunner LJ, Yoell H, Milgroom MG, Anderson JB, Morrall RAA, Kohn LM, 1995. Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus Sclerotinia sclerotiorum. Molec Ecol 4, 69-77.

Kull LS, W L Pedersen, GL Hartman, 2000. Aggressiveness and mycelial compatibility among isolates of Sclerotinia sclerotiorum. Abstract, Ann. Mtg. Am Phytopath Soc.

Manandhar, JB Kull LS, Mueller DS, Hartman GL, Pedersen WL, 1998. Sclerotinia sclerotiorum in soybeans: pathogenic variability, host resistance, and seed infection. Abstract, Int’l Sclerotinia Workshop, North Dakota State University, Fargo, USA pp36-37.

Sirjusingh C, Kohn LM, 2001. Characterization of microsatellites in the fungal pathogen, Sclerotinia sclerotiorum. Molec Ecol Notes (In press, PDF available on request).

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Sclerotinia 2001 20

S1.2

Characteristics of Kazakhstani isolates of Sclerotinia sclerotiorum

N.A.Aytkhozhina and N.K.Kolokolova

Lab. of Plant Pathology, Inst. Microbiology & Virology, Bogenbai Batyr Street 103, Almaty 480100, Kazakhstan

Abstract Isolates of Sclerotinia sclerotiorum, a casual agent of white rot of diverse economically important plants have been studied under laboratory and field conditions. Growth and development pattern under the light microscope, ultrastructural characteristics of initial and mature sclerotia, apothecia and ascospores have been revealed.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary is an important fungal pathogen of many plant hosts and is responsible for substantial losses in crop production in Kazakhstan and worldwide. Symptoms include stem, leaf, root and fruit rot or cancer. Infection of plants by S. sclerotiorum occurs primarily by mycelogenic germination of sclerotia and production of enormous amounts of inoculum. Sclerotia are specialized structures that confer the ability to survive severe unfavorable environments. They allow the fungus to effectively colonize ecologically different niches. Considerable variation in morphology and pathogenicity has been observed among S. sclerotiorum isolates (Willets, 1980). Until now little data from Kazakhstan concerning S. sclerotiorum has been published.

Materials and MethodsCultures of S. sclerotiorum were originally isolated from diseased plants from fields, orchards and greenhouses in Kazakhstan (source/isolate code: aubergine/A1,A2, a1; tomato/T1,T2; cucumber/C1,C2; carrots/Cr1,Cr2; sunflower/S1,S2; fruits /Pf1 and twigs/Pt1 of pear tree; strawberry/Sw1,Sw2; beans/B1,B2). Stock cultures for macro observations were inoculated on PDA agar in 9cm diam Petri dishes and incubated at 5,15,20,23,28°C upto 14 days. To induce sclerotia germination followed by apothecia formation, sclerotia were kept for 1-2 months to break dormancy or exposed to UV-light then placed on PDA, water, water agar, moistened sand and/or soil. Microscopic examination of isolates grown on glass slides with thin layer of agar were made daily for 7 days to study micro-development pattern. Fine mycelia, sclerotia and apothecia structures were also examined using a transmission electron microscope.

Results and DiscussionPlate observation: Cultures C1, C2, Cr1, Cr2, T2 produced abundant white lawns of fur-like mycelium while B2, Pf1, Pt1, T1, Sw1, Sw2 was more scanty. Incipient sclerotia appeared in 3-4 days and later graphite grey or black mature sclerotia were seen throughout the mycelial lawn. Sclerotia size and arrangement varied depending on isolate source, media composition, and incubation temperature. A1, T1 and Sw1 sclerotia usually formed at the plate edge; C1, Pt1, B1 sclerotia covered the substrate in concentric circles and S1 sclerotia were loosely scattered on a scanty mycelium lawn. B1 strain formed no sclerotia and always remained as a white lawn of abundant fur-like mycelia. Most isolates germinated by means of light brown cup-shaped apothecia (often frilly, 0.2-0.5 cm diam) on stalk up to 1.5-2 cm long. Most apothecial stipes were found on sclerotia exposed to UV- light or pre-conditioned at 4°C.

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Ascospores exposed for 120s to UV-light developed sclerotia nonforming mycelium. The characteristic pattern of onthogenesis was: i) Development of sclerotia components and their assembly; ii) Sclerotia germination followed by development of apothecia with ascospores. The appearance of phialids bearing microconidia in some old cultures was also noteworthy.Light microscope observation: Sclerotium forming plan comprised of fast growing initial hyphae with numerous small outgrowths blown out from the hyphal wall, coalescence in pairs, tip-to-tip and tip-to-lateral branch fusions, anastomosis and forming of loop-like structures (not previously described), finally made the overall picture like a net. The interweaving interior of net cells provided the firmness to primordia, and consequently, future sclerotia. Latterly, a cluster of individual pre-sclerotia assembled in mature visible sclerotium. Inner components of sclerotia, probably, held together by mucilage, melanin and old stahling hyphae. Our findings also showed disturbed initial and subsequent individual development steps in a mutant a1 isolate which had a different growth pattern; e.g. runner hyphae didn’t ramify extensively, only a few anastomoses were observed, and typical primordia were hardly initiated. As a result, the a1 isolate formed no sclerotia. Light microscopy of median sections of mature sclerotia showed two or three rows of morphologically identical polyhedral or round shaped and covered with melanin marginal cells of the rind. A cortical region arranged beneath the rind was composed of closely packed and pseudoparenchymatous in appearance isodiametric cells with intensive cell wall material. The next large zone revealed loosely arranged medullary cells with hyphal-like appearance. Electron microscope observation: The general organization of mycelial organization was similar to other ascomycetous groups. Average sized vacuoles filled with moderate and high density bodies acting as the main storage material in presclerotial septate hyphal aggregates and mature sclerotium cells of the rind, cortex and medulla were observed. Those of high density were of two types, fully filled with black bodies and smaller ones containing vesicle-like structures often invaginated inside protein bodies as described by Kohn & Grenville, (1989). Rind cells were heavily impregnated with melanin that easily penetrated into the septum interior. Medullar cells were embedded in copious matrix, septae and subtended Woronin bodies, numerous microbodies and well developed (stacked) endoplasmic reticulum (ER) were also observed. In field-collected sclerotia disrupted rind areas, more melanin deposits and less organelles were distinct. The mycelial cells of the a1 isolate showed very dense cytoplasm filling with cellular organelles, enormous ribosomes and polysomes and vacuoles lacking in above mentioned storage material. Ultrastructure of ectal, medullary excipulum, hymenium, subhymenium and ascospores of apothecia were similar to those described by Willets, (1980). Young excipulum cells contained short cisterns of ER, mitochondria; subhymenium hyphae characterized by nonperforated septae. Hymenium consisted of paraphyses and asci with 8 ascospores in each ascus. Some asci and ascospores were filled with high electron density granules very similar to polyphosphate ones. Ascospores contained two nuclei. This comparison of isolates from various sources showed special characteristics and general ones previously described.

ReferencesWillets HJ, 1980. The biology of Sclerotinia sclerotiorum, S. trifoliorum, and S. minor with

emphasis on specific nomenclature. The Botanical Review 46, 101-165.Kohn LM, Grenville DJ, 1989. Ultrastructure of stromatal anamorphs in the Sclerotinaceae.

Canadian Journal of Botany 67, 394-406.

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S1.3

Mycelial compatibility grouping and aggressiveness of Sclerotinia sclerotiorum

L.S. Kulla, W.L. Pedersena, and G.L. Hartmana,b

aDept. of Crop Sciences, University of Illinois; and bUSDA/ARS,1101 W. Peabody, Urbana, Illinois 61801, USA

AbstractMycelial compatibility and aggressiveness of Sclerotinia sclerotiorum isolates were assessed. Among 302 isolates, 40 mycelial compatibility groups (MCGs) were identified with the majority of the MCGs sampled once at single locations. MCGs in two Illinois soybean fields were spatially clustered. Isolate aggressiveness varied, but cultivar by isolate interactions were not observed.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary has a wide geographic distribution and a diverse host range. On soybeans, S. sclerotiorum causes Sclerotinia stem rot (SSR), an important yield-reducing disease in the United States and a major disease on soybeans in Illinois (Hartman et al., 1998). High levels of physiological resistance to S. sclerotiorum have not been reported in soybean, and correlations between field and greenhouse resistance evaluations have been low (Nelson et al., 1991; Kim et al., 2000). Problems associated with evaluating and breeding for resistance may in part be due to the influence of pathogen population structure and variation in isolate aggressiveness. The objectives of this study were to determine the spatial distribution of MCGs in soybean fields and to assess the variability in aggressiveness of S. sclerotiorum isolates.

Materials and Methods A set of geographically diverse isolates and three soybean field isolate collections (Illinois and Argentina) totaling 302 isolates were utilized. The two Illinois soybean fields were systematically sampled. Mycelial cultures were maintained on potato dextrose agar at 4oC. MCGs were determined using published protocols (Kohn et al., 1990), and isolate aggressiveness was assessed using a limited-term, plug inoculation technique (Kim et al., 2000). For cultivar by isolate tests, partial resistant cultivars NKS19-90 and A2506, intermediate Bell and Elgin, and susceptible cultivars A2242 and Williams82 were inoculated with six isolates that varied in aggressiveness. Plants were assessed over time and the area under disease progress curve (AUDPC) was calculated. Experimental design for isolate aggressiveness tests was a randomized complete block with four replications and four plants per replication. Data were analyzed using Proc ANOVA or Proc GLM (SAS Institute, Inc.), and AUDPC means were compared by least significant differences at P = 0.05. Spatial distribution of MCGs within the Illinois fields was determined by spatial autocorrelation analysis using the AutocorG program (Hardy & Vekemans, 1999).

Results and DiscussionAmong 302 S. sclerotiorum isolates, 40 MCGs were identified with 65% of the MCGs sampled once at single locations, 25% sampled locally at low to high frequencies, and the

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remaining 10% were sampled at high frequencies at multiple locations. Both Illinois soybean fields contained two or three high frequency MCGs and a larger proportion of low frequency MCGs. Three MCGs were sampled at multiple locations. MCG 8 was detected in soybean fields in several states, on soybean in Switzerland, on canola in Canada, and composed 53% and 34% of the isolates within the Watseka and DeKalb, IL fields, respectively. No common MCGs were observed between Argentina and any other location indicating little or no movement of propagules, selection for specific MCG genotypes, or a small sampling size.Field populations of S. sclerotiorum on soybean are a heterogeneous mix of MCGs with significant spatial clustering which differs from Canadian canola MCGs that show no significant spatial aggregation. These differences in spatial distribution may be due to level of sampling, endemic vs. more recent infestation, or choice of spatial analysis programs. Aggressiveness of field isolates varied (P = 0.05), and variation in aggressiveness was highly significant (P < 0.001) for isolates in MCGs composed of members from different locations. MCGs do not appear to vary in aggressiveness. Cultivar by isolate interactions were not observed (P = 0.05), but cultivar performance varied when inoculated with isolates that vary in aggressiveness. Greenhouse resistance screening programs may be conducted with one isolate of unknown relative aggressiveness and may not represent the range of pathogen variability existing in cultivated field populations. Designing field resistance evaluation experiments to eliminate or compensate for the variability in isolate aggressiveness and MCG profile may not be possible.

AcknowledgmentsThis work was supported by the Illinois Soybean Promotion Operating Board, and the North Central Soybean Research program. We thank Ron Warsaw, Katie Urish, Kristina Roemer, and Kelley Smith for field, greenhouse and laboratory assistance; Vivek Singh, Erica Bakker and Xavier Vekemans for data analysis; and various individuals for donation of isolates.

ReferencesHardy OJ, Vekemans X, 1999. Isolation by distance in a continuous population:

reconciliation between spatial autocorrelation analysis and population genetics models. Heredity 83, 145-154.

Hartman GL, Kull LS, Huang YH, 1998. Occurrence of Sclerotinia sclerotiorum in soybean fields in East-Central Illinois and enumeration of inocula in soybean seed lots. Plant Dis. 82, 560-564.

Kim HS, Hartman GL, Manandhar JB, Grief GL, Steadman JR, Diers BW. 2000. Reaction of soybean cultivars to Sclerotinia stem rot in field, greenhouse, and laboratory evaluations. Crop Sci. 40, 665-669.

Kohn LM, Carbone I, Anderson JB. 1990. Mycelial interactions in Sclerotinia sclerotiorum. Experimental Mycology 14, 255-267.

Kohn LM, Stasoviski E, Carbone I, Royer J, and Anderson JB. 1991. Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum. Phytopathology 81, 480-485.

Nelson BD, Helms TC, Olson JA. 1991. Comparison of laboratory and field evaluations of resistance in soybean to Sclerotinia sclerotiorum. Plant Dis. 75, 662-665.

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SESSION II

BIOLOGY, TAXONOMY AND MOLECULAR BIOLOGY (CONTINUED)

Chair:

Linda Kohn, University of Toronto, Canada

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S2.1

Mycelial Compatibility Groups in Sclerotinia sclerotiorum from Agricultural Fields in Argentina

S. B. Durman*, A. B. Menéndez and A. M. Godeas

Department of Biology, Faculty of Exact and Natural Sciences, University of Buenos Aires, Pabellón II, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina.

AbstractOne hundred and forty isolates of Sclerotinia sclerotiorum were obtained from agricultural fields in Argentina during 1998-2000. They were grouped in 50 mycelial compatibility groups (MCG). All isolates were morphologically characterized on solid medium and were tested for virulence on celery petioles. Results are discussed.

IntroductionIn Argentina, S. sclerotiorum is one of the most important fungal pathogen of some economically important crops, causing diseases like Sclerotinia stem rot of soybean, head rot of sunflower and stalk rot of sunflower. Also, infection of peanut is a serious problem in the province of Córdoba, which concentrates 98% of Argentinian peanut yield. In addition, Sclerotinia lettuce drop causes great losses in the Gran Buenos Aires Horticultural Belt.It has been stablished that field population of S. sclerotiorum are clonal and that several clones may infect each field (Kohn, 1994). One of the criterions for detecting clonality is the mycelial compatiblity grouping.The objective of this work was to determine the presence of different MCGs among a set of isolates from various hosts and fields from the Buenos Aires province, and to estimate diversity. We also characterized each MCG according to virulence and morphology on solid medium.

Materials and MethodsIsolates used in this study were obtained from sclerotia sampled from various hosts and from different fields along the Buenos Aires province. To test mycelial compatibility isolates were confronted following the procedure of Kohn et al. (1990). Shannon Index (Hs) was calculated for assessing MCG diversity (Robin et al., 2000). Isolates were morphologically characterized on Patterson’s medium (Patterson, 1986) in 15-mm Petri plates at room temperature in the dark. To assess isolate virulence, pieces of detached celery petioles were inoculated with agar-mycelia plugs and incubated onto water agar. Lesions were measured after 72 h.

Results and DiscussionAfter 140 isolates were paired, 50 MCGs were distinguished. 27 MCGs consisted of two or more isolates (MCG 3 had 12 isolates) and the remaining 23 MCGs were each made up of one isolate, compatible only with itself.

* Corresponding author: [email protected]

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In most pairings, mycelial incompatibility was not detected by the presence of a red reaction line, instead, there was, usually, an interaction zone of sparsely mycelium.The calculated Shannon Index for the sample was 3.6. MCG 3 was the most frequent one, as it was identified in five different localities, and ten MCGs were made up by isolates sampled in different years.None of the morphological characteristics could be related to the grouping made by mycelial compatibility. Virulence showed neither relationship to morphological characteristics nor to mycelial compatibility grouping.This is the first report of the existence of mycelial compatibility groups in S. sclerotiorum from field crops in Argentina. This study has demostrated that Argentinian populations of S. sclerotiorum from field crops are made up by various and different MCGs. These populations presented a frequency profile in which many MCGs are recovered once or twice and locally, and few MCGs ocurred at high frequency and at far-off places. Due to the heterogeneity, on the analized variables, among the Argentinian isolates within each MCG, any imposed groups based on morphological or pathological characters would not be easily related to the mycelial compatibility grouping.

Acknowledgements The research was conducted at the Soil microbiology Laboratory, University of Buenos Aires. S. Durman has been supported by a Research Grant, “Susceptibility of different Sclerotinia sclerotiorum clones against biocontrol agents”, from the National Council of Technic and Science (CONICET).

ReferencesAnderson JB, Kohn LM, 1995. Clonality in soilborne plant-pathogenic fungi. Annales

Review of Phytopathology 33, 369-393.Kohn LM, Carbone I, Anderson JB, 1990. Mycelial interactions in Sclerotinia sclerotiorum.

Experimental Mycology 14, 255-267.Kohn LM. 1994. The clonal dynamic in wild and agricultural plant-pathogen populations.

Canadian Journal of Botany 73, S12312-S1240.Patterson CL, 1986. Comparative biology, epidemiology, and control of lettuce drop caused

by Sclerotinia minor and S. sclerotiorum and the genetic analysis of vegetative and sexual compatibility in S. minor. Ph. D. Thesis, University of California, Davis.

Robin C, Anziani C, Cortesi P, 2000. Relationship between biological control, incidence of hypovirulence, and diversity of vegetative compatibility types of Cryphonectria parasitica in France. Phytopathology 90, 730-737.

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S2.2

Impact of soil moisture and temperature on viability of sclerotia of Sclerotinia minor and S. sclerotiorum

M.E. Matheron and M. Porchas

Department of Plant Pathology, Yuma Agricultural Center, University of Arizona, Yuma, AZ 85364 USA

AbstractGrowth chamber and field studies revealed that the number of viable sclerotia of Sclerotinia minor and S. sclerotiorum declined significantly in irrigated soil at temperatures normally occurring during the summer in the desert southwest lettuce production region of Arizona.

IntroductionSclerotinia minor and S. sclerotiorum are the two fungal pathogens that cause leaf drop of lettuce in Arizona. During the winter months from late December through early March, Sclerotinia leaf drop can cause significant economic losses in lettuce fields. During the same time period, 80 to 90% of the total lettuce production in the United States is harvested from the desert southwest, including southwestern Arizona and southeastern California. In the desert production region, lettuce is primarily grown on raised beds with two rows of lettuce on each bed spaced 30 cm apart. After crop harvest, the lettuce residue typically is incorporated into the soil by disking, followed by planting of wheat on non-bedded soil or sowing of melons, cotton, corn or safflower on newly constructed beds.

Since lettuce has become the dominant vegetable crop in southwest Arizona from September through March, the incidence of lettuce drop caused by S. minor and S. sclerotiorum has increased. To develop a successful management program for this disease, we need to understand the fate of sclerotia of both pathogens from the time of their production in diseased lettuce fields during the winter through the remainder of the year when lettuce is not present. To this end, laboratory and field studies were performed to evaluate the effect of soil moisture and temperature on the viability of sclerotia of S. minor and S. sclerotiorum

Materials and MethodsIn laboratory studies, sclerotia of S. minor and S. sclerotiorum were placed within nylon-mesh packets and buried 2.5 cm below the surface of a dry field soil (7-56-37 sand-silt-clay) in a series of containers 7.5 cm in diameter and 10 cm deep, each with three 2.0-mm-diameter drain-holes in the bottom. Containers with sclerotia and soil then were incubated at 15, 20, 25, 30, 35 or 40C for 1 to 4 weeks. At each temperature, the soil in half of the containers remained dry for the duration of each trial, whereas the soil in the remaining containers was irrigated with enough water to thoroughly wet the soil. Containers with irrigated soil were placed on trays maintained with a 1-cm-deep layer of water to preserve moisture in the soil. At 1, 2, 3, and 4 weeks after burial in soil, sclerotia were collected, surface-sterilized, then plated onto potato dextrose agar (PDA) to determine their viability, which was determined after 14 days by noting growth of typical Sclerotinia mycelia from sclerotia as well as formation of daughter sclerotia.

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Field trials were established at the Yuma Agricultural Center in dry silty clay loam soil typically used for lettuce production. The same soil was used in the laboratory studies. Small nylon mesh packets (7.5 cm long x 4.0 cm wide) were each filled with 15 sclerotia of S. minor or S. sclerotiorum. Sclerotia were placed on the soil surface or buried at a depth of 5 cm within furrows in the field. The soil containing sclerotia was either irrigated periodically or maintained in a dry state. Temperature sensors and gypsum blocks were placed at the soil surface and 5 cm deep to monitor temperature and soil moisture, respectively. At 2, 4, 6, and 8 weeks after burial in soil, sclerotia were collected, surface-sterilized, then plated onto PDA to determine their viability. A total of eight field tests were conducted at different times of the year and with irrigation frequencies ranging from every 2 to 14 days.

Results and DiscussionAs the incubation temperature increased from 15 to 30 C, the percentage of viable sclerotia of S. minor recovered from wet soil during the 4-week incubation period gradually declined from 74 to 38%; however, the number of viable sclerotia at 35 and 40 C was 6 and 0%, respectively. For S. sclerotiorum, incubation at 15 to 30 C in wet soil resulted in sclerotia viability ranging from 42 to 22%, respectively; however, at 35 and 40 C the percentage of viable sclerotia dropped to 14 and 0%, respectively. On the other hand, in dry soil at temperatures from 15 to 40 C, the viability of sclerotia of S. minor and S. sclerotiorum ranged from 53 to 41% and 78 to 51%, respectively.

During the 8-week period that sclerotia of S. minor were in the field at a depth of 0 or 5 cm in irrigated soil, the mean percent viability of these fungal propagules was 5, 7, 8, and 55% at mean soil temperatures of 33, 31, 26, and 19 C, respectively. The percentage of sclerotia of S. sclerotiorum that were viable in irrigated soil tended to increase as soil temperature decreased, with mean viability values of 19, 32, 45, and 73% recorded when the mean soil temperature was 33, 31, 26, and 19 C, respectively. Soil water potential in irrigated soil remained at or above -25 to -40 kPa in each field trial. In field trials where irrigated soil was compared to dry soil, the respective percentage of sclerotia that were viable was 5 and 35% for S. minor and 15 and 50% for S. sclerotiorum.

Recorded summer temperatures in irrigated soil devoid of vegetation ranged from 13 to 65 C at the soil surface to 25 to 51 C at the 5 cm depth. In the constant temperature studies, no viable sclerotia of S. minor or S. sclerotiorum were recovered after two or more weeks at 40 C in wet soil. A 2-week flood irrigation of fields infested with S. minor or S. sclerotiorum in the summer could significantly reduce the number of these pathogen propagules in lettuce production fields in southwestern Arizona and southeastern California.

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Camellia flower blight – first official European record of Ciborinia camelliae an EU quarantine listed member of the family Sclerotinaceae.

C.R. Lane and P.A. Beales

Central Science Laboratory, MAFF, Sand Hutton. York, YO41 1LZ, UK.

AbstractCamellia flower blight, caused by Ciborinia camelliae was considered absent from Europe until 1998 when suspicious symptoms were seen in Southern Europe. A survey carried out by the PHSI in England and Wales in 1999 detected the fungus for the first time in the UK, however, distribution was very limited and restricted to the south and south west of England.

IntroductionCamellias are popular ornamental plants belonging to the family Theaceae that occur naturally in southern China and adjacent countries and are grown for their evergreen foliage and colourful flowers (Huxley, 1992). The three most popular species are C. japonica, which accounts for about three-quarters of all camellia varieties grown world-wide, C. reticulata which flowers from early winter to early summer and C. sasanqua which flowers in the autumn (Taylor, 1999).Camellia flower blight is caused by the fungus Ciborinia camelliae Kohn and attacks floral parts of camellias with all species, hybrids and cultivars of camellia being susceptible. The disease was first described in Japan in 1919 by Hara as Sclerotinia camelliae Hara. It was subsequently found in North America in 1938 and described as Sclerotinia camelliae Hansen & Thomas. After some debate over the correct nomenclature Kohn & Nagasawa (1984) decided on Ciborinia camelliae Kohn. C. camelliae is an ascomycete, in the order Helotiales and family Sclerotiniaceae characterised by the production of apothecia from stroma or sclerotia. The family is subdivided into 14 genera and the genus Ciborinia is separated from Sclerotinia by incorporation of wholly or partially digested host tissue in the sclerotial medulla (Hanlin, 1998). Twenty species of Ciborinia are described and in general they attack leaves and floral parts of specific host plants (Taylor, 1999).C. camelliae lifecycle is annual with sclerotia forming in infected petal bases of fallen flowers in late spring and producing apothecia the following spring. Ascospores germinate on petals causing irregular, small brown spots that coalesce until the entire petal and flower turns brown and falls prematurely. Symptoms may be confused with cultural problems such as drought, frost or other fungi such as Botrytis cinerea. The fungus produces microconidia but no macroconidia and a ring of white or grey mycelium that can be seen when the calyx is removed. Fungal pseudoparenchyma in the base of the petals eventually forms sclerotia in fallen flowers. Sclerotia germinate producing apothecia from which ascospores are released but they may remain viable in the soil for at least four years.The disease was introduced into California in 1938 and was believed to have occurred on imported plants from Japan (Taylor, 1999). The disease spread to other states quickly and within twenty years was present in all regions where camellias were grown. In 1993, the disease was found in New Zealand and was believed to have been there for at least two seasons before being detected (Taylor, 1999). In 1998, symptoms suspicious of camellia

Sclerotinia 2001 31

S2.3

flower blight were observed by camellia growers in parts of Europe. No symptoms of the disease had been reported in the UK prior to 1999.

Materials and methodsThe Plant Health and Seeds Inspectorate of the Ministry of Agriculture Fisheries and Food carried out a survey in the spring of 1999 and 2000 to ascertain if C. camelliae was present and the extent of its distribution if present. In 1999, 218 and in 2000, 184 premises were inspected with samples being sent for diagnosis to the Central Science Laboratory from early March to May. These comprised of partially or totally rotten flowers placed in sealed plastic bags, which were examined on arrival and, if necessary, incubated in sealed damp chambers for 4-10 days in the laboratory (approx 12 h light, 12 h dark, 22°C). Flowers were checked for the presence of a ring of white to grey mycelium following removal of the sepals, for microconidia and for sclerotia.

Results and DiscussionC. camelliae was identified for the first time in the UK in 1999. However, the fungus was only present on five premises out of a total of 218 inspected (2.3%). Positive findings were limited to the south and south west of England (Cornwall, Dorset, Hampshire and West Sussex). At the beginning of the survey all samples required incubation to detect presence of the characteristic microconidia in addition to the ring of mycelium at the petal base. However, towards the end of the survey immature sclerotia were present on samples on receipt. No apothecia were observed during inspections. In 2000, 184 premises were inspected and only three (1.6%) were found infected; again all were in the south and south west of England. Further attempts were made to find apothecia but none were observed. Following finding C. camelliae in the UK, reports from other countries were forthcoming. According to the European and Mediterranean Plant Protection Organisation, C. camelliae has now been reported in France, Portugal, Spain, and Switzerland (EPPO, 2000, 2001).

AcknowledgementsWe wish to acknowledge the assistance of Drs. Christine Taylor and Peter Long of Massey University, Palmerston North, New Zealand for technical guidance. The work was carried out in support of MAFF Plant Health and Seeds Inspectorate.

ReferencesEPPO (2000). Situation of Ciborinia camelliae in Western Europe. EPPO Reporting Service

Ref 99/155.EPPO (2001). Ciborinia camelliae found in Portugal. EPPO Reporting Service Ref

2000/116.Hanlin RT (1998). Illustrated Genera of Ascomycetes, Volume II. APS Press, Minnesota.Huxley AJ, Griffiths M, Levy M 1992. The New Royal Horticultural Society Dictionary of

Gardening. MacMillan Press Ltd, London. Kohn LM & Nagasawa E, 1984. A taxonomic reassessment of Sclerotinia camelliae Hara (=

Ciborinia camelliae Kohn), with observations on flower blight in Japan. Transactions of the Mycological Society of Japan 25 146-161.

Taylor CH, 1999. Studies of camellia flower blight (Ciborinia camelliae) in New Zealand. Thesis for Master of Applied Science in Plant Health, Massey University, New Zealand.

Sclerotinia 2001 32

SESSION V

CONTROL

Chair:

Alison Stewart, Lincoln University, New Zealand

Sclerotinia 2001 33


Sclerotinia 2001 34

S5.1

Developments in the use of Coniothyrium minitans for the biocontrol of Sclerotinia sclerotiorum

John M. Whipps

Plant Pathology and Microbiology Department, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK

AbstractConiothyrium minitans is a mycoparasite of sclerotia of S. sclerotiorum and has considerable potential as a biocontrol agent. Recent work at HRI on C. minitans examined molecular characterisation, inoculum production and application, as well as integration with pesticides. Key features of this work are discussed.

IntroductionThe potential of C. minitans as a biocontrol agent of S. sclerotiorum has long been recognised (De Vrije et al., 2001). Recent work at HRI discussed below has examined the molecular characterisation and phylogenetic position of C. minitans (Muthumeenkashi et al., 2001), efficacy of C. minitans inoculum produced by solid state fermentation (Jones & Whipps, unpublished), and integrated control of S. sclerotiorum (Budge & Whipps, 2001).

Molecular characterisation and phylogenetic position of C. minitansSimple sequence repeat (SSR)-PCR amplification using a microsatellite primer (GACA)4 and ribosomal RNA gene sequencing were used to examine the intraspecific diversity in C. minitans based on 48 strains, representing eight colony types, from 17 countries. The SSR-PCR technique revealed a relatively low level of polymorphism within C. minitans but did allow some differentiation between strains. There was no relationship between SSR-PCR profiles and colony type, but there was some limited correlation between these profiles and origin. Sequences of the ITS 1 and ITS 2 regions and the 5.8S gene of rRNA genes were identical in all twenty-four strains of C. minitans examined irrespective of colony type and origin. These results indicate that C. minitans is genetically not very variable despite phenotypic differences. ITS and 5.8S rRNA gene sequence analyses showed that C. minitans had similarities of 94 % with C. fuckelii and C. sporulosum and only 64 % with C. cerealis. Phylogenetic analyses using database information suggest that C. minitans, C. sporulosum, C. fuckelii and A. terricola cluster in one clade, grouping with Helminthosporium species and ‘Leptosphaeria’ bicolor. C. cerealis grouped with Ampelomyces quisqualis and formed a major cluster with members of the Phaeosphaeriacae and Phaeosphaeria microscopica.

Inoculum quality produced by solid-state fermentationTo assess the quality of the conidia produced within a solid-state fermenter containing hemp impregnated with glucose and yeast extract, conidia samples were taken from the top (where the temperature was higher) and bottom, and compared with conidia from PDA plates. The conidia were assessed for germination on PDA after 24h and ability to infect sclerotia of S. sclerotiorum under standardised conditions when taken immediately from the fermenter, after drying and then after storage of both fresh and dried conidia at 5oC for 6 months. Germination of conidia taken immediately without storage from the top of the fermenter was

Sclerotinia 2001 35

S5.1

significantly lower than germination of sclerotia from the bottom of the fermenter and the drying process itself decreased germinability. However, sclerotial infection by the conidia was the same irrespective of fermenter position or drying. After 6 months storage at 5oC, germination was significantly reduced in all samples in comparison with the PDA control. Nevertheless, dried conidia with such reduced germinability were still able to infect sclerotia at the same level as the PDA controls, and even conidia stored without drying with only 1% germination still gave 2% infection. This clearly indicates that conidial germination per se may not be a good indicator of efficacy of this efficient biocontrol agent.

Integrated control of S. sclerotiorum using C. minitans All pesticides used in UK glasshouse lettuce production were evaluated for their effects on C. minitans mycelial growth and spore germination in in vitro agar plate tests and only the fungicides had any significant effect. Subsequently, all pesticides were assessed in soil tray tests, and despite weekly applications of pesticides at twice their recommended concentrations, C. minitans survived in the soil and infected sclerotia equally well in all pesticide-treated and untreated control soil trays. This demonstrated the importance of assessing pesticide compatibility in environmentally relevant tests. Based on these results, solid substrate inoculum of a standard and an iprodione-tolerant strain of C. minitans were then applied individually to S. sclerotiorum-infested soil in a glasshouse before planting lettuce crops, and the effects of a single spray application of iprodione on disease control in the C. minitans treatments were assessed. Disease caused by S. sclerotiorum was significantly reduced by C. minitans and was enhanced by a single application of iprodione, irrespective or not of whether the biocontrol agent was iprodione-tolerant. In a second experiment, disease control achieved by the combination of C. minitans and a single application of iprodione was shown to be equivalent to that of fortnightly prophylactic sprays with iprodione. These results indicate that integrated control of S. sclerotiorum using soil applications of C. minitans and reduced foliar iprodione applications was feasible and did not require a fungicide tolerant isolate

AcknowledgementsI would like to thank the BBSRC, MAFF and the EU for financial support.

ReferencesBudge SP, Whipps JM, 2001. Potential for integrated control of Sclerotinia sclerotiorum in

glasshouse lettuce using Coniothyrium minitans and reduced fungicide application. Phytopathology 91, 221-227.

De Vrije T, Antoine N, Buitelaar RM, Bruckner S, Dissevelt M, Durand A, Gerlagh M, Jones EE, Lüth P, Oostra J, Ravensberg WJ, Renaud R, Rinzema A, Weber FJ, Whipps JM, 2001. The fungal biocontrol agent Coniothyrium minitans: production by solid state fermentation, application and marketing. Applied Microbiology and Biotechnology (in press).

Muthumeenakshi S, Goldstein AL, Stewart A, Whipps JM, 2001. Molecular studies on intraspecific diversity and phylogenetic position of Coniothyrium minitans. Mycological Research (in press).

Sclerotinia 2001 36

S5.2

The control of Sclerotinia spp. and Sclerotium cepivorum with the biological fungicide Contans®WG – experiences from field trials and commercial use

P. Lüth

PROPHYTA Biologischer Pflanzenschutz GmbH, Inselstrasse 12, 23999 Malchow, Germany

AbstractThe efficacy of the product Contans®WG as a biological fungicide against Sclerotinia sclerotiorum has been tested in various field and greenhouse trials. The poster gives an overview of the product’s properties and its suitability for use in different crops to protect against attack by the named pathogen.

IntroductionThe fungus Coniothyrium minitans is well known as an antagonist of different Sclerotinia species (Whipps & Gerlagh, 1992) as well as of Sclerotium cepivorum (Ahmed & Tribe, 1977). The product Contans®WG, which contains the conidia of C. minitans, was developed for use as a biocontrol agent for soil treatment. For the registration procedure and in order to demonstrate the effectiveness of the product to the growers and farmers it was necessary to carry out efficacy tests. These tests took place in the field as well as in the greenhouse. They were carried out either by the distributors of PROPHYTA or by the official plant protection authorities.

Materials and MethodsThe product was dissolved in water. The resulting suspension was sprayed onto the soil surface and incorporated into the upper soil layer as homogeneously and thoroughly as possible. The incorporation of the product was intended to get all sclerotia located in the treated soil layer contaminated with the antagonistic fungus. The rate of application was dependent on the depth of incorporation, and the depth of incorporation was dependent on the crop and growing method. The application took place some months before infection might have been expected. Tests to evaluate the efficacy of Contans®WG against S. minor and Sclerotium cepivorum were carried out using other methods.

ResultsThe results of some trials to evaluate the efficacy of Contans®WG against S. sclerotiorum are presented in table 1. In a trial carried out at the “Landesanstalt für Pflanzenbau und Pflanzenschutz” (Regional Institute for Horticulture and Plant Protection) in the federal state Rhineland-Palatinate, the use of Coniothyrium coated onion seeds resulted in an up to 100 % better emergence rate of the seeds in a soil infested with Sclerotium cepivorum. In trials in Australia and the USA we achieved a 50 % reduction in infection of different crops caused by S. minor. Other tests to investigate the efficacy of Contans®WG against S. minor and S. cepivorum are still running.

Sclerotinia 2001 37

S5.2

Table 1: Reduction in cases of disease caused by Sclerotinia sclerotiorum due to the use of Contans®WG in different countries and crops

Country Institution Crop Conditions rate perha

depth of incorpo-ration

percentage of disease reduction

Poland Research Institute of Vegetable Crops in Skierniewice

lettuce glasshouse 8 kg 10 cm 79.8 %

Institute of Pomology and Floriculture in Skierniewice

gerberagerberachrysanthemumchrysanthemum

glasshouseglasshouseglasshouseglasshouse

4 kg4 kg4 kg4 kg

10 cm10 cm10 cm10 cm

100 %91.6 %84.2 %88.6 %

Spain Agrichem, S.A. iceberg lettuce open fieldopen fieldopen fieldopen field

4 kg4 kg6 kg6 kg

20 cm20 cm30 cm30 cm

93.0 %89.2 %88.6 %95.9 %

Germany Landespflanzen-schutzamt M-V

oilseed-rape open field 2 kg 5 cm 93.8 %

Landesamt f. Ernährung und Landw. Kiel

oilseed-rape open field 2 kg 5 cm 62.5 %

Switzerl. OMYA AG tobacco open field 4 kg 5 cm 93.6 %

DiscussionThe results of the tests clearly indicate that the product can be used to prevent the infection of different crops by S. sclerotiorum. Contrary to the results of McQuilken et al. (1995) who found very good reduction of the sclerotia following an Autumn application of C. minitans, but a limited reduction in the incidence of disease, the infection of oilseed rape could also be avoided by the use of Contans®WG . In our field trials we used very large plots (at least 1500 sq. m each) to avoid a spread of ascospores from outside into the central part of the plots which were used for the evaluation. Contans®WG also works against S. minor and S. cepivorum, but it seems to be more complicated to combat these pathogens. The results we have got from the trials concerning S. minor at least correspond with the results of Gerlagh (1996) who also achieved almost a 50% reduction in the amount of diseased lettuce plants. Furthermore, the improved emergence rate of Coniothyrium coated onion seeds was already observed by Ahmed & Tribe (1977) when they used C. minitans against S. cepivorum.

ReferencesAhmed HM, Tribe HAT, 1977. Biocontrol of White Rot of Onion (Sclerotium cepivorum) by

Coniothyrium minitans. Plant Pathology 26, 75-78Gerlagh M, 1996. Schimmels nemen smet onder handen. Groen + Fruit /

Vollegrondsgroenten. 36, 2McQuilken MP, Mitchel SJ, Budge SP, Whipps JM, Fenlon JS, Archer SA, 1995. Effect of

Coniothyrium minitans on survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape. Plant Pathology 44, 883-896

Whipps JW, Gerlagh M, 1993. Biology of Coniothyrium minitans and its potential for use in disease biocontrol. Mycological Research 96, 897-907

Sclerotinia 2001 38

S5.3

Effects of bacteria on sclerotia of Sclerotinia sclerotiorum

B. D. Nelsona, T. Christiansona and P. McCleanb

Departments of aPlant Pathology and bPlant Sciences, North Dakota State University, Fargo, 58105, ND, USA.

AbstractThe role of bacteria in the ecology of Sclerotinia sclerotiorum is poorly studied. Bacteria isolated from sclerotia were evaluated for effects on sclerotia in soil. Strains of three Bacillus species reduced germination and increased degradation. One strain produced β-1-3-glucanase. Soil matric potential had a significant effect on survival of sclerotia in soil.

IntroductionSclerotinia sclerotiorum causes some of the most destructive diseases in production agriculture. The fungus overwinters or survives adverse conditions primarily as sclerotia in the soil or plant debris. The soil microbial community plays an important role in the survival of S. sclerotiorum. Most studies on the microorganisms degrading sclerotia have concentrated on fungi (Adams, 1989). The role of bacteria, however, has been neglected. During a study on the survival of S. sclerotiorum in North Dakota soils, the senior author determined that 53% of 3,000 sclerotia recovered from the soil and surface sterilized then placed on agar medium were infected with bacteria. Wu (1988) studied the bacteria isolated from sclerotia in North Dakota and found strains of Bacillus that reduced germination and adversely affected medulla color and integrity in in-vitro studies. To further understand the role of these Bacillus spp in the ecology of S. sclerotiorum, the following study was conducted. The objective was to identify the species of Bacillus and to determine their effects on degradation of sclerotia in natural soil.

Materials and MethodsBacterial isolates BD3, BM108, BP9A, BD2, BN11 and BO3B, originally isolated and purified by Wu (1988), were grown in potato dextrose broth in shake cultures. Bacterial cells were then concentrated by centrifugation, washed with isotonic NaCl, then re-suspended in isotonic NaCl. All isolates were sent to Microbial ID, Inc., Newark, DE, for identification to species based on cellular fatty acid analysis. Sclerotia were produced in a corn-vermiculite medium (Nelson, et al., 1988).

A Bearden fine silty loan was adjusted to water potentials of -0.05 and -0.5 MPa and placed in petri dishes. Sclerotia were soaked for 30 min in bacterial suspensions of 2.5 x 10 8

bacterial cells/ml, then buried in the soil. Controls were soaked in isotonic NaCl. Each soil moisture was run as a separate experiment. All sclerotia were incubated at 22 C for 8 weeks then sieved out of the soil and washed in gently flowing tap water for 20 minutes. The total number of sclerotia recovered and the number of intact sclerotia (<20% of rind or medulla degraded) were counted. Sclerotia were then placed on 2% water agar and evaluated for myceliogenic germination. In another similar experiment, the soil was adjusted to water potentials of -0.5, -0.1, and -0.033 MPa. Sclerotia were soaked in a bacterial suspension of isolate BD3 and in isotonic NaCl as a control and then buried in the soil for 5 months. BD3 demonstrated strong activity against sclerotia in in vitro studies on agar. Sclerotia were evaluated as previously described. All data were analyzed with analysis of variance and

Sclerotinia 2001 39

S5.3

means were compared with Fisher=s protected least significant difference. Bacterial isolates were evaluated for production of β-glucanase activity by measuring the release of reducing sugars from laminarin.

Results and DiscussionThe isolates of Bacillus were identified to the following species: B. cereus (BO3B), B. macerans (BD2), B. polymyxa (BN11), and B. subtilis (BD3, BM108, and BP9A). At -0.5 MPa there was no effect of the bacterial isolates, but at -0.05 MPa treatment with bacteria was significant (P = 0.05 to 0.01). Three bacteria, BD2, BO3B and BN11, significantly reduced the numbers of sclerotia for all characteristics measured. For these bacteria, respectively, recovered sclerotia were 49%, 60% and 54%, intact sclerotia were 12%, 27% and 19%, and germinations were 49%, 59% and 59% of the controls. Sclerotia not considered intact had prominent areas with broken and disintegrating rinds and discolored and degraded medullas. Of the three bacteria, BN11 produced β-glucanase.

In the 5 month study with B. subtilis (BD3), the sclerotia in the controls were mostly degraded in the two highest soil matric potentials, thus there was no significant effect of bacterial treatment. However, there was a significant (P = 0.05) effect of soil matric potential on survival of sclerotia. For the three matric potentials, -0.5, -0.1, and -0.033 MPa, the percentage of sclerotia recovered from the soil after 5 months incubation was 97%, 39% and 43%, respectively. For the numbers of intact sclerotia of those recovered, the percentages were 87%, 8% and 5%, respectively.

This study demonstrated that bacteria shown to degrade sclerotia in in vitro studies also degraded sclerotia in natural soil. The role of bacteria as primary or secondary colonizers of sclerotia and specifically their ability to degrade the rind to gain entrance to the medulla, needs further investigation. Bacteria probably work in concert with many other soil organisms to reduce populations of sclerotia of S. sclerotiorum in soil. Soil matric potential is a critical factor in the natural degradation of sclerotia in soil, with high matric potentials fostering the activity of bacteria.

ReferencesAdams PB, 1989. Comparison of antagonists of Sclerotinia species. Phytopathology 79,

1345-1347.Nelson B, Duval D, Wu H, 1988. An in-vitro technique for large scale production of

sclerotia of Sclerotinia sclerotiorum. Phytopathology 78, 1470-1472. Wu H, 1988. Effects of bacteria on germination and degradation of sclerotia of Sclerotinia

sclerotiorum (Lib) de Bary. Master of Science Thesis, North Dakota State University. Fargo, ND. 73 pp.

Sclerotinia 2001 40

S5.4

Epicoccum nigrum: A biological control agent for the control of Sclerotinia sclerotiorum in New Zealand kiwifruit (Actinidia deliciosa)

P. A. G. Elmer, S. M. Hoyte, R. Marsden, F. Parry and T. Reglinski

The Horticultural and Food Research Institute of New Zealand, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand

Abstract Sclerotinia sclerotiorum is responsible for considerable crop loss in New Zealand kiwifruit orchards. Market demands for reduced pesticide use and increasing organic kiwifruit production has renewed interest in biological control. Application of Epicoccum nigrum to kiwifruit vines during flowering in 1998 and 1999 demonstrated biological suppression of sclerotinia was feasible.

IntroductionEpidemiological studies on Sclerotinia sclerotiorum (Lib.) de Bary in New Zealand kiwifruit (Actinidia deliciosa (A. Chev.)) have demonstrated that disease development is initiated by primary ascospore infection of petals during flowering and that adhering floral tissues on developing fruit are a major source of fruit disease. Disease control is based on one or two applications of iprodione (RovralTM), but market demand for reduced pesticide use and increasing organic kiwifruit production have renewed interest in biological control. An isolate of Epicoccum nigrum Link. (syn. E. purpurascens) was identified in detached petal assays as an aggressive saprophytic coloniser with potential as a biological control agent (BCA). Subsequent field bioassays in 1996 and 1997 confirmed the ability of this isolate to protect petals from ascospore infection and fruit surfaces from mycelial infection. Treatment of whole vines with E. nigrum over flowering in 1998 reduced field infection of petals from 7% to zero. The objectives of this study were to confirm these findings in a high disease risk orchard and to determine the effect of E. nigrum concentration on disease control.

Materials and MethodsIn 1999, treatments were (i) E. nigrum (concentrations ranging from 2 ´ 103 to 2 ´ 106

spores ml-1, plus Tween 80 (0.05% v/v), (ii) Rovral (37.5 g a.i./100L) and (iii) a water control plus Tween 80 (0.05% v/v), applied twice during flowering (2 days apart) to five replicate vines per treatment. Suspensions of E. nigrum were produced using a dry fermentation system with autoclaved millet as the nutrient base. Fifty petals were collected from each treated vine 24 hours after the last application and sclerotinia incidence was determined by recording the number of petals with sclerotia following incubation for 10 days in high humidity trays at 20°C. The proportion of fruit at harvest that were not exportable due to sclerotinia scarring (rejects) was determined on a 200 fruit sample per vine.

Results and DiscussionIn 1999, petal infection in the control vines was 67% (Figure 1). As the concentration of E. nigrum increased, petal infection decreased significantly (P<0.001). Rovral had the lowest incidence of petal infection (5.6%). The incidence of reject fruit at harvest due to sclerotinia

Sclerotinia 2001 41

S54

was 4.7% in the control vines (Figure 2). All E. nigrum treatments significantly (P<0.05) reduced the incidence of export reject fruit and the highest concentration was not significantly different (P>0.05) from the Rovral treatment.

0

10

20

30

40

50

60

70

80

Control 2 x 10 3 2 x 10 4 2 x 10 5 1 x 10 6 2 x 10 6 Rovral

Treatment

dcd

bc

a

bb bc

0

1

2

3

4

5

6

Control 2x10 3 2x10 4 2x10 5 1x10 6 2x10 6 Rovral

Treatment

Perc

enta

ge o

f exp

ort r

ejec

t fru

it

2 x 1061 x 1062 x 1052 x 1042 x 103

Figure 2

Effect of vine applications of a range of concentrations of E. nigrum on petal infection (Figure 1) and the incidence of export reject fruit (Figure 2). Bars with the same letter are not significantly different (P>0.05), based on LSDs.

Our research in 1999 has confirmed that this isolate of E. nigrum protected kiwifruit petals from ascospore infection. In addition, application of this BCA to flowers has provided protection of fruit against secondary mycelial infection over a three month period when fruit are susceptible. Importantly, the lowest concentration of E. nigrum (2 ´ 103 spores ml-1) significantly reduced disease at harvest, despite no reduction of petal infection. This implies that there may be a long period of interaction between the BCA and the pathogen prior to secondary infection, an added advantage compared to the fungicide. Therefore, reducing E. nigrum spore concentrations down from 2 ´ 106 spores ml-1 is feasible without a significant loss of disease control. Mode of action studies have shown that this isolate produces a diffusable anti-microbial metabolite which inhibited spore germination (Elmer et al. In press). Reduced spore concentrations, improved formulation and improved timing in high disease risk orchards will be the focus of future research.

AcknowledgementsThe authors are grateful to the New Zealand Foundation for Research Science and Technology and Technology New Zealand for providing research funds. We thank K. Stannard for valuable technical support and R. Glen and S. Steel for providing trial sites.

ReferencesElmer PAG, Hoyte SM, Reglinski T, 1999. Biological suppression of Sclerotinia

sclerotiorum in kiwifruit. In: 12th Biennial Australasian Plant Pathology Society Conference, p.63, Canberra.

Hoyte SM, 2001. Epidemiology and management of Sclerotinia sclerotiorum (Lib.) de Bary in kiwifruit (Actinidia deliciosa (A. Chev.)). PhD thesis, Massey University.

Hoyte SM, Elmer PAG, Reglinski T, Perry J, 1998. Biological suppression of sclerotinia: A future option for disease control. 1998 Kiwifruit Technical Updating Report 98/48, 39-44.

Sclerotinia 2001 42

S5.5

Integrated Soil Disease management on sclerotinia drop of salad by solarization and green manure sequence in southern France (Roussillon)

C. Martin and M. DuboisAgriphyto, Maison de l’Agriculture, 19 avenue de Grande Bretagne, 66025 Perpignan Cedex France

Abstract Nuisance threshold by Sclerotinia drop has been precisely evaluated in salad crop after Solarization sequence or combination with green manure. Organic farming compared with Integrated production including post plantation fungicide shows a slow interspecific derive of the pest responsible for the salad drop from S. minor to S. sclerotiorum.

IntroductionSalad drop by Sclerotinia sp. remains a major soil born pathogen for intensive salad crops in open-field and in greenhouses (Davet & Martin, 1979). To follow the general tendency to reduce chemical inputs in Integrated production of vegetables such as post plantation fungicide and also to check alternatives for soil disinfection purposes for Organic farming we developed a demonstration project based on technical and economical data in greenhouses.

Materials and methods Since 1994, a comparison of different soil management strategies in greenhouses which all conform to the requirements of Organic Farming and Integrated Pest Management (IPM) in separate units of greenhouses have been checked for consequences on soil pathogens for an annual rotation of lettuce- melon according the following « biophyto » protocol.

4 plastic tunnels of 400 m2 (2 tunnels in Organic farming and 2 tunnels in Integrated production) each divided in 2 plots of 200 m2 with different soil summer treatments :

Table 1. Soil summer treatments in « biophyto »Factors Conditions TimingDazomet + solarization 50g/ha + 60 days solar. mid-july to mid-septemberGreen manure Sorgho 50kg seeds/ha Mixed in soil mid septemberSolarization 60 days polyeth.film of 40 mid-july to mid-septemberGreen manure+solarization 40 t/ha fresh manure+solar 30 days July to mid septemberSteam mid-september

Results and discussion The consistent reduction of sclerotinia to very low or to zero levels in crops from 1994 to 2001 (Fig. 1) confirm the effects of solarization (Martin, 1992) in southern France, and demonstrate more precisely the practical interval between two soil disinfections to keep losses below the economic threshold. The influence of green manure with Sorgho alone or combined with solarization prove its interest different from a real biodisinfestation effect but at least with an interesting stabilizing effect on evolution of soil pathogens and antagonistic flora.

Sclerotinia 2001 43

S5.5

In organic farming plots, following solarization or green manure treatments, the proportion of S. sclerotiorum relative to S. minor has increased significantly in the last 4 years (Martin & Dubois, 2000) (Table 2).

Table 2. IPM plots Organic farming plots

Crop year Sclerotinia minor/ sclerotiorum Sclerotinia minor/ sclerotiorum 1997 0 57%1999 100% 51%2000 100% 66%2001 100% 25%

We can only suggest the following hypothesis to explain the phenomenon: in IPM plots the use of anti-bremia fungicides such as dithiocarbamates may interfere with the more active antagonistic flora on S. sclerotiorum in the soil. This effect has to be fully evaluated in the near future to develop other effective control strategies in Organic Farming.

Acknowledgements We thank A. Arrufat and D.Marty (CIVAM Bio) for their contribution to the Organic Farming side of the project.

ReferencesDavet P, Martin C. 1979. A propos de la sclérotiniose des salades en Roussillon PHM n°197.Martin C. 1992. La solarisation, méthode de désinfection des sols aux perspectives nouvelles

in Les plastiques en agriculture, ed. CPA, 553-566.Martin C, Dubois M. 2000. Gestion raisonnée du risque Sclerotinia en culture de salade

AFPP 6ème Conf. Intern. Sur les Maladies des plantes.

Sclerotinia 2001

Fig. 1. Effect of 'Biophyta' organic treatments on sclerotinia lettuce drop from 1994-2001

0

5

10

15

20

25

Lettuce 93-94 Lettuce 94-95 Lettuce 96-97 Lettuce 98 - 99 Lettuce 2000-2001

green manure / steamannual Solarization Untreatedsolarization/green manure/solarizationgreen manure+solarizationSolarization/green manure+solarization

44

SESSION VI

CONTROL (CONTINUED)

Chair:

Alison Stewart, Lincoln University, New Zealand

Sclerotinia 2001 45


Sclerotinia 2001 46

S6.1

Sclerotinia disease on canola, crambe, safflower and sunflower as influenced by previous crops

J.M. Krupinsky, D.L.Tanaka, S.D. Merrill, and R.E. Ries

U.S. Department of Agriculture, Agricultural Research Service, Northern Great Plains Research Laboratory, Mandan, North Dakota, USA 58554-0459.

AbstractCanola (Brassica napus L.), crambe (Crambe abyssinica Hochst. ex R.E. Fr.), safflower (Carthamus tinctorius L.), and sunflower (Helianthus annuus L.), were evaluated for natural infection by Sclerotinia sclerotiorum (Lib.) De Bary following ten crops (barley [Hordeum vulgare L.], canola, crambe, dry bean [Phaseolus vulgaris L.], dry pea [Pisum sativum L.], flax [Linum usitatissimum L.], safflower, soybean [Glycine max (L.) Merr.], sunflower [Helianthus annuus L.], and wheat [Triticum aestivum L.]) for two annual growing seasons. Sclerotinia head blight on safflower ranged from 0 to 3%. Sclerotinia stem rot ranged from 0 to 6% on canola and from 2 to 60% on crambe. Sclerotinia stem rot was not detected on sunflower in 1999.

IntroductionThe fungus, Sclerotinia sclerotiorum (Lib.) De Bary, causes Sclerotinia stem rot on canola and crambe, sclerotinia stem rot and head rot (head blight) of safflower and sunflower (Farr et al., 1989; Nyvall, 1999). The objective of this study was to determine the effect of previous crops and crop residues on Sclerotinia disease on canola, crambe, safflower, and sunflower.

Materials and MethodsField plots were located 8 km from the Northern Great Plains Research Laboratory, southwest of Mandan, North Dakota, USA. During the first year, ten crops (barley, canola, crambe, dry bean, dry pea, flax, safflower, soybean, sunflower, and wheat) were no-till seeded in a strip-block design (four replicates) with a no-till drill into a uniform cereal residue. During the second year, the same crops were no-till seeded perpendicular to the residue strips of the previous year’s crops. Thus, a 10 X 10 matrix with 100 treatment combinations, where each crop was grown on ten crop residues, was established. This was repeated so the crop X crop residue matrix was present in the field for two consecutive years. Each experimental unit was a 9 X 9-m plot. The ten cultivars were: ‘Stander’ barley, ‘Dynamite’ canola, ‘Meyer’ crambe, ‘Shadow’ Black Turtle dry bean, ‘Profi’ dry pea, ‘Omega’ flax, ‘Montola 2000’ safflower, ‘Jim’ soybean, ‘Cenex 803’ oilseed sunflower, and ‘Amidon’ spring wheat.

Safflower, canola, crambe, and sunflower plants were evaluated for white mold during two seasons. Safflower was rated for Sclerotinia head blight incidence using the presence of sclerotia under the necrotic head to confirm the disease. Canola and crambe plants with bleached (white) stems, stems sometimes shredding, were rated positive for Sclerotinia. During the 1999 growing season, 1500 safflower heads (50 heads X 10 treatments X 3 replicates), and 3000 canola and crambe plants (100 plants X 10 t X 3 reps) were evaluated. During the 2000 growing season, 4000 safflower heads, canola and crambe plants (100 heads or plants X 10 crop residues X 4 reps) were evaluated. In 1999, 1500 sunflower plants

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(50 plants X 10 crop residues X 3 reps) were rated for Sclerotinia stem rot. In 2000, 1000 sunflower plants (25 plants X 10 crop residues X 4 reps) were rated for Sclerotinia stem rot. An analysis of variance was conducted using the number of infected heads or plants for each field evaluation. Statistical comparisons within each evaluation were made with Student-Newman-Keuls' test and the Dunnett’s one-tailed test. Statistical differences were evaluated at a probability level of P < 0.05.

Results and DiscussionAlthough disease incidence was low, Sclerotinia head blight on safflower ranged from 0% to 3% in 1999 and from 0% to 2% in 2000, with the highest level on crambe residue for both years. For canola, Sclerotinia stem rot ranged from 0% to 6% in 1999 and from 1% to 5% in 2000 with the highest level occurring on safflower residue both years. For crambe, Sclerotinia stem rot ranged from 2% to 15% in 1999 and from 10% to 60% in 2000 with the highest level occurring on safflower residue both years. Thus, the higher levels of Sclerotinia on canola and crambe were observed when those crops followed a safflower crop for both years even though the incidence of Sclerotinia head blight on the previous safflower crop was rather low. Sunflower was rated for Sclerotinia stem rot but the data are not included because no diseased plants were identified in 1999 and few infected plants (0.01%) were found in 2000. Given the variation in disease incidence among plots, it was difficult to demonstrate significant differences among previous residue treatments at a probability level of P < 0.05. Even though there were some patterns in the incidence of Sclerotinia disease, one can speculate that the movement of ascospores among plots (interplot interference) or from other areas made it difficult to detect significant differences among the individual crop residue treatments. Crop rotation may be only partially effective because of the movement of wind-borne ascospores. In the future a uniform wheat crop will be grown over the crop X crop residue matrix, followed by a susceptible sunflower crop. Because Sclerotinia stem rot on sunflower is caused by root contact with the fungus, the use of sunflower as a Sclerotinia-indicator crop should reduce possible interplot interference and give an indication of Sclerotinia carryover associated with the various crop sequences.

AcknowledgmentsWe thank D. Wetch, C. Flakker, J. Hartel, M. Hatzenbuhler, D. Schlenker, and C. Klein for technical assistance, and M. West for statistical advice. Data were generated with the support of the Area IV Soil Conservation Districts, The National Sunflower Association, and The North Dakota Oilseed Council.

Mention of a trademark, proprietary product, or company by USDA personnel is intended for explicit description only and does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

ReferencesFarr DF, Bills GF, Chamuris GP, Rossman AY. 1989. Fungi on plants and plant products in

the United States. APS Press, St. Paul, Minnesota, USA. Nyall RE, 1999. Field Crop Diseases, 3rd ed., Iowa State Univ. Press, Ames. 1021p.

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Integrated control of Sclerotinia disease in field-grown lettuce in Scotland

M. P. McQuilken

Department of Plant Biology, The Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK

AbstractSoil incorporation of dazomet completely inhibited carpogenic germination of Sclerotinia sclerotiorum, whereas calcium cyanamide and quintozene delayed and reduced numbers of apothecia. Post-harvest application of diquat or direct flame heat to infected crop debris reduced sclerotial production. Combined soil incorporation of quintozene and foliar sprays of iprodione improved disease control compared to either treatment alone. The implications of the results for integrated disease control of Sclerotinia in field-grown lettuce are discussed.

IntroductionSclerotinia, caused by Sclerotinia sclerotiorum, is an important soil-borne disease of field-grown crisphead lettuce in Scotland. In recent years, crop losses in excess of 50% have been reported. A field pot bioassay was conducted to determine the effect of soil incorporations of dazomet, calcium cyanamide and quintozene on apothecial production. Field trials were also carried out to evaluate post-harvest applications of desiccants to infected crop debris on sclerotial production, and single and combined applications of quintozene and foliar sprays of iprodione for disease control. The aim was to develop effective integrated disease control.

Materials and MethodsField pot bioassay. Treatments included: (1) control (untreated soil); (2) calcium cyanamide fertiliser, 40 g m-2; (3) dazomet (Basamid, 97% a.i. gr.; 57 g m-2); (4) quintozene (Terraclor 20 D, 20% a.i. w.p.; 35 g m-2). Each treatment was mixed thoroughly with soil (sandy-loam, Balgour series) prior to filling ten replicate rectangular pots (11 x 10 cm). Twenty sclerotia enclosed in Terylene net bags were buried in each pot c. 1 cm beneath the surface. Pots were arranged in a randomised block design in soil outside in early April 1998. Numbers of apothecia produced from sclerotia in each pot were counted at fortnightly intervals from June to mid August. Recovery of sclerotia was assessed 24 weeks after burying.Lettuce field trials. Crops of lettuce were planted in 1997 and artificially inoculated with S. sclerotiorum to produce a natural population of sclerotia in soil. Field trials were conducted in 1998 (Trial 1) and 1999 (Trial 2) on a site at Auchincruive. Plots (2.4 x 2.4 m) separated by 1 m paths were marked out in a randomised block design (4 replicates/treatment). Blocks of crisphead lettuce (cv. Saladin) were planted (64 plants/plot) in mid May and harvested c. 10 weeks later (late July).Trial 1 - Crop desiccants. The crop was harvested, leaving diseased material on plots, and desiccants applied immediately. Treatments included: (1) control (untreated); (2) diquat (Reglone, 16.7% a.i. s.l.; 3 l ha-1); (3) glyphosate (Roundup Biactive, 41.1% a.i. s.l.; 4 l ha-1); (4) direct flame heat by crop debris burner (twice with 48 h interval). After 8 weeks, numbers of sclerotia present on the soil surface within five random quadrats (each 500 cm2) were counted in each plot.Trial 2 - Fungicides. Treatments included: (1) control (untreated); (2) pre-planting soil incorporation of quintozene (Terraclor Flo, 40% a.i. s.c., 14 ml m-2); (3) 2 sprays (4-6 true

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leaves + rosette/early-heading stages) of iprodione (Rovral WP, 50 % a.i. w.p.; 0.5 kg ha-1); (4) quintozene + iprodione. The crop was assessed for number of diseased plants at harvest.

ResultsField pot bioassay. Soil incorporation of dazomet completely inhibited apothecial production and no sclerotia were recovered from pots 20 weeks after burying (Table 1). Calcium cyanamide and quintozene significantly reduced numbers of apothecia and sclerotia recovery.

Table 1. Effect of different soil incorporations on apothecial production and percentage recovery of sclerotia of S. sclerotiorum (Mean ± SE, n = 10)

Soil incorporation Sum of apothecia % Sclerotia recovered Control (nil)Calcium cyanamideDazometQuintozene

30 ± 1.8 8 ± 2.2 0 5 ± 1.5

93 ± 1.861 ± 5.3064 ± 3.7

Field trials. Eight weeks after post-harvest application of desiccants to infected crop debris, diquat (38-62 sclerotia/500 cm2) and direct flame heat (48-65 sclerotia/500 cm2) significantly reduced the number of sclerotia produced on the soil surface, compared to the untreated control (77-132 sclerotia/500 cm2). Glyphosate was less effective (69-108 sclerotia /500 cm2). All fungicide treatments reduced disease incidence compared to the untreated control (Table 2). However, the combined soil incorporation of quintozene and foliar sprays of iprodione significantly improved disease control compared to either treatment applied alone.

Table 2. Effect of single and combined applications of soil- and foliar-applied fungicides on S. sclerotiorum-diseased lettuce plants (Mean ± SE, n = 4)

Treatment % DiseasedControl (nil)Quintozene (soil incorporation)Iprodione (2-spray foliar application)Quintozene + iprodione

41 ± 3.120 ± 3.623 ± 1.512 ± 1.3

DiscussionResults from this study have shown that it is possible to reduce S. sclerotiorum inoculum levels and disease incidence by implementing a number of control strategies. Recommendations to lettuce growers include: consider using the soil sterilant dazomet or soil-applied fertiliser calcium cyanamide to reduce apothecial production; use a soil-applied fungicide (e.g. quintozene while still available) in combination with fungicide foliar sprays; reduce numbers of sclerotia returned to soil by applying effective post-harvest desiccants to crop debris.

AcknowledgementsI would like to thank SERAD and P. P. Products, Norwich, UK for financial support.

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Fungigation with benomyl and fluazinam and their fungicidal effects in soil for white mould (Sclerotinia sclerotiorum) control on dry beans

R.F. Vieira, C.M.F. Pinto and T.J. de Paula JúniorEPAMIG, C.P. 216, Viçosa, MG 36571-000, Brazil

AbstractThe effectiveness of the fungicides fluazinam and benomyl applied through irrigation water (fungigation) onto plants or only in soil in controlling white mould of dry beans was evaluated. Fungigation provided disease control equivalent to that of a backpack sprayer; application only in soil was less effective.

IntroductionWhite mould, caused by Sclerotinia sclerotiorum, is a serious disease of sprinkler irrigated areas of dry beans during fall and winter in Minas Gerais State, Brazil. Among the fungicides used for white mould control are benomyl and fluazinam. Timing of fungicidal application is critical to protect blossoms from infection. The fungicide must be applied in sufficient water to provide thorough coverage of blossoms, stems and leaves, especially those closest to the soil surface. Fungicide application by aircraft has not given satisfactory results for white mould control since the fungicide does not penetrate deeply into the plant canopy. Fungicide application by tractor is impaired by row closure when plants reach the reproductive phase. Therefore application of fungicides through irrigation water (fungigation) is a practical method. Prescription application, reduced application costs and operator hazards, no soil compaction and vine injury are important advantages of fungigation over ground sprays (Vieira & Sumner, 1999). The high volume of water applied by irrigation means that more fungicide reaches the soil than with application by conventional methods. However, there is evidence that control of disease is also provided by fungicide that reaches soil (Vieira & Paula Júnior, 1998). The objectives of this study were to determine efficacy of two fungicides applied through irrigation water and the effectiveness of the fungicides in soil.

Materials and MethodsTwo trials were conducted at Viçosa Federal University, Minas Gerais State, Brazil, in a field naturally infested with sclerotia of S. sclerotiorum: one was sown on 16 April 1998 and the other on 29 April 1999. The vine bean cultivar Pérola (type III) was sown spaced 0.5 m apart. The trials were conducted as a (2 x 3) + 1 factorial in the randomized complete-block design replicated six times: two fungicides x three application modes + one untreated control. The fungicides were benomyl (Benlate 500WP, 1.0 kg a.i. ha-1) and fluazinam (Frowncide 500SC, 0.5 l a.i. ha-1). The three application modes were: by backpack sprayer equipped with one cone nozzle delivering 667 l ha-1 of water; by garden watering-cans with volume for 10 l of water, simulating a sprinkler irrigation using 35,000 litres of water per hectare; and by garden watering-cans applying water between the rows and near the soil surface, avoiding wet leaves and flowers, in 35,000 litres of water per hectare. In 1998, fungicides for white mould control were applied at 43 (early bloom) and 54 days after emergence (DAE). In 1999, fungicides were applied at 47 and 61 DAE. One square meter of each plot was harvested separately for disease evaluation. The plants were rated for severity of white mould on a scale of 0, 1, 2, 3,

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and 4, representing 0, 1-25, 26-50, 51-75, and 76-100% of stem and branches with disease symptoms (Hall & Phillips, 1996). Disease incidence was calculated as the percentage of plants with symptoms on stem or branches, and percentage of pods with white mould was determined. An area of 3 m2 of each plot was harvested to estimate 100-seed weight, and weight and number of sclerotia larger than 2 mm mixed with seeds. Yield data were based on weight of seeds at 12-13% moisture (w/w) harvested in 4 m2 (including the 1 m2 harvested for disease evaluation). The data were subjected to analysis of variance. Duncan’s multiple range test (DMRT) values were calculated for evaluation of application mode effects. The Dunnett’s test was used for comparison of the treatments against the untreated control.

Results and DiscussionBoth fungicides were similarly effective on white mould control when applied by either fungigation or backpack sprayer, resulting in an average yield 21% higher than untreated control (2,739 vs. 2,263 kg/ha). Fluazinam provided better disease control than benomyl for applications only in soil. This mode of application with fluazinam resulted in an average yield 14% higher than that of control. According to Oliveira et al. (1999) fluazinam is more efficient than benomyl on inhibition of myceliogenic germination of sclerotia and on stipes growth of apothecium. Fungigation provided white mould control equivalent to that of backpack sprayer in terms of incidence, severity and number of diseased pods. Consequently, yield differences between these application methods were not significant. These results agree with those reported by Vieira & Paula Júnior (1998) and Vieira & Sumner (1999). However, there was a consistent trend of less sclerotia being produced by plants treated by backpack sprayer than by fungigation. One hundred-seed weight was not affected significantly by treatments. Our results suggest that benomyl and fluazinam are efficient when fungigated, controlling white mould when kept on the plants as well as in soil.

ReferencesHall R, Phillips LG, 1996. Evaluation of parameters to assess resistance of white bean to

white mold. Annual Report of the Bean Improvement Cooperative 39, 306-307. Oliveira SHF, Recco E, Sugahara DA, 1995. Avaliação comparativa da fungigação e

aplicação convencional de fungicidas para controle de Sclerotinia sclerotiorum em feijoeiro. Summa Phytopathologica 21, 249-252.

Vieira RF, Paula Júnior TJ de, 1998. Application of fungicides with watering-cans simulating chemigation for white mold control in dry beans. Annual Report of the Bean Improvement Cooperative 41, 175-176.

Vieira RF, Sumner DR, 1999. Application of fungicides to foliage through overhead sprinkler irrigation – a review. Pesticide Science 53, 412-422.

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Effect of Microsphaeropsis ochracea, a new biological control agent, on germination of sclerotia of Sclerotinia sclerotiorum

O. Carisse

Agriculture and Agri-Food Canada, HRDC., 430 Gouin Blvd., Saint-Jean-sur-Richelieu, Quebec, Canada, J3B 3E6

AbstractMicrosphaeropsis ochracea is a known biological control agent of Venturia inaequalis, the causal agent of apple scab. M. ochracea was evaluated for the reduction of inoculum of Sclerotinia. sclerotiorum based on the viability of sclerotia produced in vitro and on bean debris. On sclerotia produced in vitro, for all incubation periods (1 to 14 days), M. ochracea significantly reduced germination. Percent germination of sclerotia treated with M. ochracea decreased with increasing incubation period from an average of 84% after 1 day to stabilize at an average of 13% after 14 days. Similarly, percent germination of debris borne sclerotia was significantly lower when treated with M. ochracea. As the period of incubation increased, the viability of sclerotia decreased to a minimum of about 9% after 4 weeks at 10°C. These observations are in agreement with the results from other tests conducted with M. ochracea at low temperature. The capacity of M. ochracea to affect S. sclerotiorum sclerotia viability at low temperature is a great advantage for post harvest treatments, especially under cold climates.

IntroductionSeveral mycoparasites were identified and tested against sclerotia germination. M. ochracea is a Coelomycetes that was isolated from dead apple leaves (Bernier et al., 1996, Carisse & Bernier, 2001). This antagonist is gaining interest as a biocontrol agent and has been found to exert a strong antimicrobial activity against V. inaequalis and Rhizoctonia solani (Benyagoub et al., 1998, Carisse et al., 2001). Recent studies showed that M. ochracea was capable of inhibiting in vitro and field produced ascospores of V. inaequalis, in vitro produced ascospores of Giberella zeae and in vitro and tuber-borne sclerotia of R. solani (Bujold et al., 2001, Carisse et al., 2000, 2001). Because V. inaequalis and G. zea produce ascospores in melanized structures (pseudothecia and perithecia), and because sclerotia of both R. solani and S. sclerotiorum are also melanized, it was hypothesized that M. ochracea could inhibit the germination of sclerotia of S. sclerotiorum.

Materials and methodsM. ochracea was maintained on potato-dextrose agar (PDA) medium at room temperature. S. sclerotiorum was isolated from sclerotia collected from green beans debris and was cultured on PDA at room temperature. S. sclerotiorum was grown on bean pods at room temperature for 5 to 8 weeks. Sclerotia were removed, surface sterilized and kept in sterile glass vials until used for the experiment (maximum of 1 week). Sclerotia, of approximately the same size were placed at the periphery of a 15 day-old culture of M. ochracea (5 sclerotia per plate) and incubated at room temperature for up to 14 days. Similarly, sclerotia were placed on a moist filter paper in a Petri dish and treated with M. ochracea applied at a rate of 50 µL per sclerotia of a spore suspension of 4.5 X 108 spores ml-1. The spore suspension was

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made from frozen spores of provided by PhilomBios (Saskatoon, Canada). For both tests, sclerotia viability was evaluated after 1, 3, 5, 7, and 14 days. In a third experiment, infected debris were produced from greenhouse grown beans. Infected plant material was sprayed with M. ochracea applied at a rate of 1 mL g-1 of material of a spore suspension of 4.5 X 108

spores ml-1 and placed in mesh sachets (20 g per sachets) maintained at 10 °C. Viability of sclerotia was evaluated after 1, 2, 3, and 4 weeks. For all tests, the viability of sclerotia was evaluated by placing sclerotia on the surface of water agar plates for 24 h at room temperature. Viability was evaluated based on hyphal growth observed at 40X. Sclerotia were considered viable when typical Sclerotinia-like hyphae grew from the sclerotia. Viability was recorded as the incidence of viable sclerotia expressed as a percentage. Completely randomized designs were used with 30 plates per treatment for the first two experiments and 5 sachets from each of which 25 sclerotia were evaluated for the third experiment. Water treated sclerotia served as controls.Results and discussionFor the in vitro tests, the percent germination of water treated sclerotia remained high for all incubation period (91 to 99%), although significant differences in percent germination over time were detected. For both tests and for all incubation periods, percent germination of sclerotia treated with M. ochracea was significantly lower (P< 0.0001) than for the water treated sclerotia. Percent germination of treated sclerotia decreased with increasing incubation period, from an average of 84% after 1 day to stabilize after 14 days at an average of 13%. On bean debris the percent germination of the water treated sclerotia varied from 67 to 92%. When sclerotia were treated with M. ochracea, percent sclerotia germination was reduced to 11% after 14 days of incubation, which further reduced to 9% for the other incubation periods. As the period of contact between the M. ochracea and the sclerotia increased, the viability of sclerotia decreased to a minimum of about 9% after 4 weeks of incubation on beans debris incubated at 10°C. These observations are in agreement with the results from other tests conducted with M. ochracea at low temperature (Carisse et al., 2000). The capacity of M. ochracea to affect S. sclerotiorum sclerotia viability at low temperature is a great advantage for post harvest treatments, especially under cold climates. ReferencesBenyagoub, M., Benhamou, N., and Carisse, O. 1998. Cytochemical investigation of the

antagonistic interaction between a Microsphaeropsis sp. (isolate P130A) and Venturia inaequalis. Phytopathology 88, 605-613.

Bernier, J., Carisse, O., and Paulitz, T.C. 1996. Fungal communities isolated from dead apple leaves from orchards in Quebec. Phytoprotection 77, 129-134.

Bujold, I., T.C. Paulitz, and Carisse, O. 2001. Effect of Microsphaeropsis sp. on the production perithecia and ascospsores of Gibberella zeae. Plant Disease (accepted).

Carisse, O., Philion, V., Rolland, D., and Bernier, J. 2000. Effect of fall application of fungal antagonists on spring ascospore production of the apple scab pathogen, Venturia inaequalis. Phytopathology 90, 1120-1125.

Carisse, O. and Bernier, J. 2001. Microsphaeropsis ochracea sp. nov. associated with dead apple leaves. .Mycologia (accepted).

Carisse, O., Bassam, S. El, and Benhamou, N. 2001. Effect of Microsphaeropsis sp. (strain P130A) on germination and production of sclerotia of Rhizoctonia solani and interaction between the antagonist and the pathogen. Phytopathology (accepted).

Sclerotinia 2001 54

SESSION VIII

RESISTANCE

Chair:

Jim Steadman, University of Nebraska, USA

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QTL-analysis of Sclerotinia sclerotiorum resistance in sunflower

V. Hahna, Z. Micica, A. E. Melchingerb and E. Bauera

a State Plant Breeding Institute, University of Hohenheim, Fruwirthstr. 21, 70593 Stuttgart-Hohenheim, Germany, b Institute of Plant Breeding, Seed Science, and Population Genetics, University of Hohenheim, Fruwirthstr. 21, 70593 Stuttgart-Hohenheim, Germany

AbstractSSR markers were used to investigate quantitative trait loci (QTL) involved in sunflower resistance to Sclerotinia sclerotiorum. Eight QTL were detected for resistance to mycelial extension in the plant tissue. Four QTL explained 65 % of the genetic variance for the speed of the growth of S. sclerotiorum in leaf and petiole tissue.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating pathogens of sunflower (Helianthus annuus L.) and can attack all parts of the plant. Yield losses can reach up to 100%. To date, no complete resistance to S. sclerotiorum is available in cultivated sunflower, but differences in susceptibility exist (Degener et al., 1999). Our objectives are to study the inheritance of resistance to mycelial extension of S. sclerotiorum in sunflower leaves and stems using the leaf test described by Degener et al. (1998) and to present the preliminary results from mapping QTL that control this resistance.

Materials and methodsA sunflower F2 population (n = 354) was developed by crossing inbred lines NDBLOS and CM625. In previous investigations NDBLOS showed less stem lesions than CM625 after artificial leaf infection with S. slerotiorum. F3 lines were developed by single seed descent. Field experiments of the F3 lines were conducted at Eckartsweier, Germany, during 1999 in a 19 x 19 lattice design with three replications. The trial was sown on May 7 and June 23 and infected in July and August, respectively. Reaction of F3 lines to S. sclerotiorum infection was assessed across both environments, which differed in temperature and precipitation. The S. sclerotiorum isolate was collected in 1995 from naturally infected sunflowers at Eckartsweier. The tip of one leaf of the fifth fully grown leaf pairs was infected with S. sclerotiorum mycel. Five plants per plot were infected. We recorded leaf length and petiole length, and the time when the first symptoms were visible at the stem, to calculate the speed of fungal growth. Furthermore leaf lesion length and stem lesion length were measured (for details see Degener et al., 1998).Up to now, the DNA of the parents was screened with 500 SSR markers. A sub-set of 135 F2 plants was screened with 45 polymorphic markers. A partial linkage map of the cross was calculated using software package G-Mendel 3.0 with a minimum LOD score of 3.0. Statistical analyses were done with computer programs PLABSTAT (Utz, 1991) and PLABQTL (Utz & Melchinger 1996).

ResultsThe parents differed significantly (P < 0.05) and the genotypic variance across F3 lines was highly significant (P < 0.01) for all resistance traits. Variances due to genotype x environment

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interactions were not significant. Heritability was highest for stem lesion (h² = 0.89) and lowest for leaf lesion length (h² = 0.55). All resistance traits showed highly significant (P < 0.01) but moderate phenotypic correlations whith each other (r = 0.45 – 0.66). The speed of fungal growth was normally distributed for the means across environments. The distribution of leaf lesion length was highly significant (P < 0.01) skewed towards higher values and the distribution of stem lesion length was highly significant (P < 0.01) skewed towards smaller values. In the analysis of means across environments two putative QTL were detected for leaf lesion length, explaining 7.2 and 9.8 % of the genetic variance, respectively. For stem lesion length, four putative QTLs were identified, explaining between 4.2 and 39.2 % of genetic variance. All four putative QTL detected for the speed of fungal growth explained 65 % of the genetic variance in a simultaneous fit.

DiscussionIndividual QTL explained 4 to 39 % of the genetic variation for resistance to S. sclerotiorum leaf infection. This is comparable to the study of Mestries et al. (1998) who found QTL that explained 9 to 48 % of the phenotypic variability. In total we detected 8 QTL for resistance. This is less than in the study of Arahana et al. (2001). They found 28 putative QTL for resistance to S. sclerotiorum in soybean. Further investigations with the whole set of 354 lines and more SSR markers covering the whole genome will enable us to use cross validation to obtain unbiased estimates of the proportion of the genotypic variance explained by QTL (Utz et al. 2000).

AcknowledgementsWe thank S. Knapp, Department of Crop and Soil Science, Corvallis, Oregon, USA for providing the SSR primers. This work was supported by the Deutsche Forschungsgemein-schaft (DFG) (Sp292/7-1, Ha 2253/3-1).

ReferencesArahana VS, Graef GL, Specht JE, Steadman, JR, Eskridge KM, 2001. Identification of

QTLs for resistance to Sclerotinia sclerotiorum in soybean. Crop Sci. 41, 180-188.Degener J, Melchinger AE, Gumber RK, Hahn V, 1998. Breeding for Sclerotinia resistance

in sunflower: A modified screening test and assessment of genetic variation in current germplasm. Plant Breeding 117, 367-372.

Holloway JL, Knapp SJ, 1993. G-Mendel 3.0: Software for the analysis of genetic markers and maps, pp. 1-130. Oregon State University, Corvallis.

Mestries E, Gentzbittel L, Tourvieille de Labrouhe D, Nicolas P, Vear F, 1998. Analyses of quantitative trait loci associated with resistance to Sclerotinia sclerotiorum in sunflowers (Helianthus annuus L.) using molecular markers. Molecular Breeding 4, 215-226.

Utz HF, 1991. PLABSTAT. Ein Computerprogramm zur statistischen Analyse von pflanzenzüchterischen Experimenten. Institut für Pflanzenzüchtung, Saatgutforschung und Populationsgenetik. Universität Hohenheim, Stuttgart, Germany.

Utz HF, Melchinger AE, 1996. PLABQTL: a program for composite intervall mapping of QTL. J. Quant. Trait Loci 2 (1).

Utz HF, Melchinger AE, Schön CC, 2000. Bias and sampling error of the estimated proportion of genotypic variance explained by quantitative trait loci determined from experimental data in maize using cross validation and validation with independant samples. Genetics 154, 1839-1848.

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Quantitative trait loci (QTL) conditioning resistance to white mould in common bean

P.N. Miklasa, R.H. Rileyb, K.F. Graftonc, and P. Geptsd

aUnited States Department of Agriculture, Agricultural Research Service, Vegetable and Forage Crop Research Unit, 24106 North Bunn Road, Prosser, WA, 99350, USA; bSyngenta, Nampa, ID, 83687, USA; cDepartment of Plant Sciences, North Dakota State University, Fargo, ND, USA; and dDep. of Agronomy and Range Science, University of California, 1 Shields Avenue, Davis, CA 95616-8515, USA

Abstract Breeding for resistance to white mould in common bean is complex because resistance is quantitatively inherited and confounded by avoidance traits. We have identified three QTL from two resistance sources that condition physiological resistance in the field. The putative QTL have major effect and may be amenable to marker-assisted selection.

IntroductionWhite mold, caused by Sclerotinia sclerotiorum Lib. de Bary, is a devastating disease of common bean (Phaseolus vulgaris L.) worldwide. The current breeding strategy to combat this disease in bean is to combine physiological resistance with avoidance mechanisms such as upright architecture, which promotes a drier microclimate less conducive to white mold infection within the plant canopy. Measurement of physiological resistance in the field is confounded by disease avoidance and vice versa. Thus, greenhouse tests are used to evaluate solely for physiological resistance. Mapping populations have enabled researchers (Miklas et al., 2001; Park et al., 2001, Kelly & Kolkman, 2001) to identify QTL that condition physiological resistance or avoidance and discern their relative importance to overall field reaction. Our current objective is to examine novel resistance sources for QTL conditioning resistance to white mold disease. A future goal is to fine map the major-effect QTL identified, with the primary purpose of efficient marker-assisted selection in mind, but also as the next step toward candidate gene discovery and cloning.

Material and MethodsQTL analyses of resistance to white mould in five different recombinant inbred populations is currently under investigation: A55/G122 (67 lines), Benton/NY6020-4 (77 lines), G122/Montcalm (98 lines), I9365-31/Raven (107 lines), and Aztec/ND88-106-04 (86 lines). Each population is to be screened for reaction to white mold in multiple greenhouse straw tests (Petzoldt & Dickson, 1996) and field trials. Disease reactions are recorded using a 1-9 scale (Miklas et al., 2001), where 1 = no disease and 9 = severe disease and plant death. Regression analysis (P<0.005) is used to identify QTL with a major effect on disease reaction. Mapmaker/EXP 3.0 is used to build linkage maps and integrate the major QTL into the core map (BAT 93/Jalo EEP 558).

Results and DiscussionQuantitative trait analysis has been completed for two populations. For A55/G122 (Miklas et al., 2001), one major-effect QTL on linkage group B7 explained 38% of the phenotypic variation for physiological resistance to white mold in the straw test. This same QTL (26%)

Sclerotinia 2001 59

S8.2

was an important component of field resistance. Another QTL (18%) on B1 contributed to avoidance via an open canopy attributable to the fin locus for determinate growth habit. Many determinate lines were undesirable for selection, however, because their avoidance was generally a result of poor vigour associated with the determinate bush habit. Reduced vigour of determinate lines is a common occurrence for progeny from such a wide cross as between a Middle American vine bean (A55) and Andean bush bean (G122). A taller plant canopy was also associated with less disease (r = -0.37, P<0.01) in this population, but a QTL for plant height was not detected.

For Benton/NY6020-4, major-effect QTL on B6 (12%) and B8 (35%) conditioned physiological resistance in the straw test. These same QTL conditioned field resistance 31% (B6) and 15% (B8), but the degree of expression was reversed. A QTL associated with disease avoidance, plant height (19%), mapped to the same general location on B6, which may explain why there was an increased effect for this QTL in the field. The decreased expression in the field for the QTL on B8 may result from a greater effect of avoidance due to low disease pressure. Less disease pressure occurred when this population was tested compared to A55/G122, suggesting that, as disease severity increases, expression of physiological resistance conditioned by the B8 QTL is induced as avoidance mechanisms are overcome. As additional field data from multiple environments are collated, the influence of disease pressure on relative expression of avoidance mechanisms versus physiological resistance should become clearer.

In similar studies, Kelly & Kolkman (2001) detected major-effect QTL on B2 and B7 that conditioned field reaction to white mould in navy bean. Park et al. (2001), in PC-50/XAN-159, detected major-effect QTL on B7 and B8 that conditioned both greenhouse (straw test) and field resistance, which is similar to the findings herein. However, plant height was associated with both QTL, and open canopy with the B8 QTL, which circumvented the clear separation of avoidance mechanisms from physiological resistance in their population. The QTL results obtained thus far support selection for both physiological resistance and avoidance to enhance field resistance of common bean to white mould. The identification of QTL conditioning physiological resistance on B6, B7, and B8 that also contribute significantly to field resistance have helped us to gain a better understanding of the complexity of field resistance to white mould disease in bean.

ReferencesKelly JD, Kolkman JM, 2001. QTL analysis of resistance to white mould in common bean.

Proceedings of the X1th International Sclerotinia Workshop, York, UK, July, 2001. Miklas PN, Johnson WC, Delorme R, Gepts P, 2001. QTL conditioning physiological

resistance and avoidance to white mold in dry bean. Crop Sci. 41,309-315.Park SO, Coyne DP, Steadman JR, Jung G, 2001. Mapping of QTL for resistance to white

mold disease in common bean. Crop Sci. 41, (in press).Petzoldt R, Dickson MH, 1996. Straw test for resistance to white mold in beans. Annu. Rep.

Bean Improv. Coop. 39,142-143.

Sclerotinia 2001 60

S8.3

Development of white mould resistant soybean

D.H. Simmondsa, P.I. Donaldsona, T. Andersonb, S. Hubbarda, A. Davidsona, S. Riouxc, I. Rajcand and E. Cobera

aAgriculture and Agri-Food Canada, ECORC, CEF, Building 21, Ottawa, Ontario, K1A 0C6 CanadabAgriculture and Agri-Food Canada, GPCRC, P.O. Box 370, Harrow, Ontario N0R 1G0 CanadacCentre de Recherche sur les Grains Inc. 2700 rue Einstein, Sainte-Foy, Quebec G1P 3W8 CanadadDepartment of Plant Agriculture, Crop Science Division,U. of Guelph, Guelph, Ontario N1G 2W1 Canada

Abstract Canadian short-season soybean, transformed with the wheat germin gene and expressing oxalate oxidase, showed superior resistance to Sclerotinia sclerotiorum in greenhouse pathogenesis tests and confined field trials. This gene imposed no yield reduction in non-infested fields and outperformed the null and parental lines in white mould infested fields.

IntroductionWhite mould, also known as sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a major and economically devastating disease of soybeans in Canada, northern United States, Argentina and China. In Canada, yield reduction results in revenue losses of ~$6 million annually. Natural crop resistance to this pathogen is inadequate. Pathogen invasion is enabled by secretion of oxalic acid, a plant >toxin= that weakens cell walls by chelating calcium pectate (Bateman & Beer, 1965) and lowering extracellular pH to activate plant cell wall-degrading enzymes (e.g. Marciano et al., 1983). Oxalic acid also disables plant defence responses by inhibiting phenol oxidases (Marciano et al., 1983) and suppressing the oxidative burst (Cessna et al., 2000). The degradation of the oxalate toxin offered a strategy to enhance resistance. This was investigated by producing transgenic soybean expressing wheat germin (Lane et al., 1991), an oxalate oxidase (OxO) that catalyses the oxidation of oxalic acid to CO2 and H2O2. This reaction serves two modes of defense, 1) destruction of oxalic acid, and 2) production of H2O2 to enhance plant defense responses.

Materials and MethodsSoybean cv.AC Colibri were transformed with A. tumefaciens EHA105 carrying wheat germin gene gf-2.8 (Lane et al, 1991) regulated by CaMV35S promoter cloned in binary vector pRD400 (Donaldson & Simmonds, 2000). Kanamycin-resistant shoots and progeny of transgenic plants were screened for germin gene expression with a modified histological OxO assay (Dumas et al 1995). OxO activity was localized by microscopic examination of tissue sections after histological assay treatment. For resistance tests, stems were severed below the first trifoliolate leaf and inoculated with 3-d old suspensions of invitro grown mycelial cultures of S. sclerotiorum. Further evaluation was conducted in confined field trials sites in Ontario & Quebec.

Results and DiscussionTransformed soybean lines were analyzed for stability of transmission of transgene expression. Southern analysis showed only 1 germin hybridizing band in line 80(30)-1 and its progeny; this line transmitted OxO activity to the T8 generation. OxO activity was prominent in vascular and epidermal cell walls, consistent with the presence of a transit peptide sequence in the gene.

Sclerotinia 2001 61

S8.3

Fig. 1. Lesion length 9 d post-inoculation.Greenhouse stem inoculation tests showed resistance in transgenic OxO+ line (Fig. 1). The pathogen invaded the null siblings, the susceptible checks, and 9 d post-inoculation resulted in lesions greater than 6 cm and plant death. The transgenic plants showed shorter lesions than S19-90, one of the best resistant commercial lines. Superior resistance of the transgenic line was confirmed in 3 years of confined field trials at a total of 8 sites (Tab.1). In white mould infested fields, transgenic lines had higher yields than the nulls but in non-infested fields there was no yield difference (Tab.2).

Table. 1. Disease severity index (0 to 100) for progeny of transgenic line 80(30)-1 & control checks from white mould infested nurseries.

Genotype Disease severity indexOttawa 1999

Ste-Foy 1999

Ste-Foy 2000

80(30)-1 (trans.) 3 5 580(30)-9 (null) 14 28 68Nattosan (susc.) 17 55 65AC Colibri ND ND 83LSD 0.05 7 21 19

Table 2. Yields (kg ha-1) of transgenic line 80(30)-1 & check cultivars grown in infested & non-infested fields.

Ottawa Ste-Foy Ste-Foy Ste-FoyGenotype Non-

infestedNon-

infestedNon-

infestedInfested

1999 1999 2000 200080(30)-1 (trans.) 1380 2444 2612 292880(30)-9 (null) 1414 2460 2531 2059AC Colibri nd nd 2477 1832Nattosan 2381 2674 2664 2035OAC Shire 2957 3588 3270 3068OAC Salem 1567 4763 3033 2920CV 6 11 6 10LSD 0.05 154 473 192 303

AcknowledgementsWe thank Ontario Soybean Growers & Ontario Research Enhancement Program for funding.ReferencesBateman DF, Beer SV, 1965. Simultaneous production and synergistic action of oxalic acid and

polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathol. 55, 204-211.Cessna SG, Sears VE, Dickman MB, Low PS, 2000. Oxalic acid, a pathogenicity factor for

S. sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12, 2191-2199.Donaldson PA, Simmonds DH, 2000. Susceptibility to Agrobacterium tumefaciens and

cotyledonary node transformation in short-season soybean. Plant Cell Repts. 19, 478-484.Dumas B, Freyssinet G, Pallett KE,1995. Tissue-specific expression of germin-like oxalate

oxidase during development and fungal infection of barley….. Plant Physiol.107,1091-1096.Lane BG, Bernier F, Dratewka-Kos E, Shafai R, Kennedy TD, Pyne C, Munro JR, Vaughan T,

Walters D, Altomare F, 1991. Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins. J. Biol. Chem. 266, 10461-10469.

Marciano P, Di Lenna P, Magro P, 1983. Oxalic acid, cell wall-degrading enzymes and pH in pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum isolates on sunflower. Physiol. Plant Pathol. 22, 339-345

Sclerotinia 2001 62

S8.4

Search for resistance to Sclerotinia sclerotiorum in common bean - screening and sources.

J.R. Steadmana, J.M. Kolkmanb, and K.M. Eskridgea

aUniversity of Nebraska, Lincoln, NE 68583, USA; bOregon State University, Corvallis, OR 97331, USA

AbstractEleven common bean genotypes were tested for putative resistance to white mould at seven sites in North and South America in 2000, using both field tests and various laboratory and greenhouse screening methods. The greenhouse straw tests and field tests were highly associated by Spearman’s rank correlation. Seven genotypes were ranked significantly lower than the susceptible control genotype, indicating that these genotypes have broad partial resistance.

IntroductionWhite mould of common bean, incited by Sclerotinia sclerotiorum, causes yield losses to both dry and succulent bean crops throughout the world. Difficulties in managing this disease result in part from persistence of sclerotia inoculum in soil, wide host range of the pathogen and lack of highly resistant germplasm. Developing resistant cultivars is the most economical disease management strategy for the grower. The objective of the study was to identify broadly resistant bean genotypes by testing putative sources of partial resistance developed by bean breeders with laboratory, greenhouse and field methods in different locations.

Materials and MethodsField tests consisted of two rows of each entry and a common susceptible genotype resulting in a three-row plot 4.6m (15 ft) long replicated three times in a randomized complete block design. The laboratory and greenhouse tests were detached leaf (Steadman et al., 1997), straw (Petzoldt & Dickson, 1996), oxalate (Kolkman & Kelly, 2000), and modified limited term (Pennypacker & Hartley, 1995). In addition to the data of the authors Kolkman and Steadman, data were generated by J. Costa (Brazil), K. Grafton (North Dakota), J. Kelly (Michigan), K. Kmiecik (Wisconsin), J. Myers (Oregon) and P. Miklas (Washington). Location and tests were as follows: oxalate - Brazil, North Dakota, Michigan; field - Michigan, Washington, Wisconsin; straw - Oregon, Washington, Wisconsin; detached leaf - Nebraska; and limited term - Brazil. Because of the differences in data sets, e.g., field disease severity, lesion size, length of stem affected and number of nodes afftected, the entries were ranked from most resistant (1) to most susceptible (12) in each test. A Spearman’s rank correlation was used to compare entry rankings in each test.

Results and DiscussionIn general, there were significant (p< 0.05) positive correlations among Michigan field, Oregon straw, Washington field and straw, and Wisconsin field and straw tests. The three most highly associated tests were between Michigan field and Washington field (r=0.746, p=0.005); Michigan field and Wisconsin field (r=0.795, p=0.002) and Washington field and Wisconsin straw (r=0.762, p=0.004). Oxalate tests were not correlated significantly with each

S8.4

other nor were they correlated with other tests. Both environmental variation between tests and variation in methodologies may have contributed to the lack of correlations between oxalate tests. The detached leaf test was similar to the oxalate test in its lack of correlation with other tests. Since only one detached leaf test and one modified limited term test were compared, it was not possible to evaluate consistency in different locations. However, the straw and field tests from Washington, Michigan, Wisconsin and Oregon produced similar rankings even though different S. sclerotiorum isolates were involved at each state location. When an ANOVA was used on ranks, with each test as a block and bean genotype as a treatment, there were significant differences (p=0.004) among genotypes (Table 1). B7354 (J. Myers) and I9365-25 (P. Miklas) had the best mean rank, but L192 and MO162 (J. Myers), G122, PC-50 and NY6020-5 (M. Dickson) all were significantly ranked lower than the susceptible control great northern Beryl.

Table 1. Mean ranking of bean lines for reaction to Sclerotinia sclerotiorum from 11 field and in vitro tests.

Entry Ranking T Grouping

BerylN97774ProsperityND8915146-02ExRico (Bunsi)NY6020-5PC-50M0162G122L192I9365-25B7354

9.7238.2738.0007.8187.7276.2736.0915.4555.2734.8184.4554.455

AABABCABCABC BCD BCD BCD CD D D D

Means with the same letter are not significantly different LSD (0.05) = 2.832.

AcknowledgementsWe thank Janelle Fenton for technical assistance and Shayne Ortmeier for word processing.

ReferencesKolkman JM, Kelly JD, 2000. An indirect test using oxalate to determine physiological

resistance to white mold in common bean. Crop Science 40, 281-285.Pennypacker BW, Hatley OE, 1995. Greenhouse technique for detection of physiological

resistance to Sclerotinia sclerotiorum in soybean. Phytopathology 85, 1178. Supplement. Petzoldt R, Dickson MH, 1996. Straw test for resistance to white mold in beans. Ann. Rep.

Bean Improv. Coop. 39, 142-143.Steadman JR, Powers K, Higgins B, 1997. Screening common bean for white mold

resistance using detached leaves. Ann. Rep. Bean Improv. Coop. 40, 140-141.

S8.5

Sclerotinia blight research in peanut

H. A. Melouk and K. D. Chenault

USDA-ARS, PSWCRL, Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK, USA

AbstractSclerotinia blight resistance research is aimed at reducing the reliance on chemical management. Greenhouse methods are being developed and improved to identify and quantify resistance in peanut to Sclerotinia minor. Standardization of fungal inoculum, host plant, and incubation conditions will be discussed in relation to obtaining reliable results that are useful in resistance breeding efforts.

IntroductionSclerotinia blight, caused by Sclerotinia minor Jagger, is an important disease of peanut in the United States. Efforts by researchers in the last 20 years were successful in developing peanut cultivars having resistance to Sclerotinia blight. Development of greenhouse methods to evaluate genotypes to Sclerotinia blight is essential to accelerate progress in peanut breeding programs. The objectives of our research are: 1) to characterize biological and environmental elements conducive to development of Sclerotinia blight under greenhouse conditions, and 2) to quantify reaction of peanut germplasm to S. minor in the greenhouse, and determine how that correlates with disease reaction in field plots.

Materials and MethodsIn greenhouse experiments, detached shoots (Melouk et al., 1992) or whole peanut plants (Goldman et al., 1995) were inoculated with mycelial plugs of S. minor, and rate of lesion expansion (LE) or area under disease progress curve (AUDPC) were calculated. Small field plots were established at Stillwater, Oklahoma, and maximum disease incidence (MDI) and rate of disease progress (R) were determined (Akem et al., 1992). Rank correlations between LE and MDI and/or LE and R were calculated.

Results and Discussion Favorable conditions required for consistent infections under greenhouse environment are: 1) use mycelial plugs from a 2-day-old cultures; 2) use 6-8 week old plants; 3) maintain 100% relative humidity, and a temperature of 21+2 oC for 3-5 days post inoculation; and 4) provide light of 25-35 μE/m2/s. Data will be presented to illustrate the importance of using mycelial plugs from 2-day-old culture for inoculation. Plugs from 2-day-old culture were more consistent in reproducing disease (as determined by AUDPC) on whole plants, which differentiated susceptible and resistant reaction under greenhouse conditions. Values of rank correlations between LE and MDI or LE and R (Table 1) strongly support the usefulness of inoculating peanut germplasm with S. minor under greenhouse conditions to augment field evaluations, and accelerating breeding programs for Sclerotinia blight resistance.

S8.5

Table 1. Rankinga of reactions of peanut to S. minor in the greenhouse and field plots.

Genotype GreenhouseLE*

FieldMDI** R***

TX 798736 5 4 3

TX 804475 1 1 4

TX 798731 3 3 1

TX 798683 2 2 2

UF 73-4022 7 5 5

TX 77174 10 9.5 10

TX 771108 6 6 6

TP107-3-8 12 11 7

TX 833829 9 7 8

TX 835841 4 8 9

TX 833841 8 9.5 11

Florunner 11 12 12* LE = Rate of lesion expansion (mm/day). Range of values for LE (0.52-1.35).** MDI = Maximum disease incidence (%). Range of values for MDI (1.7-93.3).*** R = Rate of disease progress. Range of values for R (0.0057-0.14).aRank and rank correlations: Rank of 1 designates most resistant reaction, while rank of 12 designates most susceptible reaction. Rank correlations were: LE & MDI = 0.89 (P=0.001) ; LE & R = 0.71 (P= 0.01) ; MDI & R = 0.88 (P=0.001).

ReferencesAkem CN, Melouk HA, Smith OD, 1992. Field evaluation of peanut genotypes for

resistance to Sclerotinia blight. Crop Protection. 11, 345-348.Goldman JJ, Smith OD, Simpson CE, Melouk HA, 1995. Progress in breeding Sclerotinia

blight-resistant runner-type peanut. Peanut Science 22, 109-113. Melouk HA, Akem CN, Bowen C, 1992. A detached shoot technique to evaluate the

reaction of peanut genotypes to Sclerotinia minor. Peanut Science 19, 58-62.

SESSION IX

PATHOLOGY AND EPIDEMIOLOGY

Chair:

Berlin Nelson, North Dakota State University, USA

S9.1

Apothecium development from sclerotia of Sclerotinia sclerotiorum in relation to rain and crop density

E. Twengström, J. Yuen and R. Sigvald

Department of Crop Production Science, Swedish University of Agricultural Sciences, P.O. Box 7043, SE-750 07 Uppsala, Sweden

AbstractThe influence of rainfall and crop density on apothecium formation from Sclerotinia sclerotiorum was studied with survival analysis. Amount of rain and the number of apothecia from sclerotia placed in the soil were recorded on weekly basis during 12 cropping seasons. The model can be used to quantify the risk for apothecium development depending on the status of the six covariates: weekly rainfall during each of the past four weeks, time of the season, and crop density.

IntroductionA risk point table is since several years used to predict the need for fungicide applications against Sclerotinia stem rot in spring sown oilseed rape in Sweden. The prediction model is based on field specific data and weather parameters. Six factors (number of years since last oilseed rape crop, disease incidence in last susceptible crop, rain during the two weeks before flowering, weather forecast, and regional risk for apothecium development) that influence Sclerotinia infection have been given points with regard to the risk for heavy infestation (Twengström, et al., 1998).

The regional risk value in the Swedish forecasting system is based on apothecium development in sclerotia depots. The system with depots has been run since 1987 and weekly observations of apothecium development, rainfall and data on crop density and soil type have been collected (Twengström, 1999).

To improve the Sclerotinia forecasting system, better predictions of the risk for apothecium development in individual fields are needed. The aim of this study was to correlate rain, crop canopy and soil type to apothecium development. If quantitative rain data of sufficient quality are available, the probability of apothecium development, assuming that sclerotia are present in the soil, could be estimated for individual fields. It has also been a goal to reduce the number of sclerotia depots to make predictions of the regional risk value less expensive.

Materials and MethodsPreconditioned sclerotia were placed in about 30 spring sown oilseed rape fields per year from 1987 to 1998. The number of viable apothecia were counted weekly and rainfall was registered at each occasion. Crop canopy was judged as sparse, normal or dense at the time of flowering. Soil type and organic matter was determined from a soil sample from each field.

S9.1

Statistical analysisSurvival analysis was used to model the appearance of apothecia as a function of time, rain preceding an observation (time dependent covariates), soil type and crop density (time independent covariates). The SAS procedure GENMOD (SAS Institute, 1993) was used to perform the analyses.

Results and DiscussionA dense crop canopy increased the risk for apothecium development as did increasing amounts of rain during the last four weeks. Soil type was not correlated to probability of apothecium occurrence The odds for presence of apothecia in the lowest risk fields was 0.00032 (C.I. 0.000037–0.0028). The odds of apothecia developing in a field that belongs to the highest risk group for all parameters was 4.62 which equals a probability of 0.82 (C.I. 0.65–0.92).

The study presents a method of evaluating risk factors for development of soil inoculum. In the analyses, the influence of both time independent variables such as crop density and time dependent variables such as rain as well as the influence of time itself is quantified. It has been shown in several studies that moist conditions favour apothecium development and that dry conditions inhibit apothecium formation. With the method presented here the influence of fluctuating weather on apothecium production can be estimated.

AcknowledgementsThanks are due to the extension officers at the Regional Plant Protection Centres and at the Swedish Agricultural Societies for assistance with data collection. The work was funded by the Swedish Seed and Oilseed Growers Association, the Swedish Board of Agriculture and the Swedish University of Agricultural Sciences.

ReferencesTwengström E, Sigvald R, Svensson C, Yuen J, 1998. Forecasting Sclerotinia stem rot in

spring sown oilseed rape. Crop Protection 17, 405-411.Twengström E, 1999. Epidemiology and forecasting of Sclerotinia stem rot in spring sown

oilseed rape in Sweden. Doctoral thesis. Agraria 181. Department of Ecology and Crop Production Science, Swedish University of Agricultural Sciences, Uppsala.

SAS Institute, 1993. SAS Technical Report P-243, SAS/STAT Software: The GENMOD Procedure, Release 6.09, Cary NC: SAS Institute Inc. 88pp.

S9.2

Effects of temperature and relative humidity on the colonisation of kiwifruit (Actinidia deliciosa) petals by Sclerotinia sclerotiorum ascospores

S. M. Hoytea, R. M. Beresfordb, and P. G. Longc

a HortResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand, b HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand; and c Institute of Natural Resources, Massey University, Private Bag 11222, Palmerston North, New Zealand.

AbstractDisease development in New Zealand kiwifruit by Sclerotinia sclerotiorum is dependant on the level of colonisation of floral tissues by ascospores. Controlled environment studies showed that petal colonisation is significantly reduced at relative humidities <90% and under diurnal fluctuations that simulate field conditions with <5 mm rainfall day-1 during flowering.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary can cause significant crop loss (up to 17%) in New Zealand kiwifruit (Actinidia deliciosa (A. Chev.)) orchards and primary infection involves ascospores colonising floral tissues during flowering (Hoyte, 2001). Infected floral tissues are the major source of inoculum leading to diseased fruitlets, scarring and field rot of fruit (Hoyte, 2001). Understanding the environmental conditions favourable for petal infection will assist with the development of a weather-based disease prediction system.

Materials and MethodsTen batches of ten 3–4 day-old (post-anthesis) kiwifruit petals were inoculated with S. sclerotiorum ascospores in a modified Burkard ‘Jet’ spore trap. One petal from each batch, plus three non-inoculated petals, were assigned to each of ten high humidity chambers held at 5, 10, 15, 18, 20, 22.5, 25, 27.5, 30 and 34oC for 72 h (Experiment 1). Colonisation was quantified by maceration of pairs of petals in PBS and spreading aliquots onto selective medium (Bourdôt et al., 2000). Colonies of S. sclerotiorum were counted after 14 days. In Experiment 2, inoculated petals were incubated in 20 combinations of temperature (10, 15, 20 and 25oC) and relative humidity (RH) (85, 90, 93, 98 and 100%) in chambers containing saturated salt solutions. Experiment 3 involved placing inoculated petals in saturated salt chambers at (i) 98% RH at 20oC, (ii) 85% RH at 20oC, and in dynamic controlled environment chambers at (iii) 97% RH at 20oC (iv) 85% RH at 20oC (v) diurnal fluctuation of RH (70–100%) at 20oC, and (vi) diurnal fluctuation of temperature (10–20oC) and RH (70–100%). The latter simulated field conditions with <5 mm rainfall day-1 during flowering.

Results and DiscussionIn Experiment 1, there were very low numbers of S. sclerotiorum colony forming units (cfu) on inoculated petals incubated at 5–15oC (<100 cfu/petal), and no cfu were recovered from petals at 34oC. There was a significant increase (P<0.001) in the mean cfu/petal between 15oC and 18oC. The mean cfu/petal remained high over the temperature range 18–27.5oC (1972–2255 cfu/petal). No S. sclerotiorum cfu were recovered from non-inoculated petals. In Experiment 2, the number of S. sclerotiorum cfu/petal was highest in the treatment combinations of 25oC ´ 90–100% RH (258–2530 cfu/petal) and 20oC ´ 98–100% RH (520–

S9.2

960 cfu/petal) (Figure 1). There was significantly less (P<0.05) colonisation (<30 cfu/petal) of petals incubated at 10 and 15oC regardless of the RH. The number of S. sclerotiorum cfu/petal recovered from non-inoculated petals was <20 for all treatments (Figure 1). In Experiment 3, the number of S. sclerotiorum cfu/petal recovered from petals in the static 98% RH treatment (390 cfu/petal) was significantly greater (P<0.05) than that from petals in the static 85% RH treatment (80 cfu/petal) (Figure 2). Treatments in the waterbath chambers, other than the 97% RH treatment, all had <5 S. sclerotiorum cfu/petal. No S. sclerotiorum cfu were recovered from the non-inoculated petals.

Figure 1 Figure 2

0

10

100

1000

10000

1015

2025

85

90

95

100

Mea

n cf

u/pe

tal

Temperature (oC)

Relative humidity (%

)

Incubation conditions

Mea

n cf

u/pe

tal

0

10

100

1000

= Maximum sed

Fig. 1. Effect of temperature and relative humidity on colonisation of kiwifruit petals (cfu/petal) inoculated with S. sclerotiorum ascospores and incubated for 72 h. Fig. 2. Mean S. sclerotiorum cfu/petal recovered from inoculated petals incubated in a range of conditions during Exp. 3.

The sharp increase in petal colonisation between 15oC and 20oC in Experiments 1 and 2 overlaps with the mean daily temperature range during flowering of kiwifruit (Morley-Bunker & Salinger, 1987). Therefore small changes in the prevailing weather conditions could have a substantial affect on disease development. The significant reduction in colonisation at <90% RH is common among plant pathogens and indicates that without free moisture, high RH is required for infection of floral tissues. Experiment 3 showed that typical field conditions during flowering without rainfall are not conducive to colonisation of petals by S. sclerotiorum. Other ranges of environmental conditions need to be studied to more completely understand infection responses under field conditions.

ReferencesBourdôt GW, Saville GA, Hurrell GA, Harvey IC, de Jong MD, 2000. Risk analysis of

Sclerotinia sclerotiorum for biological control of Cirsium arvense in pasture: sclerotium survival. Biocontrol Science & Technology 10, 411-425.

Hoyte SM, 2001. Epidemiology and management of Sclerotinia sclerotiorum (Lib.) de Bary in kiwifruit (Actinidia deliciosa (A. Chev.)). PhD thesis, Massey University.

Morley-Bunker MJ, Salinger MJ, 1987. Kiwifruit development: the effect of temperature on budburst and flowering. Weather and Climate 7, 26-30.

S9.3

Ascospores, petals and infection of oilseed rape by Slerotinia sclerotiorum

Alastair McCartneya, Arwinderpal Herana, Jackie Freemana and Qiangsheng Lib

aPlant Pathogen Interactions, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK.bAnhui Academy of Agricultural Science, Hefei, Anhui, People’s Republic of China.

NB: For paper please see P5.4 (page 183-184) and P5.5 (page 185-186).

Abstract P5.4Infection of oilseed rape (Brassica napus) by petals containing ascospores of Sclerotinia sclerotiorum

Alastair McCartneya, Arwinderpal Herana and Qiangsheng Lib


AbstractIn oilseed rape crops infection by Sclerotinia sclerotiorum is usually via ascospore-bearing petals. At petal-fall ascospore-infected petals stick to leaves initiating infections that can develop into stem rot lesions. The effects of ascospore numbers and environmental conditions on infection of oilseed rape leaves by ascospore-bearing petals are reported.

Abstract P5.5Petal fall, petal retention and petal duration in oilseed rape crops

Alastair McCartneya, Arwinderpal Herana, Qiangsheng Lib and Jackie Freemana


AbstractOilseed rape petals bearing ascospores of Sclerotinia sclerotiorum play an important role in initiating stem rot disease in oilseed rape crops. Patterns of flowering, petal fall and petal retention measured in oilseed rape crops in the UK and china are reported.

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S9.4

Factors contributing to the adhesion of petals to oilseed rape leaves and the risk of sclerotinia stem rot

P. Gladdersa, C. Youngb, J. Smithb, M. Watling a and L. Hirona

a ADAS Consulting Limited, Boxworth, Cambridge, CB3 8NN, UK and bADAS Consulting Limited, Woodthorne, Wergs Road, Wolverhampton, WV6 8TQ, UK

AbstractDecision making for sclerotinia control in oilseed rape requires an understanding of inoculum and the processes which lead to plant infection, particularly the adhesion of petals to the foliage. Conditions favourable for petal sticking occurred infrequently and may be limiting stem rot development in oilseed rape in the UK.

IntroductionIn the UK, stem rot (Sclerotinia sclerotiorum) is a common disease in winter oilseed rape affecting, on average, about 2% plants each year (Turner et al., 2000). Severe attacks which cause economic damage occur infrequently (<10% crops). Fungicide use to control stem rot increased after a general epidemic in 1991 and currently, about 40% of crops receive a fungicide during flowering. Many of these sprays are unnecessary, although it is accepted that farmers are looking for long term benefits from preventing a build up of sclerotia of S. sclerotiorum in soil, rather than direct yield benefits. With improved risk assessment, there is scope to reduce these fungicide treatments by at least 50 percent (Davies et al., 1999).

Problems tend to recur on farms which have experienced severe attacks (>20% plants affected) with 41% crops at risk on these farms during 1993-1998 compared with 9% crops at risk on all farms with histories of <20% plants affected. Testing petals for the presence of sclerotinia using agar media would improve decision making compared with consideration of farm histories of stem rot problems. Under UK conditions, it is unlikely that petal testing alone will provide satisfactory guidance for spray decisions. The disease cycle in oilseed rape requires petal adhesion to plant surfaces and this is poorly understood. This paper presents observations on the behaviour of petals, which may ultimately assist in decision making.

Materials and MethodsTwo cultivars of spring oilseed rape, cv. Hyola 401 and ZNA 005 (an apetalous type provided by the John Innes Centre (JIC), Norwich) were sown at three seed rates (60, 120 and 180 seeds/m2) at Crowland, Lincolnshire in replicated plots (40 m2) on 30 March 1999. A similar experiment on winter oilseed rape with cv. Apex and an experimental type with reduced petals (supplied by JIC) was sown at Syerscote, Staffordshire in early September 1999. The crops were inspected at 3-7 day intervals to record the number of open flowers on tagged main and secondary racemes and the number flowers which had shed their petals. Petals deposited and stuck on the leaves were assessed on five plants per plot as % leaf area affected on selected leaf layers. Petal sticking was monitored in a farm crop of winter oilseed rape cv. Pronto at ADAS Boxworth, Cambridge in 2001. The crop was used as a source of detached leaves and

S9.4

recently fallen petals for laboratory experiments to establish if petal sticking could be produced by lightly misting or by dipping leaves or petals with water.

Results and DiscussionIn 1999, the apetalous ZNA 005 had 0.4 petals/main raceme compared with 259.6 for Hyola 401. Petal sticking was apparent on 11 June (GS 4,7) and 15 June (GS 4,9) when there was significantly higher petal deposition and petal sticking on cv. Hyola than on ZNA 005 (5.4% and 0.2% leaf area with petal deposits and 3.4% and 0.1% area with stick petals on 15 June). Although the numbers of petals deposited increased with increasing seed rate, the differences were not always significant. On cv. Hyola, there were fewer sticking petals on 15 June at the lowest rate (1.7% leaf area with petals compared with 3.8% and 4.8% area at 120 and 180 seeds/m2 respectively). Sclerotinia stem rot did not develop at this site even though up to 43% petals were carrying the pathogen.At Syerscote in 2000, flowers opened at a linear rate (2.5% per day) and petal fall also appeared to be linear (3.7% flowers lost per day). Flower life decreased from 10.5 days at GS 4,1 to <3 days by GS 5,0. Petal sticking was considered efficient on 24 April and 8 May because of the high ratio of stuck to fallen petals (0.7-0.9). This was associated with periods of rain on successive days. In general, petal sticking occurred repeatedly at low levels, rather than as discrete events. Sclerotinia affected 3% stems of cv. Apex and 6% stems of the reduced petal cultivar, despite only 8% petal infection. In 2001, no petal stick was recorded until 15 and 18 May (GS 4,8) at Boxworth. This was associated with periods of rain (0.8-7.2 mm/day) giving several hours of surface wetness on successive days. Tests on detached leaves and fallen petals showed that petal sticking could occur if leaf and/or petal surfaces were moist with various combinations of wet and dry leaves and petals, but it was difficult to get petals to stick firmly to leaves after the petals had been dipped in water. This suggests that lightly wetted surfaces are most conducive to petal sticking.Under UK conditions, many crops have a low risk of sclerotinia problems because inoculum is low, temperatures during flowering are sub-optimal and petal sticking occurs infrequently. An understanding of the factors affecting petal sticking will be required to develop improved risk assessment schemes to accompany rapid diagnostic tests. Petal sticking varies within the plant canopy and is influenced by plant population as well as by weather factors. There remains a challenge to understand the processes involved and to develop predictive schemes for use on farms.

AcknowledgementsFunding for this project from the Ministry of Agriculture, Fisheries and Food is gratefully acknowledged. We thank Dr E Arthur for providing seed of apetalous cultivars.

ReferencesDavies JML, Gladders P, Young C, Dyer C, Hiron L, Locke T, Lockley D, Ottway C, Smith

J, Thorpe G, Watling M, 1999. Petal culturing to forecast sclerotinia stem rot in winter oilseed rape: 1993-1998. Aspects of Applied Biology 56,129-134.

Turner JA, Elcock SJ, Hardwick NV, Gladders P, 2000. Changes in fungicide use on winter oilseed rape in England and Wales during the 1990s. Proceedings of the BCPC Conference - Pests and Diseases 2000, 2, 865-870.

SESSION X

EPIDEMIOLOGY

Chair:

John Whipps, Horticulture Research International, UK

S10.1

Epidemiology of Sclerotinia sclerotiorum on lettuce

J.P. Clarksona, J.M. Whippsa and C.S. Youngb

a Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UKbADAS Consulting Limited, ‘Woodthorne’, Wergs Road, Wolverhampton, WV6 8TQ, UK

AbstractApothecia of Sclerotinia sclerotiorum were produced at an optimum temperature of 15°C and only above a threshold soil water content. Ascospores survived a wide range of conditions but high temperature and humidity reduced viability. There was no clear relationship between wetness or humidity duration and lettuce infection.

IntroductionLettuce drop caused by Sclerotinia sclerotiorum continues to be a problem in field grown lettuce in the UK. Currently the disease is controlled with iprodione but timing is problematic as the disease is difficult to predict. To address this problem, a collaborative project between ADAS and HRI was set up to determine key environmental factors affecting aspects of Sclerotinia epidemiology on lettuce through field and laboratory studies. This paper presents results from experiments examining apothecial production, ascospore survival and infection of lettuce plants under controlled conditions.

Materials and MethodsSclerotia of two S. sclerotiorum isolates from Cheshire and Norfolk soils were assessed for apothecial production at a range of soil water contents either in the glasshouse (15°C min, 20°C max) or in growth cabinets at 10-20°C. Ascospores of S. sclerotiorum were collected on statically charged acetate sheets and exposed to different relative humidity (RH) levels between 5 and 30°C. At intervals, spores were removed by ‘spreading’ an acetate piece on tap water agar flooded with sterile distilled water. Spore germination was assessed after incubation overnight at 20°C. Infection of lettuce plants was examined at different temperatures, RH or durations of leaf wetness. Plants were inoculated either by spraying ascospore suspensions to run off, or by allowing mature apothecia to puff directly onto lettuce plants. At the end of the infection period under study, plants were removed to the glasshouse and disease assessed every week.

Results and DiscussionIn the glasshouse, stipes developed after 3 weeks and apothecia after 6 weeks in both the Cheshire peat soil and the Norfolk silty clay loam soil. Apothecia were only produced in large numbers when soil water content was above 50% (approx. -12kPa) for the Cheshire soil and 15% (approx. -32kPa) for the Norfolk soil. Some stipes but few apothecia were produced in the peat soil at 40% water content. At water contents close to field capacity in the Norfolk soil (35% and 40%) apothecial production was reduced, but this was not apparent for the Cheshire soil at up to 90% water content. When the Norfolk Sclerotinia isolate was tested for apothecial production at different temperatures and 20% soil water content, stipes appeared after three weeks at 15 and 20°C with apothecia produced after four weeks. However, only

S10.1

17% of sclerotia germinated at 20°C compared to 70% at 15°C. At 10°C stipes appeared after 6 weeks and apothecia after 7 weeks. It would therefore appear that these Sclerotinia isolates have a narrow range of temperature and soil water conditions under which large numbers of apothecia are produced and this was confirmed in field studies where bi-weekly burials of sclerotia often produced apothecia simultaneously (Young et al., 2001).

When ascospores were exposed to RH >80% at 30°C, only 50% survived between 2 and 4 weeks, which increased to 5-6 weeks at 15-25°C. Ascospores at 5 and 10°C at >80% RH did not show any significant decrease in viability for the 16 week duration of the experiment. At 58% RH, ascospores survived longer with a decrease to 50% viability at 30°C after 6-8 weeks and after 13 weeks at 25°C. There was no significant decrease in viability at lower temperatures. Preliminary experiments on plants at 10°C and 25°C at both 60% and 85% RH were in agreement with these results. Significantly, ascospore survival in these experiments was much greater than that found by workers in the USA (Caesar & Pearson, 1983) and may explain why diseased lettuce is observed apparently in the absence of suitable preceding conditions for apothecial production or infection by ascospores.

Lettuce plants inoculated with ascospore suspensions of S. sclerotiorum and exposed to wetness periods between 2 hours and 4 days at temperatures between 5 and 25°C all resulted in some infection but in general, longer wetness periods and temperatures of 15 or 20°C resulted in more disease (up to 90% plants infected) than at 10 or 25°C (up to 70% plants infected). However, results were variable and shorter periods of wetness sometimes resulted in more disease. Plants also became infected in control treatments when fan-dried immediately after spray inoculation. Moreover, when dry ascospore inoculum was applied to plants exposed to a range of RH between 50% and 98% for up to 5 days, infection still occurred in all treatments even though microscopic examination of leaf samples showed that there was no spore germination below 98% RH. However, disease was always seen at the lettuce stem base and leaf infections only occurred after prolonged periods of wetness. It is therefore likely that conditions in this region are quite different to the measured aerial environment and this may explain the inability to determine a clear relationship between wetness or RH duration and lettuce infection. Further work is required to examine this phenomenon. AcknowledgementsWe gratefully acknowledge DEFRA for funding.

ReferencesCaesar AJ, Pearson RC (1983) Environmental factors affecting survival of ascospores of

Sclerotinia sclerotiorum. Phytopathology 73, 1024-1030.Young CS, Smith JA, Watling M, Clarkson JP, Whipps JM (2001) Environmental conditions

influencing apothecial production and lettuce infection by Sclerotinia sclerotiorum in field conditions (this conference).

S10.2

Polymerase chain reaction (PCR)-based assays for the detection of inoculum of Sclerotinia spp

Jacqueline Freeman, Carmen Calderon, Elaine Ward and Hugh Alastair McCartney

Plant Pathogen Interactions Division, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK.

AbstractPCR-based assays for detecting inoculum of Sclerotinia spp. are described. Untreated spores were successfully used for these assays, but DNA purification was required when large numbers of non-target spores were present. DNA from as few as 10 spores could be reliably detected. The assay detected inoculum of S. sclerotiorum in field-based air samples and from oilseed rape petals.

IntroductionAirborne ascospores produced by carpogenic germination of sclerotia are the major source of inoculum for infection of crop plants by Sclerotinia sclerotiorum. Infection occurs when ascospores germinate on non-living or senescent plant parts, and then infect healthy plant tissue. For example, in oilseed rape crops ascospore-bearing petals stick to leaves causing initial infections. Monitoring of airborne or, for oilseed rape crops, petal-borne inoculum offers a direct measure of the risk of crop infection. Methods of detection of airborne inoculum based on microscopy or culturing are time-consuming, labour intensive and subjective. Methods based on DNA analysis have the potential to detect airborne fungal spores more reliably and rapidly. We report development and testing of a PCR-based assay for the detection of inoculum of S. sclerotiorum in air samples and on oilseed rape petals.

Materials and MethodsDNA sequences unique to S. sclerotiorum were determined by analysing nuclear rDNA sequences of relevant fungi available in the EMBL/Genbank databases. Primer/probe design and DNA sequence analysis was performed using the programs PILEUP, MELT, and FASTA available in the GCG package (Genetics Computer Group, 1994). The program Net Primer was used to analyse likely secondary structures of PCR primers.

DNA, for use in PCR assays, was purified from ascospore suspensions, field samples and oilseed rape petals. The samples were violently agitated in 0.1% Nonidet P-40 containing Ballotini balls using a FastPrep® machine (Savant Instruments, Holbrook, New York, USA). This treatment disrupted more than 99% of the ascospores in each sample. DNA was purified from the disrupted samples as described by Williams et al. (2001).

Results and DiscussionTwo regions, designated SSFWD and SSREV, were identified as potential specific primers. The primers amplify a region within that amplified by the consensus fungal primers ITS4/ITS5. FASTA searches of the EMBL/Genbank databases revealed that the primers were identical to sequences of the very closely related species S. trifoliorum, S. minor, and S. glacialis. The primers would therefore be likely to detect DNA from these species as well

S10.2

as S. sclerotiorum. PCR assays using SSFWD/SSREV were done using DNA from a range of different fungal species including pathogens of oilseed rape, cereal pathogens and other air-dispersed fungi. The primers amplified DNA from S. sclerotiorum but not from any of the other species tested. They also amplified S. sclerotiorum DNA specifically, even in the presence of large amounts of DNA from the closely related species Botrytis cinerea.The PCR assays were tested using untreated ascospores, disrupted ascospores and DNA purified from ascospores. Similar ranges and sensitivities of detection (1-104 spores per PCR) were observed when using either untreated spores or purified DNA. The sensitivity of the PCR assay appeared to be inhibited when disrupted spore preparations were used: S. sclerotiorum was only detected at the highest spore concentration tested (104 spores in PCR). However, when mixtures of suspensions of S. sclerotiorum and B. cinerea were used, the PCR assay detected about 10 spores in purified DNA samples, but did not even detect 104

spores when the other two treatments were used. DNA was also purified from pieces of Burkard spore-trap tape (Burkard Manufacturing Co. Ltd., Rickmansworth, UK) containing known numbers of ascospores. The sensitivity of the PCR assay was the same whether the DNA was purified from the tape or from spore suspensions. A spore-trap was operated in an oilseed rape field for five weeks. Microscopic examination of the tapes revealed numerous spore types including small numbers of S. sclerotiorum-like ascospores on some of the tapes. DNA was purified from the tapes. For all samples, multiple bands were obtained in PCR assays using consensus fungal primers. A number of samples collected towards the end of May and beginning of June gave a single PCR product of expected size when used in the Sclerotinia-specific PCR assay.The SSFWD/SSREV PCR assay was also used to detect ascospores on oilseed rape petals. The results obtained suggested that the sensitivity of the assay was between about 50 and 200 ascospores per petal.The experiments reported here demonstrate that it is feasible to detect ascospores of S. sclerotiorum in air samples and on oilseed rape petals, by purifying DNA and using specific PCR assays to detect it. The primers should also detect other Sclerotinia species, but do not detect B. cinerea, a closely related fungus, common in the UK. In field crops cross reactivity with other Sclerotinia species may not be a problem as only the crop pathogen is likely to be present. The PCR-based assays, therefore have the potential to be incorporated into risk assessment systems for Sclerotinia diseases by enabling airborne inoculum to be relatively easily monitored. Also, in the case of S. sclerotiorum on oilseed rape, they could be used for assessments of percentage petal infestation at early bloom. However, work is still needed to further assess the sensitivity under field conditions, to determine detection thresholds for epidemic development, and to assess the risk of cross contamination by non-target Sclerotinia species in the field.

AcknowledgementsThis work was supported by a grant from the European Union (No. ERBIC18CT970173).

ReferencesWilliams, R.H., Ward, E. and McCartney, H.A. (2001) Methods for integrated air sampling

and DNA analysis for the detection of airborne fungal spores. Applied and Environmental Microbiology, 67, (6), 2453-2459.

S10.3

Relationships among environmental, pathogen and crop variables and their influence on sclerotinia rot of carrot [Sclerotinia sclerotiorum (Lib.) de Bary].

C. Koraa, C., M.R. McDonaldb and G.J. Bolanda

aDepartment of Environmental Biology and bDepartment of Plant Agriculture, University of Guelph, Guelph, ON, N1G 2W1, CANADA

AbstractSelected environmental, pathogen and crop variables were investigated in experimental and commercial carrot crops in southwestern Ontario during 1999 and 2000, and evaluated for their influence on sclerotinia rot of carrot caused by Sclerotinia sclerotiorum. Preliminary correlation analyses indicated a significant positive relationship between ascospore concentration and soil moisture, and a significant negative relationship between foliar disease and soil temperature.

Introduction Sclerotinia rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is among the most important storage diseases of carrot (Daucus carota L.) worldwide (Lewis & Garrod, 1983). In Canada this disease is regarded as a major limiting factor to long-term storage of carrot because substantial yield losses have been documented (Finlayson et al., 1989, Pritchard et al., 1992). Geary (1978) demonstrated that sclerotinia rot infection occurred in stored carrot as a direct consequence of infection in the field and that air-borne ascospores were the primary inoculum responsible for infection in growing plants. Finlayson (1989) indicated that infection of carrot by S. sclerotiorum occurred more readily when mycelial inoculum was placed near foliage in contact with soil, and that leaf wetness must be maintained for 11 days for foliar-applied ascospores to induce disease on foliage and stored roots. The purpose of the present study was to identify temporal and quantitative relationships among selected environmental, pathogen, and crop variables that influenced the development of the pathogen and disease in field conditions. Identification of seasonal patterns in these variables as well as anticipation of inoculum onset and the start of epidemics in the field can be useful tools for disease prediction.

Materials and MethodsEpidemiological studies were conducted during 1999 and 2000 in experimental (Site 1) and commercial (Site 2) carrot crops grown in organic soil (pH 6.4, organic matter 60%) naturally infested with S. sclerotiorum, in Bradford Holland Marsh, Ontario. Environmental variables included air temperature and relative humidity, soil temperature and moisture, leaf wetness duration and rainfall, and were continuously monitored using appropriate sensors attached to a CR 21X data logger (Campbell Scientific, Inc. Logan, UT, USA). Occurrence of apothecia within the crop was visually assessed each week throughout the growing season. Presence of air-borne ascospores was monitored weekly using sclerotinia semi-selective medium (SSM) (Steadman, 1994) plates as spore traps. The average count of colony forming units per plate

S10.3

was considered as an estimate of ascospore concentration within the crop. Foliar disease was estimated each week as the percentage of leaf explants that tested positive for S. sclerotiorum. Randomly selected leaflet and petiole sub-samples were surface-sterilized, plated on SSM and incubated for 7-14 days at 20-21oC. The presence of S. sclerotiorum was identified by the change of the medium color from blue to yellow and was confirmed by sclerotia formation. Selected crop growth stages, including canopy closure and presence of senescing leaves in contact with the soil, were also monitored.

Results and discussionsEtiological observations during three epidemics revealed that apothecia within the carrot crop appeared on 15 Sep 1999 (Site 1), on 12 Aug 1999 (Site 2) and 4 Aug 2000 (Site 1), always after canopy closure. During the two weeks preceding emergence of apothecia, mean soil temperature was always 19°C and mean soil moisture ranged between 35.6 to 36.7 % (v/v). Viable ascospores were first detected on 16 Aug 1999 (Site 1), 9 Aug (Site 2) and 18 Jul 2000 (Site 1). Ascospores were always observed in low concentration prior to emergence of apothecia, suggesting the existence of external sources of air-borne inoculum. No disease was observed in Site 1 in 1999 while in Site 2, visible symptoms were first observed on 18 Sep and disease reached 21.9% within one week. In 2000 the disease in Site 1, started on 18 Aug and reached 22.6 % within 2 weeks. Preliminary correlation analyses indicated a significant positive relationship between ascospore concentration and mean soil moisture during the preceding 2-week period (r = 0.48, P = 0.0022) and significant negative relationship between foliar disease and mean soil temperature during the preceding 2-week period (r = -0.55, P = 0.006). Foliar disease usually developed 4 to 6 weeks after ascospore occurrence within the crop and after the presence of senescing leaves on the ground. Senescing foliar tissues on the ground were readily colonized and accounted for >85% of the disease within the crop. Summarizing temporal observations, we may conclude that canopy closure, presence of ascospores and presence of senescing leaves on the ground may be important factors in disease initiation and development in the field. Quantitative relationships between the selected variables and their application to disease prediction need further investigation.

AcknowledgementsFinancial assistance from Natural Sciences and Engineering Research Council of Canada (NSERC), Bradford & District Vegetable Growers Association (B&DVGA) and Ontario Fruit and Vegetable Growers Association (OFVGA) is gratefully appreciated.

ReferencesFinlayson JE, Rimmer SR, Pritchard MK, 1989. Infection of carrots by Sclerotinia

sclerotiorum. Canadian Journal of Plant Pathology, 11(3), 242-246.Geary JR, 1978. Host-Parasite interactions between the cultivated carrot (Daucus carota L.)

and Sclerotinia sclerotiorum (Lib.) De Bary. Ph.D.Thesis, University of East Anglia UK, 230pp.

Lewis BG, Garrod B, 1983. Carrots. In: Post-harvest pathology of fruits and vegetables, Pp. 103-124. C. Dennis (Ed). Academic Press, New York.

Steadman JR, Marcinkowska J, Rutlege S, 1994. A semi-selective medium for isolation of Sclerotinia sclerotiorum. Canadian Journal of Plant Pathology, 16, 68-70.

S10.4

Risk analysis of Sclerotinia sclerotiorum as a mycoherbicide for pasture weed control in New Zealand

G. W. Bourdôt a, G. A. Hurrell a and M.D. De Jong b

a AgResearch Limited, PO Box 60, Lincoln 8152, New Zealand and b Dept. Biological Farming Systems, Wageningen University, Marijkeweg 22, 6709 PG Wageningen, The Netherlands

AbstractCrop disease risk after using Sclerotinia sclerotiorum for weed control in pasture is defined as the ratio of added to natural inoculum. Taking 1.0 to be a risk-averse value for this ratio, perimeter safety zones 0 and 50 m wide are predicted for dairy and sheep-grazed pastures respectively using a Gaussian plume model.

IntroductionThe disease risk to crops due to the use of Sclerotinia sclerotiorum (Lib.) de Bary as a mycoherbicide for weed control in pastures may be defined as the ratio of “added” to “natural” inoculum. Evaluation of this ratio in time and space enables estimation respectively of a “withholding period” for planting a susceptible crop at a biocontrol site and a “safety zone” around a biocontrol site (de Jong et al., 1999). An exponential decay model fitted to 3- and 4-year sets of survival data for sclerotia in pastures in Canterbury, New Zealand, predicted a 4-year withholding period (Bourdôt et al., 2000). Here we estimate the width of safety zones at three levels of risk averseness for pasture treated with S. sclerotiorum.

Materials and MethodsA Gaussian plume model, STACKS (van Ham et al., 1998), was used to estimate the atmospheric concentration, C, of ascospores at distances, x (m) downwind of a hypothetical 100 m x 100 m biocontrol pasture source (added inoculum) and within a hypothetical market garden area of 49 adjacent 100 m x 100 m sources (natural inoculum) by

c*e*e+e*u2

Q P=C ls2y-

2)H+-(z

2)H--(z

zyH)z,y,(x, 2

y

2

2z

2

2z

2

where y is horizontal distance (m) from the plume axis, z is height (m) above ground, H is source height (m), P is the inversion layer penetration fraction, Q is the emission rate of the source (spores s-1), û is mean wind speed (m s-1), Cls is a reflection term and σy and σz are respectively horizontal (cross-wind) and vertical dispersion terms and are functions of atmospheric stability and x. A plume was calculated for every hour from 1 Sept 1996 - 30 Nov 1996, the time of year when sporulation occurs in pasture (Bourdôt et al., 2001), using 1996 Canterbury weather records to estimate P, û, sy and σz. Using all plumes (2,184 for pasture and 107,016 for market garden), contour plots of 91-day average spore concentrations within and beyond the pasture and within the market garden area were calculated. The contours (distances) beyond the biocontrol site equal to, or 50% and 10% of, the median concentration for the market garden area, defined safety zones of increasing risk averseness. The source term was calculated as

,EaRQ spor ´´where Rspor is the release rate of ascospores from apothecia at the source (spores m -2 ground sec-1), a is the area of the source (m2), and E is the proportion of the released spores escaping

S10.4

the pasture or market garden crop canopy. Rspor was calculated as,fASRspor ´´

where S is density of sclerotia (# m-2) in the soil in the autumn, A is the size of the sporulating apothecial disc surface (mm2 sclerotium-1) and f is the flux of ascospores from the apothecia (spores mm-2 disc surface sec-1). S was set to 125 for the biocontrol and 8.8 for the market garden sources (Bourdôt et al., 2000) and A was varied with time as in Fig. 5d in Bourdôt et al. (2001). f followed a diurnal pattern differing between frosty and frostless days according to the data in Fig. 9 in Bourdôt et al. (2001). The escape proportion, E, was a mathematically derived function of mean wind speed and pasture leaf area index, LAI (leaf area/ground area) (de Jong et al., 2001). LAI was set to 1.0 in the market garden area and varied with time in the biocontrol pasture simulating 25-day grazing rotations in sheep (LAI 0.2-3.5) and dairy (LAI 3.2-6.5) pastures respectively.

Results and DiscussionThe spore concentration contour around the sheep-grazed biocontrol pasture representing the 1:1 ratio of added to natural spores occurred 50 m N and S and 25 m E and W. The contour for the 1:2 ratio occurred 150 m N and S and 75 E and W, and for the 1:10 ratio at 600 m N, 450 m S, 200 m E and 150 m W. Accepting the 1:1 ratio, 50 m in any direction would suffice as a safety distance between a sheep pasture treated with a S. sclerotiorum mycoherbicide and an area of market garden crops; 600 m would be exceptionally risk averse. For pasture grazed by dairy cattle, the spore contour lines representing the 1:1 and 1:2 ratios occurred within the biocontrol pasture and the contour representing the 1:10 ratio occurred 200 m N and S and 75 m E and W. For dairy pasture, because of the greater spore-trapping effect of its denser vegetation, no safety zone is required if a 1:1 or 1:2 ratio is accepted, while 200 m would be an exceptionally risk averse distance.

AcknowledgementsThe authors wish to thank the Foundation for Research Science and Technology, New Zealand, for providing funds and Dave Saville and David Baird, AgResearch, NZ, for helpful discussion.

ReferencesBourdôt GW, Hurrell GA, Saville DJ, de Jong MD, 2001. Risk analysis of Sclerotinia

sclerotiorum for biological control of Cirsium arvense in pasture: ascospore dispersal. Biocontrol Science and Technology, 11, 121-142.

Bourdôt GW, Saville DJ, Hurrell GA, Harvey IC, de Jong MD, 2000. Risk analysis of Sclerotinia sclerotiorum for biological control of Cirsium arvense in pasture: sclerotium survival. Biocontrol Science and Technology, 10, 411-425.

de Jong MD, Aylor DE, Bourdôt GW, 1999. A methodology for risk analysis of plurivorous fungi in biological weed control: Sclerotinia sclerotiorum as a model. BioControl, 43, 397-419.

de Jong MD, Bourdôt GW, Powell J, Goudriaan J, 2001. A model of the escape of Sclerotinia sclerotiorum ascospores from pasture. Ecological Modelling, Submitted.

van Ham J, van den Bosch K, Erbrink H, van Hoof H, van Jaarsveld H, Pulles T, Verver G. 1998. Het Nieuwe Nationaal Model. In: Lucht, pp. 25-29.







Posters

1. BIOLOGY, TAXONOMY AND MOLECULAR BIOLOGY

P1.1

Biochemical changes in abnormal sclerotia of Sclerotinia sclerotiorum

Hung-Chang Huanga and Jupiter M.Yeungb

aAgriculture and Agri-Food Canada, Research Centre, P.O. Box 3000, Lethbridge, Alberta, Canada T1J 4B1; and bNational Food Processors Association, 1350 I Street NW, Washington, DC 20005, USA

AbstractBiochemical analysis of normal and abnormal sclerotia of Sclerotinia sclerotiorum revealed a significant (P< 0.05) reduction in tryptophan (Trp) and serotonin (5-HT) but a significant (P< 0.05) increase in 5-hydroxyindole acetic acid (5-HIAA) in abnormal sclerotia. These results suggest that a defective serotoninergic pathway may be involved in the formation of abnormal sclerotia.

IntroductionAbnormal sclerotia of S. sclerotiorum were found in samples collected from diseased sunflower heads in Manitoba and Alberta (Huang,1982; Huang & Kozub,1994). They are different from normal sclerotia by the appearance of wrinkled surface and discoloured medulla (Huang, 1982). Survival of abnormal sclerotia was poor (Huang & Kozub, 1994) due to severe leakage of nutrients and destruction of medullary tissues (Huang, 1983). Formation of abnormal sclerotia is not an inherited character as their daughter sclerotia are normal (Huang, 1982).Little is known about the biochemical aspects of formation of abnormal sclerotia. The objective of this study was to investigate the possible biochemical pathway involved in the formation of abnormal sclerotia of S. sclerotiorum.

Materials and MethodsSclerotia of S. sclerotiorum used in this study were collected from diseased sunflower heads in 1977 and 1979 in Manitoba (Huang,1982) and in 1985 and 1986 in Alberta (Huang & Kozub,1994). Samples of normal and abnormal sclerotia for the amino acid analysis were prepared by the method of Yeung et al. (1986), while samples for the bioactive amines were analysed according to the method of Yeung & Friedman (1991). Amino acids were determined by GC analyses and the biogenic amines, 5-HT (5-hydroxytryptamine), 5-HIAA (5-hydroxyindole-3-acetic acid) and 5-HTP (5-hydroxy-L-tryptophan) were determined by HPLC analyses.To study formation of abnormal sclerotia in cultures, daughter sclerotia of S. sclerotiorum were harvested from 3-week-old cultures grown on PDA (potato dextrose agar) or PDA amended with 100 or 1000 ppm of 5-HIAA, 5-HT, pargyline (monoamine oxidase inhibitor, MAOI), 5-HTP, or L-tryptophan. They were cut open and examined for discoloration of medullary tissues under a stereomicroscope.

Results and DiscussionTwenty one free amino acids were detected in normal and abnormal sclerotia of S. sclerotiorum collected from diseased sunflower heads. With the exception of tryptophan (Trp), there were no significant differences (P> 0.05) for the amount of free amino acids

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between normal and abnormal sclerotia. The amount of Trp in the abnormal sclerotia was only 62% of that in the normal ones.Of the sclerotia produced in cultures grown on PDA or PDA amended with 100 or 1000 ppm of 5-HTP, L-Try, MAOI, 5-HT, or 5-HIAA, only sclerotia from 5-HIAA cultures had high frequency (>81.0%) of abnormal sclerotia showing brown discoloration of medullary tissues.Biochemical analysis of field samples showed higher concentrations of 5-HT, and lower to undetectable levels of 5-HIAA in normal sclerotia, while abnormal sclerotia had substantially less 5-HT and much higher concentrations of 5-HIAA (Table 1). The over conversion of neurotransmitter 5-HT to its metabolite 5-HIAA is only evident in abnormal sclerotia. The depletion of 5-HT and Trp and the increase in 5-HIAA in abnormal sclerotia suggests that the serotoninergic pathway is involved in the formation of abnormal sclerotia of S. sclerotiorum.

Table 1: Amount of 5-HT and 5-HIAA in sclerotia of S. sclerotiorum collected from diseased heads of sunflower in Manitoba in 1977 and 1979 and Alberta in 1985 and 1986.

5-HT2 5-HIAA 2 Ratio (5-HT/5-HIAA)2

Sclerotia 1 Normal Abnormal Normal Abnormal Normal Abnormal1977 80.3 59.1 27.1 31.6 nd 60.3 18.3 - -3 0.5 0.61979 43.8 50.9 nd 4 nd 8.3 9.6 - - - -1985 83.3 24.9 nd 22.5 8.7 69.2 16.7 3.7 1.3 - -1986 89.0 28.3 nd 43.0 5.5 98.3 43.7 2.1 0.7 - -

1 Sclerotia were stored in paper bags at room temperature (20 2C) for the 1977 and 1979 samples and at –4C for the 1985 and 1986 samples.2 All values are mean in ng/g S.D. (n=4).3 Undefined values are signified by “-“4 nd = not detected

AcknowledgmentsWe thank Dr. Glen Baker of the Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alberta, Canada for allowing us to use the facilities for this research. We also thank L. M. Phillippee and K. Zanewich for technical assistance.

ReferencesHuang HC, 1982. Morphologically abnormal sclerotia of Sclerotinia sclerotiorum. Canadian

Journal of Microbiology 28, 87-91.Huang HC, 1983. Histology, amino acid leakage, and chemical composition of normal and

abnormal sclerotia of Sclerotinia sclerotiorum. Can J.Bot. 61, 1443-1447.Huang HC, Kozub GC. 1994. Longevity of normal and abnormal sclerotia of Sclerotinia

sclerotiorum. Plant Disease 78, 1164-1166.Yeung JM, Friedman E. 1991. Effects of aging and diet restriction on monoamines and amino

acids in cerebral cortex of Fischer-344 rats. Growth Dev & Aging 55, 275-283.Yeung JM, Baker GB, Coutts RT, 1986. A simple automated gas chromatographic analysis of

amino acids in brain tissue and body fluids. Journal of Chromatography and Biomedical Applications 378, 293-304.

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Breeding, genetics, and mapping of QTL for architectural avoidance and physiological resistance to white mold in common bean.

Dermot P. Coyne1, Soon O. Park1, James R. Steadman1, and Paul W. Skroch2

1University of Nebraska, Lincoln, NE USA 68583; 28274 Andy Road, Waterloo, IL USA 62298

AbstractWhite mold avoidance due to canopy porosity (CP), partial physiological resistance (PPR), and partial field resistance (PFR) are inherited quantitatively and were mapped using RAPD markers in recombinant inbred lines of the cross ‘PC-50’ (R) x Xan-159 (S). QTL for PPR and PFR were detected on linkage groups 4, 7, and 8, and the latter with CP, of the integrated map.

IntroductionWhite mold (WM), incited by Sclerotinia sclerotiorum (Ss), is a major disease of common bean (Phaseolus vulgaris L.) (Steadman, 1979). A nondense canopy and upright and open bean plant architecture provide less favorable conditions for WM (Coyne et al., 1976). Low heritability of resistance to WM was reported by Coyne et al. (1976), whereas Miklas & Grafton (1992) reported high heritability. A recurrent selection method is recommended for improvement of resistance to this disease (Lyons et al., 1987). Molecular markers associated with resistance to WM offer prospects for marker assisted selection. Our purpose was to identify RAPD molecular markers associated with QTL affecting partial physiological resistance (PPR), partial field resistance (PFR), porosity over the furrow (POF) and plant height (PH) to Nebraska Ss isolates in a molecular marker-based linkage map using a recombinant inbred line (RIL) bean population.

Material and MethodsSixty-five RILs of the cross PC-50 (PPR) x Xan-159 (S) and parents were planted in a RCBD with four replications in the greenhouse in Lincoln, NE (1997-98) for inoculation with Ss isolates 152 (expts. 1 and 2) and 279 (expts. 3 and 4). The straw inoculation test and rating scale developed by Petzoldt and Dickson (1996) was utilized. The progress of infection on each plant was recorded 10 days after inoculation. Sixty-three of the RILs and parents were planted in a RCBD with four replications in the field in Scottsbluff, NE (1999). Severity of infection, plant architecture, and plant height were recorded for each plant. The RAPD marker-based linkage map was developed by Jung et al. (1997) using 70 RILs of the same cross. Subsequently, to facilitate integration of this map with other RAPD and RFLP maps in bean, the segregation data were reanalyzed by Skroch (1998) with some markers being added and some dropped from the different linkage groups (LGs). LG names reported by Jung et al. (1997) were changed to reflect their correspondence with the integrated common bean linkage map. The modified linkage map spanned 404 cM compared to 426 cM in the original map reported by Jung et al. (1997). The analysis of QTL affecting PPR to isolates 152 and 279 was conducted on the basis of the means from expts. 1-2, and 3-4, respectively. Composite interval mapping (CIM) analysis was applied to each trait mean and marker data to more precisely identify the locations of QTL (Zeng, 1994). CIM analysis was performed using QTL Cartographer software version 1.13g. Cofactors were chosen based on the results of a forward

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stepwise multiple regression analysis (MRA) using the QTL Cartographer program “SRmapqtl” (Model 6).

Results and DiscussionContinuous distributions for the disease reactions of the RILs to both S s isolates in the straw test (PPR) and in the field (PFR) suggested quantitative inheritance of these traits. Low h2

(0.24, 0.23) for disease reaction as detected in the greenhouse experiments. Significant correlations were found between the reactions to the two Ss isolates. CIM results indicated strong evidence for QTL on LGs 4, 7, 8 affecting PPR to both Ss isolates, on LGs 7, 8 for PFR and PH, and on LG for POF on LG 8. Overall six of the seven regions were associated with PPR to one or both isolates. These results will be published in detail in Crop Science in August, 2001. The data strongly suggest that these RAPD markers could be used for simultaneous selection for both WM traits. Miklas, et al. (2001) recently published strong evidence for a QTL for PPR and PFR on LG 7. QTL for common blight (CB) susceptibility (Jung et al., 1997), large seed size (Park et al., 2000), and PPR and PFR were located on LGs 7 and 8. Where QTL for CB and WM are linked they are generally in repulsion because XAN-159 and PC50 contributes resistance to CB and WM, respectively. QTL for larger seed weight, PPR, and leaf pubescence (Pu-a) were located on LG3. Thus breeders face difficulties in combining resistance to CB, WM, and large seed size due to unfavorable linkages.

AcknowledgementsWe thank E Arnaud Santana, G Jung, B Higgins, L Sutton, C Carlson, and J Reiser for assistance.

ReferencesCoyne DP, Steadman JR, Magnuson S, 1976. Breeding for white mold resistance avoidance

due to ideotype in beans. Ann. Rpt. Bean Improv. Coop., 19, 21-22.Jung G, Skroch PW, Coyne DP, Nienhuis J, Arnaud-Santana E, Ariyarathne HM, Kaeppler

SM, Bassett MJ, 1997. Molecular-marker-based genetic analysis of tepary bean-derived common bacterial blight resistance in different developmental stages of common bean. J. Amer. Soc. Hort. Sci. 122, 329-337.

Lyons ME, Dickson MH, Hunter JE, 1987. Reccurent selection for resistance to white mold in Phaseolus species. J. Amer. Soc. Hort. Soc. 112, 112-152.

Miklas PN, Grafton KF, 1992. Inheritance of partial resistance to white mold in inbred populations of dry bean. Crop Sci. 32, 943-948.

Miklas PN, Johnson WC, Delorme R, Gepts P, 2001. Qtl conditioning physiological resistance and avoidance to white mold in dry bean. Crop. Sci. 41, 309-315.

Park SO, Coyne DP, Jung G, Skroch PW, Arnaud-Santana E, Steadman JR, Ariyarathne HM, Nienhuis J, 2000. Mapping of QTL for seed size and shape traits in common bean. J. Amer.

Soc. Hort. Sci. 125, 466-475.Petzoldt R, Dickson MH, 1996. Straw test for resistance to white mold in beans. Ann. Rpt.

Bean Improv. Coop. 39, 142-143.Skroch PW, 1998. Random amplified polymorphic DNA based germplasm and genetic

mapping studies in common bean. PhD Diss., Univ. of Wisconsin, Madison.Steadman JR, 1979. Control of plant diseases by Sclerotinia species. Phytopathology 69,

904-907.Zeng ZB, 1994. Precision mapping of quantitative trait loci. Genetics 136, 1457-1465.

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Intraspecific Variation within North American Sclerotinia trifoliorum isolates characterized by nuclear Small Subunit rDNA introns.

K. S. Powers, J. R. Steadman, B. S. Higgins and T.O. Powers

Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722

AbstractIntraspecific variation within a population of 50 USA isolates of Sclerotina trifoliorum was examined by PCR-amplification and nucleotide sequencing of the nuclear small subunit ribosomal DNA (nSSU rDNA). Five distinct length polymorphisms due to DNA insertions (group I introns) were observed in the nSSU rDNA of S. trifoliorum. No introns were detected in the nSSU rDNA of S. minor or S. sclerotiorum.

IntroductionPopulation structure reflects evolutionary processes and phylogenetic history of an organism. On a practical level, the population structure of a pathogen has broad implications for disease management strategies such as a heterogeneous response to resistance genes. Wide cultural, morphological and pathogenetic intraspecific variability has been observed in S. trifoliorum, S. sclerotiorum and S. minor and genetic intraspecific variation in S. sclerotiorum has been well documented (Carbone et al. 1995, Holst-Jensen et al. 1999). However, relatively little is known of the genetic intraspecific variability of S. trifoliorum and S. minor. The aim of this study was to characterize the DNA insertions detected in the nSSU rDNA in S. sclerotiorum, S. minor and S. trifoliorum.

Materials and MethodsIsolates were obtained from single ascospores or hyphal tips when the perfect stage was not available or could not be induced. All isolates were identified using Kohn’s delimitation of Sclerotinia species (1979). Isolates were selected from different geographic regions or were tested by a system of somatic pairing (all S. trifoliorum isolates were paired against each other) to guarantee that each isolate represented a distinct vegetative clone. Amplification of DNA was done directly from a culture by scraping a pipette tip about 1 cm across the advancing mycelial margin of the culture (Harrington & Wingfield 1995). The nSSU rDNA was amplified by the polymerase chain reaction (PCR) using the universal primers NS1 and NS8 (White et al. 1990). After initial denaturation at 94 C for 165s, PCR used 40 cycles of annealing at 53 C for 45s, extension at 72 C for 45s and denaturation at 92 C for 60s with a final extension step of 72 C for 5 m. The amplified product was separated on a 1% agarose gel in a 0.5X TBE solution. PCR products were sequenced by the Iowa State Sequencing Lab. We used SeqWeb version 1.2 to access Wisconsin Package version 10.1 (Genetics Computer Group, Madison WI) for similarity searches, alignment, secondary and tertiary structure models and comparative sequence analysis programs. GenBank and EMBL databases searches were conducted using Basic Local Alignment Search Tool (BLAST). Insertion sites within nSSU rDNA are numbered according to the position of the 5' flanking nucleotide in Saccharomyces cerevisiae nSSU rDNA (Gargas et al. 1995).

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Results and DiscussionPCR amplification of the nSSU-rDNA of 50 isolates of Sclerotinia trifoliorum revealed 5 distinct length polymorphisms due to group I introns. Insertion sites and sizes of the introns are shown in Fig. 1. We examined an 84-isolate sample of Sclerotinia sclerotiorum, collected from 5 continents and 21 different hosts, and detected no introns in the nSSU rDNA nor were any introns detected in a 7-isolate Sclerotinia minor sample. The apparent lack of group I introns in the nuclear SSU rDNA of S. sclerotiorum and S. minor contrast with the genetic variation observed in S. trifoliorum. This may be associated with the differing modes of reproduction of the three species. The presence or absence of group I introns in the nuclear ribosomal repeat has been previously suggested as a potential species diagnostic for Sclerotinia (Holst-Jensen et al. 1999). Their conclusion, however, was based on a limited number of isolates. Our examination of 141 isolates strongly supports the potential use of group I introns as diagnostic markers.

ReferencesCarbone I, Anderson JB, Kohn LM, 1995. A group I intron in the mitochondrial small

subunit ribosomal RNA gene of Sclerotinia sclerotiorum , Curr. Genet. 27, 166-176.Gargas A, DePriest PT, Grube M, Tehler A. 1995. Multiple origins of lichen symbioses in

fungi suggested by SSU rDNA phylogeny. Science 268(5216):1492-1496.Harrington TC, Wingfield BD, 1995. A PCR-based identification method for species of

Armillaria. Mycologia 87, 280-288.Holst-Jensen A, Vaage M, Schumacher T, and Johansen S, 1999. Structural characteristics

and possible horizontal transfer of Group I introns between closely related plant pathogenic fungi. Mol.. Biol. Evol. 16, 114-126.

Kohn LM, 1979. A monographic revision of the genus Sclerotinia. Mycotaxon 9, 365-444.Perotto S, Nepote-Fus P, Saletta L. Bandi C, Young JPW, 2000. A diverse population of

introns in the nuclear ribosomal genes of ericoid mycorrhizal fungi includes elements with sequence similarity to endonuclease-coding genes. Mol. Biol. Evol. 17, 44-59.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genomes for phylogenetics. In: PCR protocols: A guide to methods and Applications (Innis MA, et al.,) pp. 315-322. Academic Press, San Diego, CA.

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Isolate variability amongst Sclerotinia sclerotiorum from New Zealand kiwifruit orchards

S. M. Hoytea, I. Carboneb and L. M. Kohnb a The Horticulture and Food Research Institute of New Zealand, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand; andb Department of Botany, University of Toronto, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L 1C6.

AbstractBased on multilocus DNA sequences, Sclerotinia sclerotiorum genotypes from New Zealand kiwifruit have evolved, from ancestors common to North America and Europe, to have unique DNA fingerprints and MCGs. Population structure is comparable to that from other agricultural crops. Biological control studies and immunological assays should factor in genetic diversity.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary can cause significant crop loss (up to 17%) in New Zealand kiwifruit (Actinidia deliciosa (A. Chev.)) orchards. Disease control is dependant on the dicarboximide fungicide, iprodione (Rovral™). However, two biological control methods are being investigated which disrupt the colonisation of floral tissues using saprophytic fungi (Elmer et al., 1999) and which utilise the mycoparasite Coniothyrium minitans Campbell to degrade sclerotia (A. Stewart, Lincoln University, pers. comm.). It is possible that the genetic variability of this pathogen may influence the success of such control measures. Immunological techniques, which are being developed for the prediction of disease risk, may also be affected by pathogen variability. The objective of this study was to investigate genetic variability by DNA fingerprinting and mycelial compatibility grouping and to integrate with larger scale studies of genetic diversity in North America and Europe by means of DNA sequencing of more slowly evolving genes and an intergenic region (IGS of the nuclear rDNA repeat) (Carbone et al., 1999; Carbone & Kohn, 1999; Carbone & Kohn, 2001).

Materials and MethodsThirty-eight strains of S. sclerotiorum (sample A) were cultured from diseased tissues taken from 15 kiwifruit orchards in four geographical regions (four Waikato, three Katikati, six Tauranga and two Te Puke orchards, maximum three strains per orchard) during November and December 1997. MCGs and DNA fingerprints were determined as described in Kohn et al. (1991). DNA sequence data was analysed using phylogenetic and coalescence-based methods as described by Carbone & Kohn (2001) to infer gene genealogies. In addition, a further 40 strains of S. sclerotiorum (sample B) were cultured from individual colonies growing on selective medium exposed for 24 h in a single block of kiwifruit vines (50 m x 70 m) in a Waikato orchard from 6–10 December 1997. Each of these strains were paired with each other on MPM and compatibility groups determined as described above.

Results and DiscussionAll self-self pairings were compatible. All incompatible pairings of strains produced an interaction zone with either thin or tufted mycelium and a pink line in the agar. There were

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29 MCGs in sample A and 22 MCGs in sample B. Within these samples there were 25 and 14 MCGs, respectively, that consisted of a single strain. Strains from sample A belonged to three distinct genetic populations based on the DNA sequences from the IGS region. These populations share a common ancestry with a high proportion of S. sclerotiorum strains sampled from agricultural crops in North America and Norway, including one population that is probably thousands of years old (Carbone & Kohn, 2001). Strains from each of the three populations were present in all of the four kiwifruit growing regions sampled (each separated by 30–80 km), indicating wide dispersal. Given the limited sample size we still found two orchards which had one strain from each population.The evidence of recent evolution in New Zealand was that DNA fingerprints were distinct from those of North American and European strains and that four MCGs had variation in DNA fingerprints, indicating that transposition had occurred relatively recently in clonal lineages. Two MCGs were distributed over 20 km. This evidence of clonality is similar to that found within S. sclerotiorum populations in North American cropping systems, where many single strain genotypes were recovered and where some clones were found in high frequency within a field and across fields separated by as much as 2,000 kilometres (Kohli et al., 1992). This New Zealand study recovered fewer high frequency clones than is typical from North American samples, perhaps because of the sampling design or other factors. These results suggest that variability within S. sclerotiorum from North Island kiwifruit is high, similar to that reported for South Island populations from cauliflower and buttercup (Carpenter et al., 1999) and comparable to that in Canadian canola crops (Kohli et al., 1992). The significance of such genetic variation on disease development through affects on apothecial emergence, pathogen aggressiveness and disease carry-over should be investigated. Evaluation of chemical or biological control agents and immunological assays should incorporate genetic diversity in S. sclerotiorum in screening and evaluation, as efficacy may be affected by variation at population, clone or individual genotype level.

ReferencesCarbone I, Anderson JB, Kohn LM, 1999. Patterns of descent in clonal lineages and their

multilocus fingerprints are resolved with combined gene genealogies. Evolution 53, 11-21.Carbone I, Kohn LM, 1999. A method for designing primer sets. Mycologia 91, 553-556.Carbone I, Kohn LM, 2001. A microbial population-species interface: nested cladistic and

coalescent inference with multilocus data. Molecular Ecology 10, 947-964.Carpenter MA, Frampton C, Stewart A, 1999. Genetic variation in New Zealand populations

of the plant pathogen Sclerotinia sclerotiorum. New Zealand Journal of Crop and Horticultural Science 27, 13-21.

Elmer PAG, Hoyte SM, Reglinski T, 1999. Biological suppression of Sclerotinia sclerotiorum in kiwifruit. In: 12th Biennial Australasian Plant Pathology Society Conference, pp. 63, Canberra.

Kohli Y, Morall RAA, Anderson JB, Kohn LM, 1992. Local and trans-Canadian clonal distribution of Sclerotinia sclerotiorum of canola. Phytopathology 82, 875-880.

Kohn LM, Stasovski E, Carbone JR, Royer J, Anderson JB, 1991. Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum. Phytopathology 81, 480-485.

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Diagnostic assays for Monilinia brown rot species; members of the family Sclerotiniaceae

K.J.D. Hughes, C.R. Lane and J.N. Banks

Central Science Laboratory, DEFRA, Sand Hutton, York, YO41 1LZ, U.K.

AbstractThe brown rot species Monilinia fructicola, M. fructigena and M. laxa cause major economic losses to stone and pome fruit. The first two are quarantine organisms in Europe and North America respectively whilst M. laxa occurs worldwide. The application of cultural, serological and molecular protocols for their identification from cultures and fruit is discussed.

IntroductionMonilinia brown rot of Prunus, Malus and Pyrus species is caused by three morphologically similar members of the family Sclerotiniaceae, M. fructicola, M. fructigena and M. laxa. The first of these is an EC 1A1 quarantine listed organism, which is absent from Europe unlike the other two species which occur widely in Europe. In North America, only M. fructicola and M. laxa are present and M. fructigena is a quarantine pest (EPPO, 2000). Fruit traded between these continents may contain quiescent infections, which are notoriously difficult to detect. Species identification is also time consuming and needs considerable mycological experience. Recently however, quick, simple and conclusive methods have been developed which aid identification.

Materials and methodsCultural protocol: Representative Monilinia isolates (6 M. fructicola, 6 M. laxa and 6 M. fructigena) from the 21 described in Hughes et al, (2000) (isolates Jap1535, Aust 5 & 6 excluded) were grown on 4% potato dextrose agar (PDA) for 10 days at 22°C, 12h near UV light/12h dark. These were then identified in a ‘blind evaluation’ to species using a simple cultural protocol (Corazza, et al 1998).

Serological protocol: The 18 Monilinia isolates described above, 8 Sclerotinia sclerotiorum (SS1-8), 1 Botrytis cinerea isolate (cc1106; anamorph of Botryotinia fuckeliana) were grown as described above for 6 days. Isolates were also placed into cuts in healthy plums and incubated on the laboratory windowsill (20°C) along with a negative control plum inoculated with PDA. All samples were then tested in an indirect ELISA using a monoclonal antibody (MAb) raised to M. fructicola (cc 778) and described by Hughes et al, (1998). MAb reactivity was then assessed based on thresholds for cultures and fruit; set at twice the absorbance (405nm) for a water control (0.102) and from the negative control plum (0.097) respectively.

Molecular protocol: Twenty-one Monilinia isolates and control species (Fusarium, Colletotrichum, Lambertella and Alternaria) were tested by PCR with primer sets to identify either individual Monilinia species or to detect simultaneously either M. laxa and M. fructicola or M. laxa and M. fructigena (Hughes et al, 2000).

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Results and discussionCultural protocol: This simple, low cost, 10 day test requiring pure cultures and specific conditions was reasonably discriminatory; correctly identifying all M. fructigena isolates, but incorrectly identifying 2 out of 6 M. laxa as M. fructigena, and 2 out of 6 M. fructicola isolates as M. laxa.

ELISA protocol: All M. fructicola cultures gave positive absorbances (0.251-1.061) as did 5 out of 7 S. sclerotiorum, although these were generally much lower (0.207-0.264), while all M. fructigena (0.117-0.144), all M. laxa (0.082-0.186), and 2 out of the 8 S. sclerotiorum (0.194&0.201) and the B. cinerea (0.151) culture gave negative absorbances. These results on 6 day old cultures showed improved M. fructicola specificity compared to using 3 week old cultures (Hughes et al, 1998), some cross reactivity to S. sclerotiorum however was shown. All M. fructicola infected fruit gave very high absorbances (0.947-1.921) but this was offset by low positives by 5 of the 6 M. fructigena (0.202-0.310), 5 of the 6 M.laxa (0.213-0.272), all 8 S. sclerotiorum (0.256-0.478) and the B. cinerea (0.226) infected fruit.

PCR protocol: The Monilinia isolates were correctly identified using the PCR primers and there was no cross-reaction with any non-Monilinia species. Identical specificity was observed in preliminary tests using natural and artificially infected fruit.

In conclusion, PCR was the most reliable diagnostic assay allowing identification from cultures and infected fruit. Whilst the cultural protocol although simple and requiring basic mycological skills and experience, led to some misidentifications. However, development of a synoptic key is in progress which should reduce errors. The serological protocol was the least reliable, but with minor protocol development it is hoped that it can also be a useful tool for Monilinia identification.

AcknowledgementsThis study was funded by the Plant Health Division of the Ministry of Agriculture Fisheries and Food and by the EC under contract FAIR CT95-0725. Quarantine organisms were held under licence PHL 100 / 2959.

ReferencesCorazza L, Cook RTA, Lane CR, Fulton CE, Van Leeuwen GCM, Pereira AM, Nazare-

Pereira A, Melgarejo P, De Cal A, 1998. Identification of Monilinia (Brown Rot) species. http://www.bspp.org.uk/icpp98/3.3/72.html

EPPO, 2000. Plant Quarantine Retrieval System (PQR) Version 3.10.Hughes KJD, Banks JN, Rizvi RH, McNaughton J, Lane CR, Stevenson L, Cook RTA 1998.

Development of a simple ELISA for identification of Monilinia fructicola and Monilinia spp. www.bspp.org.uk/icpp98/abstracts/6/146.html

Hughes KJD, Fulton CE, McReynolds D, Lane CR, 2000. Development of new PCR primers for the identification of Monilinia species. OEPP/EPPO Bulletin 30, 507-511.

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Regulation of lytic enzyme production upon plant infection by Sclerotinia sclerotiorum

C. Bruel, P. Cotton, V. Girard, R. Létoublon, N. Poussereau, G. Billon-Grand, M-B. Martel, C. Rascle, and M. Fèvre.

Laboratoire de Biologie Fongique – ERS 2009. Université Claude Bernard. Bat. Lwolf RdC. 43 Bd du 11 nov 1918. 69622 Villeurbanne, France.

AbstractSclerotinia sclerotiorum, a necrotrophic fungus, produces many different lytic enzymes. This production responds to several input signals among which are plant contact, pH, nitrogen and carbon sources. In order to understand the mechanisms that underlie the set up of the plant degrading machinery, we used molecular and biochemical tools to study the regulation of polygalacturonase and protease production during plant infection.

IntroductionDuring interaction with its host, S. sclerotiorum secretes multiple lytic enzymes that facilitate penetration, colonization and maceration of the plant tissues, hence generating assimilable nutrients for the fungus growth. In vitro tudies were undertaken to determine the effects of environmental signals on the production of two classes of these enzymes, namely the polygalacturonases and the proteases.

Materials and MethodsLiquid cultures of S. sclerotiorum were as described in Riou et al. (1992) and Poussereau et al. (2001); RNA extraction and Northern Blot analyses were as described in Fraissinet-Tachet et al. (1995); Infection of sunflower cotyledons was as described in Reymond-Cotton et al. (1996); Protein extraction was as described in Poussereau et al. (2001); Isoelectrical focusing was as described in Riou et al. (1992).

Results and DiscussionSecretion of Polygalacturonases and Proteases during Sunflower Infection – In planta infection of sunflower cotyledons was performed and the production of proteases and polygalacturonases (PGs) was monitored over a period of 48 hours. Protein extracts from infected plant tissues were analyzed by zymography and by using polyclonal antibodies. Five proteases were detected that belong to three different groups, namely the serine proteases, the aspartyl proteases and the acid non-aspartyl proteases (unpublished data). Multiple endo- and exo-PGs were also detected that fall into the group of neutral or acidic PGs (Riou et al., 1992). Genes that code for two proteases and five PGs were cloned and derived probes were used to confirm the biochemical results. All fungal genes were expressed in the infected tissue with some being induced more than others following the contact with the plant.

Secretion of Polygalacturonases and Proteases in vitro – Parallel experiments to the ones described above were carried out in which S. sclerotiorum was grown in liquid cultures in the presence or in the absence of crude sunflower extracts. The production of proteases and PGs,

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as well as the expression of selected corresponding genes, were found to be nearly identical to the results observed in planta (Riou et al., 1992, Fraissinet-Tachet et al., 1995, Reymond-Cotton et al., 1996 and unpublished data). In the presence of sunflower extract, the same proteases and PGs observed in planta were detected in the culture medium and shown to accumulate over time. Also, the corresponding selected genes were characterized by an induced transcription in the presence of plant extract. As in vitro conditions can be manipulated rationally, detailed molecular studies of the regulation of the lytic enzyme genes became possible. The large amounts of material obtained in vitro also allowed the purification of several proteases and PGs to near homogeneity and their characterization (Martel et al., 1998 and unpublished data).

Global Regulation of Polygalacturonases and Proteases encoding Genes – The impact of glucose, nitrogen and pH on the production of PGs and proteases was studied at the level of gene expression by using two probes specific to one acid non-aspartyl protease and three neutral endo-PGs, respectively (Reymond-Cotton et al., 1996 and Poussereau et al., 2001). pH exhibited a strong and dominant influence on the expression of both selected genes ; the protease gene was only transcribed under conditions where the pH was inferior to five and the neutral endo-PG genes were not transcribed if the pH was inferior to 4 or superior to 6. Interestingly, the endo-PG genes were expressed in complete synthetic medium whereas the protease gene was not. Finally, the induction of the protease gene expression by sunflower extract could be modulated by glucose or nitrogen. The presence or absence of pH, glucose and/or nitrogen regulatory sequences in the promoter region of the selected genes is in accordance with the observed behaviours.

Conclusion – By using lytic enzymes, or their encoding genes, as reporters, we have defined in vitro conditions that mirror those encountered by S. sclerotiorum in planta. We have shown that glucose and nitrogen starvation, together with acidification of the medium, are important factors in the control of the expression of these genes during pathogenesis. The cues that suppress lytic enzyme production are beginning to be elucidated. Activation of this production is now being studied.

ReferencesFraissinet-Tachet L, Reymond-Cotton P, Fèvre M, 1995. Characterization of a multigene

family encoding an endopolygalacturonase in S. sclerotiorum. Current Genetics 29, 96-99.Reymond-Cotton P, Fraissinet-Tachet L, Fèvre M, 1996. Expression of the S. sclerotiorum

polygalacturonase pg1 gene : possible involvement of CREA in glucose catabolite repression. Current Genetics 30, 240-245.

Martel MB, Létoublon R, Fèvre M, 1998. Purification and characterization of two endopolygalacturonases secreted during the early stages of the saprophytic growth of S. sclerotiorum. FEMS Microbiology Letters 158, 133-138.

Poussereau N, Creton S, Billon-Grand G, Rascle C, Fèvre M, 2001. Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of S. sclerotiorum. Microbiology 147, 717-726.

Riou C, Fraissinet-Tachet L, Freyssinet G, Fèvre M, 1992. Secretion of pectic isoenzymes by S. sclerotiorum. FEMS Microbiology Letters 91, 231-238

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Sclerotinia stem infection in flax in western Canada

K. Y. Rashid

Agriculture and Agri-Food Canada, Cereal Research Centre, Morden Research Station, Morden, Manitoba, Canada R6M 1Y5

AbstractThis is the first report of Sclerotinia sclerotiorum infection in flax in Manitoba and Saskatchewan in western Canada. This fungus causes stem shredding, fiber disintegration, and stem breakage. Sclerotinia can be potentially destructive when flax is subjected to heavy lodging and is grown in fields heavily infested with Sclerotinia.

IntroductionFlax (Linum usitatissimum L) is a major field crop in western Canada, grown on 1-1.5 million ha annually. Sclerotinia sclerotiorum (Lib.) De Bary is a widespread pathogen affecting many crops including canola, field peas, beans, lentils, and sunflower. Sclerotinia infected flax has been observed only in irrigated flax crops in southern Alberta but has not been observed in any flax crops during annual disease surveys conducted in Manitoba and Saskatchewan prior to 1999.

Materials and MethodsA total of 61 flax crops in southern Manitoba and 41 in central and eastern Saskatchewan were surveyed in August-September, 1999. Crops surveyed were selected at random along pre-planned routes in the major areas of flax production. Two persons surveyed each crop by walking 100 m in opposite directions in the field following an "M" pattern. Sclerotinia infected plants were identified by symptoms, and the incidence and severity of the disease were recorded. Lodging of plants was rated on a scale of 1 to 5 (1 = no lodging, and 5 = heavy lodging)

Results and DiscussionSymptoms of the disease: The early signs of this disease were observed on the stems of heavily lodged flax at the late green-boll stage of the plant growth in mid-August. Symptoms were light discoloration of the stems, which turned gray and became white-bleached after two to three weeks of infection. Shredding of the stems, disintegration of the fiber, and stem-breakage were observed as the infections progressed. Cylindrical-shape and black-colored sclerotia of 1-2 mm in diameter and 5-30 mm in length were commonly observed inside the infected area of the stem.

Disease situation in 1999: Sclerotinia infected flax was observed in 12 fields out of the 60 surveyed in Manitoba, and in 4 fields out of the 40 surveyed in Saskatchewan in the summer of 1999 (Table 1). This disease has not been observed in any disease survey conducted prior to 1999 in either Manitoba or Saskatchewan. The incidence of this disease ranged from 1 to 30% infected plants, and severity ranged from 5 to 60% stem-area affected. Sclerotinia infected plants were only found in heavily lodged flax caused by strong wind and/or heavy rain. This is due perhaps to the direct contact between the lodged stems and the Sclerotinia-

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infested soil, and favored by high humidity for disease development inside the canopy of heavily lodged plants. No signs of Sclerotinia infections were observed in any normal standing flax next to the heavily lodged and infected flax in the same field or in adjacent fields. Some heavy lodged flax crops had no signs of Sclerotinia infections due perhaps to the absence of Sclerotinia inoculum in the soil. A few of the heavily infected flax crops were in fields with high soil moisture levels, which resulted in poor vigor and weak stems causing the heavy lodging.

These observations suggest that lodging brought the flax stems in direct contact with the soil where they picked up the infections where the Sclerotinia inoculum is present in the soil.

Table 1. Incidence and severity of Sclerotinia infections and lodging severity in 16 flax crops in Manitoba and Saskatchewan in 1999._____________________________________________________________________ No of Incidence Severity LodgingCrops* % Plants % Stem Infection (1-5) . 4 <1% 10% 39 5% 10-20% 42 10% 10-20% 41 30% 30-60% 5 . Four crops out of 40 in Saskatchewan and 12 out of 60 in Manitoba. Lodging scale 1 = no lodging, and 5 = complete lodging.

Disease situation in 2000Stem infections with Sclerotinia were observed only in four crops, 3% of the 122 crops surveyed in 2000. The Sclerotinia infected crops were only observed in heavily lodged flax, and the incidence and severity were much lower than in those observed in 1999.

Conclusions and remarksThese findings show that S. sclerotiorum can be a potential disease problem in flax when the crop is grown in fields with heavy inoculum pressure and is subjected to heavy lodging. The recommendations to minimize the impact of this disease are: use flax varieties with good lodging resistance; avoid excessive soil moisture conditions; and avoid fields with high Sclerotinia inoculum from previously infected crops.

Acknowledgments:The technical assistance of L.J. Wiebe and M. Penner is gratefully appreciated.

2. CHEMICAL CONTROL

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Management of Sclerotinia Blight, Sclerotinia minor, in Texas, USA Peanut fields.

T. A. Lee, Jr.

Department of Plant Pathology & Microbiology, Texas A&M University, Research & Extension Centre, Stephenville, TX 76401.

Sclerotinia Blight caused by the fungus Sclerotinia minor infects about 10,000 ha of peanut in Texas, USA each year. Damage varies from 5% to 90%. Long term rotations, 3-5 years, with a grass crop of some type gives adequate control in most cases. New fungicide chemistry from ISK Biosciences and BASF provide control in the 80% range.

N.B. Unfortunately the paper to go with this abstract was not available at the time of going to press. A copy of the complete paper may however be obtained from the author or a member of the Sclerotinia 2001 workshop organising committee.

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Plant densities and fungicide effects on the intensity of white mould (Sclerotinia sclerotiorum) of dry beans

R.F. Vieiraa, C.M.F. Pintoa and E.S.G. Mizubutib

aEPAMIG, C.P. 216, Viçosa, MG 36571-000, Brazil; and bViçosa Federal University, Departament of Plant Pathology, Viçosa, MG 36571-000, Brazil

AbstractKeeping constant the space between rows in 0.5 m, reduction of planting densities from 15 plants/m to 5 plants/m decreased white mould incidence and severity and increased yield, with or without fungicide applications. Two applications of fluazinam increased yield 118%.

IntroductionS. sclerotiorum is a ubiquitous and destructive pathogen, inciting disease on up to 408 plant species (Boland & Hall, 1994). Diseases induced by this pathogen, often referred to as white mould, cause yield losses in numerous cultivated crops, such as beans, sunflower, canola, and carrots. In sprinkler irrigated areas of dry beans cultivated during fall-winter in Minas Gerais State, Brazil, white mould is one the most destructive disease. Before infecting healthy plant tissues, ascospores of S. sclerotiorum require an exogenous source of energy. In beans, the most common energy source are senescing flower parts. After colonization of such parts, the pathogen invades adjoining living tissues and initiates disease. Several factors are known to influence the colonization of senescing flower petals by the fungus. Air temperature, relative humidity, water potential, and duration of plant surface wetness affect the germination of ascospores and growth of mycelium, and influence the ability of the fungus to colonize senescing petals. Therefore increasing distance between plants as well as between rows could reduce conditions favourable for disease. Nevertheless, the benefits of this cultural practice have not been quantified in Brazil. The objective of this study was to quantify the benefits of spacing plants at wider intervals within row with or without applications of fungicide.

Materials and methodsA trial was conducted at Viçosa Federal University, Minas Gerais State, Brazil, in a field naturally infested with sclerotia of S. sclerotiorum. The vine bean cultivar Pérola (type III) was sown spaced 0.5 m apart on 2 May 2000. The trial was conducted as a 3x2x2 factorial in the randomized complete-block design replicated six times: three plant densities (5.0, 7.5 and 15.0 plants per meter), two modes of sowing (plants equidistant and three plants together), and two fungicide treatments (with and without). In the modes of sowing with three plants together the set of plants was distributed in 20 to 20 cm, 40 to 40 cm, or 60 to 60 cm. Weeds were controlled manually with hoe and with a commercial mixture of the herbicides fomesafen (250 g a.i. ha-1) and fluazifop-p-butil (200 g a.i. ha-1). Insects were controlled, when needed, with monocrotophos (400 ml a.i./ha). The fungicide azoxystrobin (60 g a.i. ha-

1) was applied before flowering to prevent anthracnose (Colletotrichum lindemuthianum) and angular leaf spot (Phaeoisariopsis griseola). The fungicide fluazinam (0.5 l a.i. ha-1) was applied at 49 (early bloom) and 62 days after emergence for white mould control. Trial was sprinkler irrigated and the weather was favourable to disease development. An area of 1.2 m2

of each plot was harvested separately for disease evaluation. The plants were rated for severity of

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white mould on a scale of 0, 1, 2, 3, and 4, representing 0, 1-25, 26-50, 51-75, and 76-100% of stem and branches with disease symptoms (Hall & Phillips, 1996). Disease incidence was calculated as the percentage of plants with symptoms on stem or branches. Yield data were based on weight of seeds at 12-13% moisture (w/w) harvested in 2.8 m2 (including 1.2 m2

harvested for disease evaluation). The data were subjected to analysis of variance. Linear regression analysis between yield and plant densities was performed.

Results and DiscussionPlanting density influenced the development of white mould. When severity was analysed, there was a significant interaction between planting densities and modes of sowing. With 15 plants/m severity was higher with three plants together (severity = 3.33) than with plants equidistant (2.99). Fungicides reduced the severity index from 3.35 to 2.23 and disease incidence from 98.9% to 86.3%. Disease incidence was over 95% for both 15 and 7.5 plants/m, but dropped to 85.7% for 5 plants/m. Without fungicide, the yield was 1,040 kg ha-1; with fungicide applications, 2,273 kg ha-1. Modes of sowing and interactions were not significant for yield. There were significant differences (P<0.01) in yield among the planting densities with the density of 5 plants/m recording the highest yield (Y=2,077.6 – 45.97X, R2

= 0,99). Number of pods/plant, number of seeds/pod, and 100-seed weight increased as planting densities decreased. Comparing treatments without fungicide with those with fungicide applications, number of pods/plant, number of seeds/pod and 100-seed weight were 11.4 and 18.0, 3.93 and 4.51, and 18.2 and 21.2, respectively. In Canada, results of Saindon et al. (1995) with upright cultivars differed from those in this study in which a vine cultivar was used. They tested four planting densities (25, 35, 50, and 60 plants/m2) in rows spaced 0.23-m apart and concluded that upright beans can be grown at high planting densities without greatly increasing the risk of a white mould outbreak. Ours results show that reduction of planting density decrease white mould incidence and severity and increase yield, with or without fungicide applications.

AcknowledgmentsMy participation in this workshop was supported in part by Fundação de Amparo à Pesquisa de Minas Gerais (Fapemig).

ReferencesBoland GJ, Hall R, 1994. Index of plant hosts of Sclerotinia sclerotiorum. Can. J. Plant

Pathol. 16, 93-108.Hall R, Phillips LG, 1996. Evaluation of parameters to assess resistance of white bean to

white mold. Annual Report of the Bean Improvement Cooperative 39, 306-307. Saindon G, Huang HC, Kozub GC, 1995. White-mold avoidance and agronomic attributes of

upright common beans grown at multiple planting densities in narrow rows. J. Amer. Soc. Hort. Sci. 120, 843-847.

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Investigation of the mechanisms by which the post-harvest modification of the internal gas atmosphere of top fruit, helps sustain the inate resistance of host tissues to attack by fungal pathogens

Olivier Belet

Natural Resources Institute, Food Systems Department, Medway University Campus, Chatham Maritime, ME4 4TB, UK

AbstractImposed controlled atmosphere gas regimes have permitted to replicate the impact of Semperfresh on the internal gas atmosphere of ‘Conference’ pears. The comparison of coates fruits inoculated with P. expansum and M. fructigena with infected fruits held under controlled atmosphere, showed no significant difference between the treatments. This might suggest the existence of a gas effect in sustaining the resistance mechanisms of host tissues to attack by fungal pathogens.


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P2.4

Seed treatment for the control of Sclerotinia basal-stalk rot/wilt in sunflower

K. Y. Rashida and J. Swansonb

a Agriculture and Agri-Food Canada, Cereal Research Centre, Morden Research Station, Morden, Manitoba, Canada R6M 1Y5; and b Croplan Genetics, Land O’Lakes Inc., Mentor, Minnesota, USA 56736.

Abstract In three years of field studies, seed treatment with fungicides significantly reduces the incidence of early root and basal-stem infections in sunflower in Sclerotinia-inoculated plots, and prevents the 60-84% yield losses caused by the disease.

Introduction Sclerotinia sclerotiorum is the causal organism of one of the most common and destructive diseases affecting sunflower worldwide. This fungus is pathogenic on many crops including canola and field peas, two of the major crops in western Canada. Mycelia from germinating sclerotia infect roots and basal-stems causing stalk rot and wilt. Ascospores, produced on apothecia formed by carpogenic germination of sclerotia, infect stems and heads causing stem breakage and head rot. Sclerotinia wilt has been the most common in Manitoba in spite of the increased incidence of head rot in recent years. Sunflower hybrids are susceptible with different levels of susceptibility (Rashid & Dedio 1992). The objective of this study was to assess the efficacy of seed treatment to protect sunflower from early infections by Sclerotinia. Preliminary data was previously reported (Rashid & Swanson 1998)

Materials and Methods Field trials were conducted at the Agriculture and Agri-Food Canada, Morden Research Station from 1998 to 2000. The oilseed hybrid CLOL 803 and the confection CLOL 110 were used. The following fungicides were used with rates as g ai per kg of seed: Topsin M 4.5F (thiophante-methyl 70%) at 0.36 g, Apron FL (metalaxyl 32%) at 0.6 g, Captan at 1 g, Ronilan (vinclozolin 50% ai) at 1.5 g, Quadris (azoxystrobin 30% ai) at 2.5 g, and Maxim (fludioxonil 48% ai) at 0.2 g (Table 2). Each treatment was used with and without Sclerotinia inoculation. A split-plot design was used with hybrids as main plots and seed treatments as sub-plots. Plots were 2 rows of 3 m long and .75 m apart. Inoculum of S. Sclerotiorum was increased in the laboratory on pearl millet seed and was drilled with the seed at planting. Data was collected on emergence, seedling survival, and incidence of wilt. Yield was obtained and kernel weight, kernel density, and oil content were measured after harvest.

Results and Discussion Sunflower emergence was reduced by 56-84% in the Sclerotinia-inoculated plots, and yield was reduced by 60-80% in the three years of testing (Table 2). All seed treatments in 1998 & 2000, and Ronilan in 1999 significantly improved stand and yield in Sclerotinia-free plots due perhaps to the protection from other micro-organism in the soil affecting sunflower emergence and seedling vigour.

In 1998, Topsin significantly improved the stand from 36% to 66% in Sclerotinia-inoculated plots, and reduced the yield losses from 60% to 22% of the healthy control plots (Table 1). In

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1999, Ronilan was the most effective seed treatment that improved the stand from 31% to 85% in Sclerotinia-infected plots and reduced yield losses of 78% caused by the Sclerotinia infections (Table 1). In 2000, Ronilan treatments reduced the Sclerotinia infections from 84% to 25% and reduced the yield losses of 80% caused by Sclerotinia infections. Maxim was also effective in reducing Sclerotinia infection from 84% to 17% and reducing yield losses from 80% to 9% in Sclerotinia-inoculated plots. The various treatments had no significant effect on Sclerotinia wilt beyond the seedling stage as well as on kernel weight, kernel density, and oil content. This study demonstrated that Ronilan, Maxim, and Topsin proved to be effective in significantly reducing Sclerotinia infections in sunflower seed/seedling, and significantly improving yield.

Table 1. Effects of seed treatment in sunflower on early infections by Sclerotinia sclerotiorum, and on yield.

1998 1999 2000 % of Control % of Control % of ControlTreatment Stand Yield Stand Yield Stand Yield Control 100 100 100 100 100 100Control + Sclerotinia 36- 40- 31- 22- 16- 20-Topsin 122* 180* 109 39- 140* 165*Topsin + Sclerotinia 66* 78 46* 32 38* 63* Ronilan NT NT 135* 176* 156*Ronilan + Sclerotinia NT NT 85* 110* 75* 115*Quadris NT NT 136* 123 147* 143*Quadris + Sclerotinia NT NT 41 44 18 37Maxim NT NT NT NT 159* 161*Maxim + Sclerotinia NT NT NT NT 83* 91*LSD (P=0.05%) 20 57 13 57 19 35 * Significantly better than the checks. - Significantly more diseased than the checks.Compare the fungicide seed treatments to the control; and the fungicide seed treatments + Sclerotinia to theControl + Sclerotinia treatment.

AcknowledgementSpecial thanks to E. Kabanuk, and M. Haugin from Croplan Genetics/Land O'Lakes for the generous supply of treated seed, and for the Chemical Companies for providing free samples of fungicides. The technical assistance of L. Wiebe, M. Penner and T. Walske, AAFC Morden Research Station is gratefully appreciated. The financial supported from the National Sunflower Associations of Canada and the USA, and the Matching Investment Initiative of the AAFC is greatly appreciated.

ReferencesRashid KY, Dedio W, 1992. Differences in the reaction of sunflower hybrids to Sclerotinia

wilt. Can. J. Plant Sci. 72:925-930.Rashid K Y, Swanson J, 1998. Update on seed treatment for the control of Sclerotinia wilt in

sunflower. Pages 87-91 In: Proceedings of the 20th Sunflower Research Workshop, USA National Sunflower Association, January 1998, Fargo ND.

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Chemical control of Sclerotinia sclerotiorum with acetylsalicylic acid

A. Moret, M. Nadal, N. Canti, and S. Sánchez

Dept. de Biologia Vegetal, Fac. de Biologia; Av. Diagonal, 645, E-08028 Barcelona

AbstractExperiments were conducted to study the inhibitory effect of acetylsalicylic acid (ASA) on the growth of Sclerotinia sclerotiorum. To determine the growth rate of the fungus we tested the following concentrations: 0, 47.04, 56.84, 66.64, 76.44, 86.24 and 96.07 ppm. The average growth rate of control was 11.47 mm/day and the average lethal dose (DL50) was 50.19 ppm.

IntroductionSclerotinia sclerotiorum (Lib.) de Bary is a soil-borne pathogenic fungus that causes a serious disease known as Sclerotinia wilt in a wide variety of crops. It is widespread in the Mediterranean country, where it damages horticultural plants, ornamentals and numerous vegetables. The symptoms of the disease are wilting and progressive soft rot of non-lignified tissues, and as infection progresses, the plant collapses. A white mat of cottony mycelium forms and covers many aerial portions of the plants during periods of high moisture and favourable temperature conditions. Black sclerotia (2-20 x 3-7 mm), which are largely produced in infected plants, are the main structures responsible for fungus overwintering and dissemination. In culture, sclerotia are found at the growing margins of the colony, usually forming concentric rings.

Sclerotinia blight is controlled with dicarboximide fungicides, which are ineffective once the fungus is established in a field. On the contrary, repeated application of these fungicides may prove less than satisfactory (Tu, 1989; Yarden et al., 1986). The aim of this study was to determine the sensitivity of S. sclerotiorum to acetylsalicylic acid in vitro and the efficacy of this product in restraining the disease in the field.

Materials and MethodsAn isolate of S. sclerotiorum, obtained from lettuce naturally infected in Alt Empordà (Catalonia country) in 2000 was used throughout the study. It was maintained in the dark at 22 ºC on potato dextrose agar (PDA). Fresh mycelia of S. sclerotiorum were produced by placing 5 mm diameter mycelial plugs, removed from the margin of a culture grown on PDA, in the centre of Petri dishes, 90 mm in diameter containing 15 ml of medium. The Petri dishes were then sealed and incubated in the dark at 22 ºC for six days before use (Moret & Nadal, 1.993). To obtain the 1M stock solution of acetylsalicylic acid, the necessary amount of ASA was dissolved in 5 ml of ethanol (98% pure), and its initial acid pH was neutralized by addition of KOH 6M to achieve physiologic pH ( 7.2-7.3). From this concentrated stock, doses of 0, 240, 290, 340, 390, 440, 490 ml (corresponding to 47.04, 56.84, 66.64, 76.44, 86.24 and 96.07 ppm respectively) were added to the flasks containing PDA, which were autoclaved for 20 minutes at 120 ºC and cooled to 45 ºC. Ten ml of amended medium was then dispensed into Petri dishes. To determine the effect of the selected chemical product on the growth of mycelia, a fresh mycelia plug (5 mm diameter) of the S. sclerotiorum isolate

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taken from the leading edge of a six day old-culture was placed at the centre of the Petri dishes with amended medium. The experiment was repeated five times. These plates were incubated in the dark at 22 ºC, and growth was measured periodically for seven days after plating.

Results and DiscussionAcetylsalicylic acid reduced mycelial growth at medium concentrations and abolished it at the highest concentrations tested (96.07 ppm). While mycelial growth in the control was white and dense, progressively covering the plate in a homogenous manner and forming sclerotia in the external margin, the colour of the mycelium and the medium at higher concentrations varied from brown-yellowish on the first days of incubation to orange and matt. Moreover, the mycelium presented limited growth forming lobulate, non-homogenous colonies with prominent cottony margins, in which the sclerotia develop.

The effects of the ASA concentrations tested were determined by linear regression. The growth rate of the control (0 ppm of the product tested) was 11.47 mm/day. At lower acid concentrations, growth was slightly inhibited but the highest concentrations clearly showed gradual inhibition of growth. The DL50 was also calculated, it was 50.19 ppm. The variation in the colour of the mycelium may be due to the pH decrease in the culture medium or to the production of some substance by the fungus that is activated by ASA. Acetylsalicylic acid is thus a useful tool to control S. sclerotiorum in experimental conditions, and its impact on the environment is relatively small, since it is degraded faster than most conventional fungicides.

ReferencesMoret A, Nadal M., 1993. Control químico de Sphaeropsis sapinea (Fr.) et Sutton. Actas da

Area de Ciencias Agrarias do S.E.G.; N.:13, 237-241 Santiago de Compostela.Tu JC, 1989. Management of white mold of white beans in Ontario. Plant Disease, 73, 281-

285 Yarden O, Ben-Yephet Y, Katan J, Aharonson N, 1986. Fungicidal control of Sclerotinia

sclerotiorum in soil with a combination of benomyl and thiram. Plant Diseases 70,738-742.

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Benzimidazole resistance of Sclerotinia sclerotiorum in French oilseed rape crops

A. Penaud a, B. Huguet b , V. Wilson b and P. Leroux c

a CETIOM, Centre de Grignon, B.P. 4, 78850 Thiverval-Grignon, FR;b SRPV Ile de France, 10 rue du Séminaire, 94516 Rungis cedex, FR;c INRA, Unité de Phytopharmacie et Médiateurs Chimiques, Route de St Cyr, 78026 Versailles cedex, FR

AbstractSclerotinia stem rot on oilseed rape is one of the major diseases which can be responsible for severe yield losses in French crops. For many years, effective control has been achieved by a preventive and systematic application of fungicide at the beginning of flowering. Because of its effectiveness and its cheapness, carbendazim has been usually sprayed but its wide application has led to fungi resistance.

Materials and methods Since 1994, surveys were conducted in open fields in different parts of France. Each year, about one hundred samples of sclerotia of S. sclerotiorum are collected. Their carbendazim sensitivity or resistance were characterized according to the method described by Souliac & Leroux (1995). The method is based on colony radial growth of S. sclerotiorum on malt agar medium amended or not with carbendazim. Isolates are considered as resistant when mycelium does not grow at all at the rate of 1 and/or 10 mg/l. AFC analysis was used to investigate relationships between cultural practices and occurence of resistance to carbendazim.

Results and discussionThe carbendazim resistance of S. sclerotiorum was first observed in 1994 in oilseed rape in Burgundy. Only four years later in 1998, two other resistant isolates were detected in Paris area. In 1999, 20% of tested isolates were resistant to carbendazim (Kaczmar & al, 2000).In 2000, due to severe attacks of S. sclerotiorum in oilseed rape and failure of its control through fungicide applications, more than 250 tests were performed. The frequency of resistant isolates increased dramatically to reach 72%, particularly in the north-eastern part of France.

An analysis of the cultural practices has suggested that the development of the resistance of S. sclerotiorum to carbendazim in oilseed rape can be explained by short crop rotations (oilseed rape every 2 or 3 years) and at least 5 carbendazim sprays during the last 10 years.

The occurence of S. sclerotiorum resistant to carbendazime has led us to develop resistance management strategies. Although in 2000, we recommanded to apply no more one spray of carbendazim to reduce the selective pressure on the pathogen population, the developement of resistance increased. So, in 2001 our recommandations concerning chemical control are to avoid carbendazim spray in the area where resistance is prevalent and to choice alternative fungicides without cross-resistance, such as dicarboximides fungicides which are very

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effective but expensive. To prevent new kind of resistance, a dicarboximide monitoring is carrying out. In the same time, we try to develop decision making systems to apply chemical control only when it is needed. Such systems can help to reduce the fungicide input and thus the selection pressure on the pathogen.

ReferencesKaczmar MJ, Wilson V, Leroux P, 2000. Sclérotiniose du colza : le carbendazime en sursis?

Phytoma-LDV 529, 31-33.Souliac L, Leroux P, 1995. Sclérotiniose du colza: Faut-il revoir la stratégie de lutte?

Phytoma-LDV 474, 29-31.

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Effect of cycloheximide on curing of the hypovirulence of Sclerotinia sclerotiorum strain Ep-1PN

Guoqing Li1, Daohong Jiang1, Daoben Wang1, Bin Zhu2 and S. R. Rimmer2

1Department of Plant Protection, Huazhong Agricultural University, Wuhan, 430070; 2 Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, SK, S7N 0X2, Canada

AbstractCycloheximide was used to cure hypovirulent Sclerotinia sclerotirum strain Ep-1PN and the growth rate, cultural characteristics, pathogenicity and double-stranded (ds) RNAs of the hyphal tip derivatives (HTD) were characterized in this research. The results showed that compared with Ep-1PNA5, a virulent strain of S. sclerotiorum, completely-cured HTDs were not isolated after three successive treatments with cycloheximide ranging from 5 to 40 μg/ml in PDA. The partially-cured HTDs, which were similar to Ep-1PNA5 in colony appearance, but still different in growth rate and pathogenicity from Ep-1PNA5, and the unaffected HTDs, which were similar to Ep-1PN in the two characteristics, were observed among the 400 HTDs. Seven partially-cured HTDs were detected to contain two dsRNA bands with the sizes of 7.4 and 1.0 kb, whereas seven unaffected HTDs contained three dsRNA bands with the sizes of 7.4, 6.4, and 1.0kb as in Ep-1PN. Ep-1PNA5 contained no dsRNAs. When the partially-cured HTDs C22 and C29 were paired with Ep-1PN or the uncured HTDs C3 and C5

on media, they became hypovirulent and the dsRNA of 6.4 kb size was present. This study suggests that cycloheximide can only partially eliminate the hypovirulence of S. sclerotiorum strain Ep-1PN and that the dsRNA of 6.4kb was more important to the hypovirulence of Ep-1PN than the other two dsRNA molecules.


3. BIOLOGICAL CONTROL

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Study of the fungal flora of sunflower varieties with different response to Sclerotinia sclerotiorum.

M.A. Rodríguez, N. Venedikian and A.M.Godeas

Dept. de Biología, F.C.E.y N, Universidad de Buenos Aires, Ciudad Universitaria, pab. II. 1428 Buenos Aires.

AbstractThe role of microorganisms from different floret parts of tolerant and susceptible (Helianthus annuus) sunflower varieties to S. sclerotiorum attack was investigated. Our results show that tolerant varieties might have an indirect defense mechanism that protects flower from pathogen growth.IntroductionThe fungus Sclerotinia sclerotiorum is a pathogen, which can cause two types of attack in sunflower: infection of the inflorescence or the stem. Head rot is the more common attack in the south and southeast of Buenos Aires (Pereyra & Bazzalo, 1988). Little is known about the susceptibility mechanisms of different sunflower cultivars. Many recent studies have analysed the use of saprophytic fungi developing on plant surfaces (Dix & Webster, 1995) as an interesting alternative to the use of chemical agents, which involve environmental alteration and pollution (Chet et al, 1997). The aims of this study were to isolate saprophytic fungi from different floret parts of susceptible cultivars of sunflower (SV) and tolerant cultivars (TV), to establish the differences between them.Materials and MethodsIsolation from H. annuus susceptible cultivars (SV) (HA 300 and Z 20028) and tolerant varieties (TV) (HA 302, Z AV 8410 and Z 30629). The heads were harvested 8 days after flowering, from plants grown at field in Balcarce (Bs As, 1997). Saprophytic surface-colonizing strains were isolated (Parkinson & Williams, 1961) from different floret parts as: interfloral bracts; pappus; corolla; nectary (N); anther; stigma and ovary. For each variety and each floret part, 5 replicas were placed in Petri dishes of agar Malta (AM) with antibiotics (T: 25°C). Frequency of appearance and several indices were calculated (richness and relative abundance of each species for each variety). The diversity of fungal community at each variety was assessed by calculating values for the Shannon-Wiener function (Krebs, 1994). We present the results of the examination of trends in community structure among the varieties based on the use of principal component analysis (PCA) (Kneel & Booth 1992). Interaction between each isolate and S. sclerotiorum was studied by dual culture on AM agar plates. For each confronted pair we established percentage of radial growth inhibition (%RGI) (Fairer & Bertoni, 1988). By using frequency and degree of inhibition for each specie, we determined the degree of protection from the pathogen in each floret part as: frequency of appearance x % ICR of all species colonizing each flower part.Results and DiscussionWe obtained 27 fungal species from 146 isolations. Only Alternaria alternata and Cladosporium cladosporoides were found on all varieties. A. citri and A. raphani were also fairly widespread. Yeasts and Penicillium species distinguished SV and TV. Both, species richness and Shannon –Wiener index rating increased at VT. PCA revealed distinctive patterns of saprophytic fungi between samples from SV and TV. The variance explained by the first factor was 16.13% and by the second 15.61%. The first ordination axes separated

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samples of the TV Z30629 (characterized by A. clamydospora, yeast AB and F. sambucinum) and the second axes of ordination separated samples of both SV, characterized by the absence of F. graminearum, Penicillium sp 4 and Penicillium sp 6. Most of the filamentous fungi reacted with degrees D2 and D2+ (hyphal contact). D2 was the most frequent, while D2+ was found only for certain Fusarium strains. Degree D3 (antibiosis) was found for certain Alternaria species. Most yeasts had potential antagonism type D5 (antibiosis). Flower parts are modified leaves and as such are colonized by a series of fungi, which may be either symbiotic endophytes, or saprophytic or weak parasitic fungi restricted to the surface (Dix & Webster, 1995). They compete through different mechanisms (McGregor, 1976). The mechanism producing antagonistic capacity in yeasts differed from the mechanism in most filamentous fungi. The degree of protection is a result of antagonist frequency and antagonistic capacity; therefore higher values reflect either higher frequency, higher antagonistic capacity or both. The flower parts with greatest anthosphere saprophytic colonization are style, anthers and corolla, these are also the most exposed to pathogen attack and pollen deposition. According to our results, the flower parts in closest contact with pollen, such as anthers, style and corolla, have greatest colonization from saprotrophic fungi on both varieties. This is particularly so on TV, where increased colonization has the additional property of providing protection from the pathogen. There seems to be competition among microorganisms for the consumption or control of access to a limited anthosphere resource (space and nutrients) (Widden, 1997). The potential antagonism of certain common fungi from plant surfaces has been studied (Brame & Flood 1983; Dickinson & Bottomley 1980). We found significant differences between the values for degree of protection between SV and TV (LSD p>0.05) (McGregor, 1976), which shows that TV might have an indirect defense mechanism (Heath, 1997) thanks to the establishment of a fungus population, which colonizes the most exposed floret parts and prevents the disease from becoming established. Acknowledgments CONICET (PIP 812) and UBA (TY02) supported this workReferencesBrame C, Flood J, 1983. Antagonism of Aureobasidium pullulans towards Alternaria solani. Trans Brit Mycol

Soc; 81, 621-4.Chet I, Inbar J, Hadar Y, 1997. Fungal antagonists and mycoparasites. In: Esser K, Lemke PA, eds. The

Mycota IV. Berlin: Springer-Verlag,:165-184.Dickinson CH, Bottomley D, 1980. Germination and growth of Alternaria and Cladosporium in relation to

their activity in the phylloplane. Trans Brit Mycol Soc, 74, 309-19.Dix NJ, Webster J, 1994. Fungal ecology. London. Chapman & Hall, 1995.Faifer G, Bertoni MD,1988. Interactions between epiphytes and endophytes from phyllosphere of Eucalyptus

viminalis III. Nova Hedwigia; 47, 219-229.Kenkel NC, Booth T, 1992. Multivariate analysis in fungal ecology. In: Carroll GC, Wicklow DT , eds. The

fungal community. Its organization and role in the ecosystem. New York: Marcel Dekker,:209-227.Krebs ChJ, 1994. Ecology. New York. Harper & Row. Heath MC, 1997. Evolution of plant resistance and susceptibility to fungal parasites In: Esser K, Lemke PA,

eds. The Mycota V part B. Berlin: Springer-Verlag: 257-276.Parkinson D, Williams ST, 1961. A method for isolating fungi from soil microhabitats. Plant and soil 13, 347-

355.Pereyra VR, Bazzalo ME, 1988. Podredumbre del girasol [Dissertation]. Balcarce. Buenos Aires: INTA.Widden P, 1997. Competition and the fungal community. In: Esser K, Lemke PA, eds. The mycota IV.

Berlin: Springer-Verlag: 135-148.

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Comparative study of fungal antagonist against Sclerotinia sclerotiorum

M. A Rodríguez and A.M. Godeas.

Departamento de Biología, F.C.E.y N, Universidad de Buenos Aires, Ciudad Universitaria, pab. II. 1428 Buenos Aires. E-mail: arodrig@ bg.fcen uba.ar.

AbstractA comparative study of different antagonist strains against S. sclerotiorum pathogen was carried out. Saprophytic fungi were isolated from soybean and lettuce cultured soils of Buenos Aires province (Argentina). The interactions between the isolated strains were tested by “in vitro” and greenhouse test.IntroductionSclerotinia sclerotiorum (Lib.) de Bary is a very important soil-borne plant pathogen that attacks a wide range of hosts. In Argentina, it affects economically valued crops: sunflower, soybean, lettuce and peanut (Bazzalo, 1986). This fungus produces over wintering structures: the sclerotia, which resist on the soil for several years. They germinate and infect directly through hyphae or produce apothecia, whose ascospores infect aerial plant parts. Its control is not successful, neither cultural nor even chemical treatments. Moreover, host resistance has been inadequate (Lumsden, 1979). Biological control is an interesting tool, which has been studied over last years (Dix and Webster, 1995; Chet el al, 1997). The aim of the present study was to select the best antagonists from different soils of Buenos Aires and investigate their activity against S. sclerotiorum by in vitro and greenhouse tests.Materials and methodsThere were isolated soil-borne strains of: Gliocladium roseum, Talaromyces stipitatus, Tal. flavus, Talaromyces sp, Trichoderma koningii, Tri. harzianum, Tri. hamatum, Fusarium camptoceras and Aspergillus sp following Parkinson & Williams (1961) method, from 3 fields of Buenos Aires province (Argentina). Interaction between each isolate and S. sclerotiorum was studied by dual culture on malt agar (MA). To evaluate the effect of metabolites over the pathogen grown, cellophane test were performed (Faifer and Bertoni, 1988). To study the potential mycoparasite activity, sclerotia were placed into spore suspensions (2 x 105 esp/ml) of each isolate (Gracia-Garza et al, 1997) and transferred to Petri dishes (9 cm) containing moist sterile sand or soil. Twenty sclerotia were placed into the surface and were incubated for 3 weeks at 24°C (Whipps & Budge, 1990). To test the effect of antagonist on germination and seed protection, superficially sterilized soybean seeds were soaked into spore suspensions of each strain (variable concentration from 107 to 104 esp/ml). Dual cultures in Petri dishes (14 cm diam) with AM and sterile vermiculite in equal parts were performed; the S. sclerotiorum inoculum was placed on the AM and the seeds on the vermiculite. They were incubated in growth chamber (14 h light; 21°C-29°C) for 10 days. In greenhouse test with T. koningii and G. roseum strains, we did four treatments: antagonist+pathogen, only pathogen, only antagonist and control. Five days old soybean seedlings, grown in 200 ml pots with sterile soil, were inoculated with spore suspensions and rice grains colonized by the isolate (3.67 x 107 for T. koningii and 1.68 x 107 for G. roseum esp/gr dry soil). After 5 days they were placed in 500 ml pots with the addition of infected soil. This infected soil by S. sclerotiorum was prepared from sterile soil with a mixture of rice: wheat brand: water (20:20:100-V:V:V) colonized by the pathogen in a relation of 10% (w:w

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dry soil). The plants were harvested after 25 days and the percentage of survival plants was registered. Results and DiscussionIn dual cultures and in cellophane test most of the isolates were antagonistic and some of them strongly inhibited the development of S. sclerotiorum. Necrotic areas where both half of dual cultures in presence of G. roseum and F. camptoceras contacted, alterations of the S. sclerotiorum mycelium were observed. They were the most active antagonists whose antibiosis as main antagonism mechanism. The alterations of the pathogen observed in the contacted line of dual cultures are probably due to the action of diffusible antibiotic substances. Also Tri. harzianum, Aspergillus sp and all the strains of Talaromyces sp caused an important inhibition mediated by antibiosis. Concerning to seed protection, we found that the best concentration was 104 esp/ml for all the isolates. Tri. koningii, Tri. harzianum and G. roseum were the most effective strains against the pathogen growth, with high level of protection. . Although Gliocladium sp and Trichoderma sp are believed to be successful tools for plant disease control (Michereff et al, 1995; Sivan et al, 1984), Tri. koningii and G. roseum strains, studied under greenhouse conditions, showed partial efficiency of disease control. These are only preliminary tests and these strains should be used for additional biocontrol experiences with the use of other formulations.Acknowledgements We thanks to Lic. C. Laporta for help with the English. Conicet (PIP 812) and UBA (TY02) supported this work.ReferencesBazzalo ME, 1986. Mecanismos de defensa de H. annuus L. Frente al ataque del hongo

Sclerotinia sclerotiorum (Lib.) De Bary. Tesis. FCEN. UBA.Dix NJ, Webster J, 1995. Fungal ecology. London. Chapman & Hall pp: 549.Chet I, Inbar J, Hadar Y, 1997. Fungal antagonists and mycoparasites. In: Esser K, Lemke

PA, eds. The Mycota IV. Berlin: Springer-Verlag:165-184.Faifer G, Bertoni MD, 1988. Interactions between epiphytes and endophytes from

phyllosphere of Eucalyptus viminalis III. Nova Hedwigia; 47, 219-229.Gracia-Garza JA, Reeleder RD, Paulitz TC, 1997. Degradation of sclerotia of Sclerotinia

sclerotiorum by fungus gnats (Bradysia coprophila) and the biocontrol fungi Trichoderma spp.. Soil Biol. Biochem. 29, 123-129.

Lumsden RD, 1979. Histology and physiology of pathogenesis in plant diseases caused by Sclerotinia species. Phytopath 69, 890-896.

Michereff SJ, da Silveira NS, Reis A, Mariano R, 1995. Greenhouse screening of Trichoderma isolates for control of Curvularia leaf spot of jam. Mycopathologia 130, 103-108.

Parkinson D, Williams ST, 1961. A method for isolating fungi from soil microhabitats. Plant and soil; 13, 347-355.

Sivan A, Elad Y, Chet I, 1984. Biological control effects of a new isolate of Trichoderma harzianum on Phytium aphanidermatum. Phytopath. 74: 498-501.

Whipps JM, Budge SP, 1990. Screening for sclerotial mycoparasite of Sclerotinia sclerotiorum. Mycol. Res. 94, 607 –612.

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The biological fungicide Contans®WG – a preparation on the basis of the fungus Coniothyrium minitans

P. Lüth

PROPHYTA Biologischer Pflanzenschutz GmbH, Inselstrasse 12, 23999 Malchow, Germany

AbstractThe biological fungicide Contans®WG is based on the conidia of fungus Coniothyrium minitans. The preparation is formulated as a water dispersible granule containing only glucose as carrier and the purified conidia of the antagonistic fungus. One gram of the product contains 1 x 109 viable conidia of C. minitans. The product can be used to combat Sclerotinia sclerotiorum. For S. minor and Sclerotium cepivorum PROPHYTA is still working on the development of an appropriate application technology.

For the recommended application to combat S. sclerotiorum in the soil the product has to be dissolved in water. The resulting spraying broth has to be sprayed onto the soil surface and incorporated into the upper 5 cm soil layer. Here C. minitans attacks the sclerotia of Sclerotinia spp. in destroys them in accordance to the soil moisture and the soil temperature. Under optimum condition the decay of the sclerotia takes 3 months.

The product is currently registered in Germany, the USA, Austria, Poland, Switzerland and Luxembourg. The inclusion of the active ingredient in Annex I of Directive 414/91/EEC is expected until the end of this year.

In the contrary to other biocontrol agents the use of the product is not restricted to niche markets like vegetable and ornamental production. Contans®WG is also sold to farmers producing e.g. oilseed rape and beans on large areas.

In the first year (2000) PROPHYTA already sold 27.000 kg especially in Germany and the USA. In 2001 the company is planning to sell 70.000 kg altogether. Reaching this amount Contans®WG would be the most sold biological fungicide world-wide.


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Control of sclerotinia within carrot crops in NE Scotland: the effect of irrigation and compost application on sclerotia germination

Gordon Coupera, Audrey Litterickb and Carlo Leifertc

aAberdeen University Centre for Organic Agriculture, MacRobert Building, King Street, Aberdeen, AB24 5UA; bLand Management Dept., SAC, Land Management Dept., Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA; cTesco Centre for Organic Agriculture, King George VI Building, Newcastle University, Newcastle, NE1 7RU

Abstract Carrots are susceptible to attack from Sclerotinia sclerotiorum. This pot study showed that irrigation encouraged, and compost inhibited, carpogenic germination.

IntroductionYield and quantity of conventional carrots produced in NE Scotland is often significantly reduced by sclerotinia (Sclerotinia sclerotiorum) infection of foliage and root. In this study, the effects of irrigation and compost on sclerotia germination are investigated.

Materials and MethodsPreparation: Twenty square, 10 litre pots, (surface area 300 x 300 mm), were filled with sieved topsoil, (sandy loam, pH 6.4, Dalcross, NH 3774 8511). Two rows of eight carrot seeds, (variety Nairobi) were sown in each pot, with spacing representative of a commercial drilling pattern, (34 mm between each carrot within a row, 150 mm between rows). Pots were inoculated with field-collected sclerotia (source; commercial carrot crop, medium sandy loam, pH 6.6, Mains of Ravensby, near Carnoustie, NO 3535 7353) at 55 sclerotia m -2, giving five sclerotia per pot.

Treatments: S (sclerotia inoculation only); NS (no sclerotia); C (compost applied at 35 t ha-1); I (soil was maintained at pot capacity by daily irrigation). Each treatment and control had five replicates and the pots were laid out in a randomised block design. The compost was manufactured by TIO Ltd., according to the CMC process (Lubke, 1995), and was applied immediately before sowing and inoculation of sclerotia. It was mixed into the top 25 mm of soil. Irrigation began in the third week after planting and stopped in the eleventh week after planting.

Recording: The number and position of each germinated sclerotium and associated apothecia were recorded weekly.

Results and DiscussionDuring the first 8 weeks after inoculation, more sclerotia carpogenically germinated in the irrigation treatment (I) than in the positive control treatment (S), and more germinated in the positive control treatment (S) than in the compost treatment (C), (Figs. 1 & 2). In the compost treatment, germination peaked in week 12, and the other treatments peaked in week 6.

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Fig. 1 The effect of irrigation and compost application on sclerotia Fig. 2 The effect of irrigation and germination with time. compost on sclerotia germination1 in the first 8 weeks after inoculation. 1Means with different letters are significantly different (P0.05) according to Fisher’s individual error rate test.

Regular irrigation to pot capacity resulted in greater volumes of moisture on the soil surface and higher relative humidity in the foliar canopy, compared to non-irrigated plots. These conditions are known to promote carpogenic germination (Abawi & Grogan, 1975). Application of compost affects the physical, chemical and biological composition of the soil. Compost increases the matric potential of the soil, creating a wetter environment for both crop and soil organisms including pathogens. Results from the irrigation treatment suggest that this may have led to increased germination. Compost also adds available nitrogen to the soil. This has been shown to increase subsequent sclerotinia infection of carrot foliage and root (G. Couper, unpublished data). However, in this experiment, addition of compost to soil resulted in significantly reduced carpogenic germination. The biological component of the compost may have been responsible for the inhibition of carpogenic germination. The germination peak in week 12 supports this hypothesis. The microbial population profile of a freshly manufactured compost can change significantly after 8 weeks in a cool, agricultural soil (Lalande et al., 1998). In this study, the microbes present in the first 8 weeks after inoculation may have inhibited carpogenic germination, but as ambient soil conditions influenced the compost microflora over time, apothecia production was less affected by the microbial profile present in the compost. A study to confirm this hypothesis would involve the addition of a sterilised compost treatment.

Acknowledgements We thank TIO Ltd., Culblair Farm, Dalcross for the supply of organic soil, compost and carrot seeds, and Mr Chris Ward for advice on composts and compost use.

ReferencesAbawi GS, Grogan RG, 1975. Source of primary inoculum and effects of temperature and

moisture on infection of beans by Whetzelinia sclerotiorum. Phytopathology 65, 300-309.Lubke U, 1995. Microorganisms for controlled composting of organic materials. 4th

International Conference on Kyusei Nature Farming, Paris.Lalande R, Gagnon, B, Simard RR, 1998. Microbial biomass C and alkaline phosphatase

activity in two compost amended soils. Canadian Journal of Soil Science 78, 581-587.

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Biocontrol of Sclerotinia sclerotiorum by film-coating Coniothyrium minitans onto seed and sclerotia

M. P. McQuilkena and J. M. Whippsb

aDept. of Plant Biology, The Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK; bPlant Pathology and Microbiology Dept., Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK

AbstractConidia of the mycoparasite Coniothyrium minitans were film-coated onto Sclerotinia sclerotiorum-infected sunflower seed and sclerotia by polymer film-coating, using a fluidised-bed seed treater. C. minitans had little effect on seedling disease in a peat-soil mix, but completely suppressed apothecial production of sclerotia when placed in soil. The potential for developing C. minitans as a commercial seed treatment for biocontrol of seedling diseases is discussed.

IntroductionC. minitans is a well documented mycoprasite with considerable potential as a biocontrol agent of S. sclerotiorum. Solid substrate preparations of the mycoparasite have often been incorporated into potting mixes and soil prior to sowing and planting. However, the development of this delivery method for commercial use has limitations because it is potentially expensive. Application of C. minitans by commercial seed-coating may be a more economical method of delivery. This paper describes the successful application of conidia of C. minitans to both sunflower seed and sclerotia, using a commercial fluidised-bed film-coating process. It also describes the effect of such film-coatings on the control of seed-borne S. sclerotiorum and the germination of sclerotia in a simple pot bioassay.

Materials and MethodsTo mimic treatment of S. sclerotiorum-infected sunflower seeds (30% (w/w) infection level), batches of seed (100 g) were film-coated (kg-1) with thiram (3.4 g) + fenpropimorph (1.9 g) and iprodione (9.8 g) by polymer film-coating, using a fluidized bed seed system (Maude & Suett, 1986). Batches (100 g) were also film-coated with a conidial suspension of C. minitans (94 ml; 1.5-1.7 x 107 conidia ml-1) to achieve 5.0-5.3 x 106 conidia g-1. Batches of sclerotia (30 g) were also film-coated (kg-1) with thiram (3 g) + fenpropimorph (2 g) and iprodione (9.6 g), and a conidial suspension of C. minitans (McQuilken et al., 1997). Polyvinylacetate (1% w/w) was used as a sticker for all applications. Survival and germinability of conidia removed from coated seeds was assessed after 1 week, 6 months and a year.To determine the effect of film-coating treatments on the control of seed-borne S. sclerotiorum, film-coated sunflower seeds were sown in a peat/loam (1:1 v/v) mix in compartmented seed trays (60 compartments/tray, one seed/compartment) placed on a greenhouse bench (22oC & 80% RH). Counts of surviving seedlings were made after 14 days.The effect of film-coating treatments on carpogenic germination of sclerotia was assessed using a field pot bioassay based on the method of McQuilken et al. (1997). Numbers of apothecia produced from sclerotia in each pot were counted at fortnightly intervals from late

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April until early June. Recovery and infection of sclerotia by C. minitans was assessed 34 weeks after the start of the experiment according to McQuilken et al. (1997).

ResultsIn seedling tests carried out with S. sclerotiorum-infected sunflower seeds, C. minitans failed to increase seedling survival. Film-coating thiram + fenpropimorph and iprodione onto infected seeds gave consistently better results than C. minitans. Conidia of C. minitans obtained from film-coated seeds 1 week after coating gave a lower germination rate (59-76%) compared with those in the inoculum used previously for film-coating (73-87%). Germinability of conidia declined from 77 to 54% over the 1 year period of storage.

In the field pot bioassay, C. minitans and fungicide treatments completely inhibited production of apothecia (Table 1). Thirty-four weeks after burying sclerotia, no sclerotia were recovered from C. minitans treatment pots, and only 9 and 1% were found in fungicide treatment pots, compared to over 70% recovered in control pots.

Table 1. Effect of film-coating sclerotia of S. sclerotiorum with C. minitans and fungicides on apothecial production and subsequent sclerotia recovered (Values are means ± SE, n =10)

Treatment Sum of apothecia Sclerotia recovered (%)ControlSticker onlySticker + C. minitansSticker + iprodioneSticker + thiram + fenpropimorph

16 ± 3.825 ±6.9

000

74 ± 8.358 ± 7.3

09 ± 5.31 ± 1.0

DiscussionThe ability to survive a commercial seed-coating process is an important attribute for any potential biocontrol agent applied to seed. The conidial inoculum of C. minitans clearly has this property with the mycoparasite demonstrating excellent survival on seed for at least one year. C. minitans film-coating of sunflower seeds infected with S. sclerotiorum failed to provide any control of disease reflecting the high level of pathogen present in the seed due to the artificial system used. However, film-coating S. sclerotiorum sclerotia with C. minitans successfully inhibited all apothecial production and killed the sclerotia. The results demonstrate considerable potential for clean-up of seed lots contaminated with sclerotia of S. sclerotiorum.

AcknowledgementsWe would like to thank the BBSRC, MAFF, SERAD and the EU for financial support.

ReferencesMaude RB, Suett DL, (1986) Application of fungicide to brassica seeds using a film-coating

technique. British Crop Protection Conference Pest and Diseases 1986, 1, 237-242.McQuilken MP, Budge, SP, Whipps JM, (1997) Biological control of Sclerotinia

sclerotiorum by film-coating Coniothyrium minitans on to sunflower seed and sclerotia. Plant Pathology 46, 919-929.

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Prolonged control of Sclerotinia sclerotiorum with Sporidesmium sclerotivorum

C. A. Martinsona and L. E. del Riob

aDepartment of Plant Pathology, Iowa State University, Ames, IA 50011-1020, USA; and bDepartment of Plant Pathology, North Dakota State University, Fargo, ND 58105-5102, USA

AbstractBiological control of Sclerotinia stem rot of soybean after soil incorporation of Sporidesmium sclerotivorum was effective up to five years later. The sclerotial parasite not was detected in some of the originally infested soils but it had spread to adjacent areas.

IntroductionSclerotinia stem rot of soybean [Glycine max (L.) Merrill], incited by Sclerotinia sclerotiorum (Lib.) de Bary, has emerged as a very important disease of soybean plants in the USA. A sclerotial parasite, Sporidesmium sclerotivorum Uecker, Adams and Ayers, was infested into fields by del Rio Mendoza (1999) after Sclerotinia-diseased soybeans were harvested and when soybeans were planted 2 or 3 years later in 1998, Sclerotinia stem rot was controlled. The current research was performed in 2000 to assess any residual disease control in the same plots and to evaluate S. sclerotivorum populations in the soil. Adams & Fravel (1990) controlled lettuce drop, incited by S. minor Jagger, for five seasons with a soil infestation of S. sclerotivorum.

Materials and MethodsSeven of the nine fields studied by del Rio Mendoza (1999) in 1998 were planted to soybeans in 2000. The Ames site and one Humboldt site were 40 m x 52 m experimental areas infested in fall 1995 and spring 1996 and the entire area became infested by fall 1997. The other sites were duplicate 10 m square macroplots in farmers fields with at least 35 m between macroplots. Macroplots were established in spring 1996 near Humboldt, in fall 1996 near Conrad and Nora Springs, and in spring 1997 near Alexander and a second site near Conrad (named Conrad-2). In late August and early September 2000, Sclerotinia stem rot incidence was determined in the centre of the areas originally infested with S. sclerotivorum and areas 20 or more meters outside of the infested area in at least two directions. After the soybean crop was harvested, soil samples were taken in the centre of the original infested area and at points 10 to 30 m outside of the infested area in two directions. Soil in a sample was blended, and three 50-g subsamples were baited with 10 sclerotia of S. sclerotiorum for 4 weeks. Washed sclerotia were surface sterilized in 0.5% NaOCl and then placed on sterile moist filter paper in a petri dish (del Rio Mendoza, 1999). S. sclerotivorum grew out of the sclerotia in 7 to 14 days.

Results and DiscussionDrier and warmer than normal weather in 2000 limited Sclerotina stem rot development in the soybeans. A full plant canopy along with timely rains are necessary and conducive for apothecia production, and infection of senescent flowers by ascospores; in 2000 this situation

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evidently occurred only in those fields where row spacing was 38 cm or less. Sclerotinia stem rot was not observed in the Ames and Nora Springs fields where row spacing was 76 cm.

Average disease incidence in areas of the fields at least 20 m from the areas intentionally infested with S. sclerotivorum was 11% at Conrad, 18% at Conrad-2, 16% at Alexander, and 7% at the two Humboldt sites (but only uphill from the infested areas). Within the centre of the originally infested areas, the disease incidence was 1% at Conrad, 3% at Conrad-2, 3% at Alexander, and 0% at the two Humboldt sites, which resulted in 81% to 100% disease control. Disease control was prolonged for up to 5 years after soil incorporation of the sclerotial parasite, S. sclerotivorum, and in most instances disease control was better in 2000 than in 1998 (del Rio Mendoza, 1999). It is likely that the S. sclerotivorum infested sites were recontaminated with sclerotia of S. sclerotiorum from external sources during harvest of the 1998 soybean crop and subsequent tillage, ascospores could have blown into treated areas from adjacent non-treated areas, and spores of S. sclerotivorum may have spread into the adjacent control areas. These interplot interferences may have compromised the experiments and potential disease control may have been better than experienced.

Assays for the presence of S. sclerotivorum in soil samples from these fields were based on the percentage of sclerotial baits yielding S. sclerotivorum colonies. The antagonist was not isolated from any of the soil samples taken at the two Conrad sites. At the Ames site 16% of the baits from the experimental area and 17% of the baits from outside of the experimental area yielded the antagonist. At Alexander S. sclerotivorum was not recovered from soil samples from the originally infested sites but was baited from every soil sample taken outside of the originally infested site at an average of 24% of the baits colonized (range of 3 to 73%). At Nora Springs the antagonist grew from 18% of the baits from soils from the infested areas, 21% of the baits from soil sampled downhill from the infested areas, but was not detected in soil samples taken uphill from the infested areas. S. sclerotivorum was baited from some soil samples taken at the two Humboldt sites, both inside and outside of the infested areas, and especially downhill. Failure to bait the antagonist from some the soils infested in 1995, 1996 and 1997, indicates that S. sclerotivorum may not survive well after it has consumed all of the sclerotial food sources in and on the soil.

AcknowledgementsThanks to The Leopold Center for Sustainable Agriculture for financial support, Miralba Agudelo and Rocio van der Laat for technical support and to the cooperating farmers Stephen Holl, Donald Latham, Phillip Naeve, and Larry Bortz.

ReferencesAdams PB, Fravel DR, 1990. Economical biological control of Sclerotinia lettuce drop by

Sporidesmium sclerotivorum. Phytopathology 80, 1120-1124.del Rio Mendoza LE, 1999. Biological control of Sclerotinia stem rot of soybean with

Sporidesmium sclerotivorum. Ph.D Dissertation, IA State Univ. Ames, 129p.

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Solarization in the management of lettuce drop (Sclerotinia spp.).

V. Gepp, E. Silvera, S. Casanova and D. Tricot.

Facultad de Agronomía, Avda. Garzón 780, 12900 Montevideo, Uruguay, E-mail: [email protected]

AbstractIn three experiments (1999-2000) in the rural areas of Montevideo, beds were covered with transparent plastic for 20-90 days. In the following lettuce crop, solarization reduced the incidence of lettuce drop by 82 and 67 % in the field, where it was mainly effective against Sclerotinia minor, and by 76 % in a greenhouse (S. sclerotiorum).

IntroductionIn the rural areas of Montevideo, Uruguay, most farms produce vegetables, principally lettuce, throughout the year and their small size means that rotations are too short to maintain the density of sclerotia of Sclerotinia spp. at acceptable levels. In a study carried out in autumn of 1998 by the Faculty of Agronomy and the Municipality of Montevideo, losses detected in lettuce crops due to these fungi reached 38% and were on average 7,5%. Solarization has been used successfully against Sclerotinia spp. in different parts of the world (Porter & Merriman, 1985; Troilo et al., 1985; Phillips, 1990 and Pereira et al., 1996). In Uruguay it has been used to control other pathogens for some years in the warmer north of the country (Cassanello, 1990), but it had never been evaluated in the cooler south. The following experiments were designed to test the effect of solarization on the disease caused by Sclerotinia sclerotiorum and S. minor of lettuce in southern Uruguay.

Materials and MethodsTwo experiments were carried out in the field (1998-99; 1999-00 ) and one in a greenhouse (1999-00) in the rural area of Montevideo (35º latitude). Moistened beds were covered with transparent 60 thick plastic PVC. The periods of solarization tested were: 60 and 90 days (field, 1998-99), 30 and 45 (field, 1999-00) and 20 and 30 days (greenhouse, 1999-00). In the last experiment an extra treatment consisted in incorporation of 3.5 Kg/m2 of poultry litter (+ rice husks) prior to solarization, in the rest of the plots it was applied after removing the plastic cover. Experimental design was a randomised block with four replicates (blocks = beds) in the first and last experiment, the 1999-2000 field experiment had two replicates in a random design. Soil temperatures at 5 and 18 cm depths were recorded in solarized and untreated beds at 8 am and 3 pm once or twice a week.After the longest period tested in each experiment, the untreated plots were hand weeded and then lettuce seedlings were transplanted into the beds (9 - 12 / m2). Diseased plants were counted and removed on weekly visits until harvest. Results were subjected to analysis of variance.

Results and DiscussionSolarization increased mean soil temperatures by 8ºC at 5 cm depth, and 6ºC at 18 cm. Porter and Merriman (1985) found similar differences at 15 cm but greater differences at 5 cm, in

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south east Australia, while Triolo et al. (1985) detected differences like those in this research, in Pisa, Italy.S. minor was the main pathogen in the field experiments, causing 87 and 84 % of the losses in each of the experiments. In the greenhouse only S. sclerotiorum was found. Solarization significantly reduced the incidence of lettuce drop in all three experiments (Table 1). Duration of treatment did not significantly affect disease incidence in the field, but in the greenhouse 30 days was better then 20 days. The results confirm those of Porter & Merriman (1985), Troilo et al. (1985), Phillips (1990) and Pereira et al. (1996) as to the effectiveness of solarization in controlling Sclerotinia sp. Adding poultry litter before solarization had no effect on the disease, although Gamiel & Stapleton (1993) have reported benefits obtained from organic matter incorporation before solar treatment.

Table 1. Effect of solarization on incidence of lettuce drop (Sclerotinia sp.) in the three experiments.

Field, 1998-1999 Field, 1999-2000 Greenhouse, 1999-2000Solarization

(days)% diseased

plantsSolarization

(days)% diseased

plantsSolarization

(days)% diseased

plants0 31.04 a* 0 28.33 a 0 5.42 a60 5.42 b 30 10.78 b 20 2.36 b90 5.02 b 45 7.62 b 30 0.78 c

30 + org.** 0.93 c* means followed by different letters in the same experiment are significantly different (P<0.05)** with poultry litter added prior to solarization

AcknowledgementsWe thank Mr. Carlos Ferrari and Mr. Paulo Camargo, the farmers who let us use their crops and participated in the experiments. We also thank the BSPP for financial support to allow V. Gepp to attend this Workshop.

ReferencesCassanello ME, Carrato AC, Franco J, 1990. Efecto de la solarización en almácigos de

brásicas. Abstracts of the III Congreso Nacional de Horticultura. Salto, Uruguay. p.19.Gamiel A, Stapleton JJ, 1993. Effect of chicken compost or ammonium phosphate and

solarization on pathogen control, rhizosphere microorganisms and lettuce growth. Plant Disease 77, 886-891.

Pereira JCR, Chaves GM, Zambolim L, Matsuoka K, Silva-Acuña R, Do Vale FXR, 1996. Controle integrado de Sclerotinia sclerotiorum. Fitopatología Brasileira 21, 254-260.

Phillips AJL,1990. The effects of soil solarization on sclerotial populations of Sclerotinia sclerotiorum. Plant Pathology 39, 38-43.

Porter IJ, Merriman PR, 1985. Evaluation of soil solarization for control of root disease of row crops in Victoria. Plant Pathology 34, 108-118.

Troilo E, Vannacci G, Scaramuzzi G, 1985. Possibilita di applicazione della solarizzazione del terreno in Italia: indagini sul binomio lattuga - Sclerotinia minor Jagger. La difesa delle piante 2, 127-140.

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Wounding of weeds enhances Sclerotinia sclerotiorum as a mycoherbicide

G. A. Hurrell and G. W. Bourdôt

AgResearch Limited, PO Box 60, Lincoln 8152, New Zealand

AbstractIn field studies a mycelium preparation of Sclerotinia sclerotiorum reduced the biomass productivity in wounded Cirsium arvense plants by a greater proportion than in intact plants. This interaction, while not evident in similarly treated Senecio jacobaea plants under glass, indicated that wounding had facilitated infection.

IntroductionS. sclerotiorum has potential as a mycoherbicide for the pasture weeds C. arvense and S. jacobaea but variable efficacy under field conditions prevents its general acceptance in weed control (Hurrell et al., 2001). Increased levels of S. sclerotiorum disease have been reported in peanut, cabbage, lettuce and brussel sprout when the leaf cuticle is breached by mechanical injury (Porter & Powell, 1978; Dillard & Cobb, 1995). In other species the cuticle, peripheral cortex and vascular tissues have proven to be effective barriers to infection by S. sclerotiorum (Lumsden & Dow, 1973; Green et al., 1998). These studies led us to consider that the low levels of disease in C. arvense and S. jacobaea that sometimes occur following applications of S. sclerotiorum may be attributable to the intactness of such barriers in these species. In a preliminary study the probability and extent of disease were greater when S. sclerotiorum was applied to crush wounds on C. arvense stems than when applied to intact stems (Hurrell and Bourdôt, unpublished data). In the current contribution the effects of wounding by crushing are examined further.

Materials and MethodsS. sclerotiorum used in two experiments was formulated as a water-miscible powder consisting of mycelial fragments, an organic food source and other additives. The first experiment was conducted in the summer of 1997-98 at Templeton, Canterbury, New Zealand, in a grazed pasture containing a uniform population of C. arvense. There were four treatments in a 2 x 2 factorial structure; (a) no wounding or S. sclerotiorum, control, (b) no wounding plus S. sclerotiorum (c) wounding without S. sclerotiorum, and (d) wounding plus S. sclerotiorum. Plots measuring 6 m x 1.5 m were replicated five times for treatments a, c & d and 10 times for treatment b in a randomised block arrangement. The treatments were imposed on December 8th 1997. Wounding was achieved by hitting all shoots with the back of a spade creating contusions on the stem and foliage. The formulated S. sclerotiorum was then applied using a low-pressure 1.5 m-wide boom equipped with “dripper” nozzles spaced 75 mm apart and calibrated to apply 120 litre ha-1. The application rate of 60 kg ha-1

of S. sclerotiorum was achieved by four passes of the boom giving a droplet density of 1400 droplets m-2 at an average droplet size of 34 µl. The effects of the treatments were measured by harvesting and drying the C. arvense shoots from two randomly located 0.25 m2 quadrats per plot on 27 April (autumn) 1998.The second experiment was conducted in the AgResearch glasshouse at Lincoln, Canterbury, using S. jacobaea plants grown from seed. On 3 February 1999 48 rosette plants averaging

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28 cm diameter were allocated to four treatments each with twelve replicate plants; (a) not wounded, no S. sclerotiorum, (b) not wounded plus S. sclerotiorum, (c) wounded plus oven-killed S. sclerotiorum and (d) wounded plus S. sclerotiorum. Wounding was achieved by pressing the base of a 50 mm diameter plant pot onto the centre of each rosette using a twisting motion to cause contusions to the leaves. Plants treated with S. sclerotiorum received a 10 ml dose of the formulation placed with a syringe onto the centre of the rosette. After application, the potted plants were transferred to metal trays enclosed within polythene-film covered tents to increase the humidity and promote the disease. Water was supplied continuously. On 22 March 1999, 7 weeks after treatment, the living foliage above ground was removed from each plant, dried and weighed.

Results and DiscussionReductions in C. arvense biomass due to S. sclerotiorum occurred despite the intermittent occurrence of externally derived moisture on leaves and stems, indicating that another source could have supported the infections, possibly exudates from the damaged tissue.In the absence of wounding or S. sclerotiorum the yield of the aerial shoots of C. arvense measured in the autumn was 188 g m-2. Wounding alone halved this yield. S. sclerotiorum reduced the yield by 26% in the absence of wounding and by 49% in the presence of wounding. This interaction was statistically significant (P < 0.10), providing support for the hypothesis that the infection process of S. sclerotiorum in C. arvense is limited by the intactness of leaf and/or stem cuticle. We conclude that wounding is a possible means of enhancing the efficacy of S. sclerotiorum as a mycoherbicide for C. arvense control in pastures.By contrast, wounding had no effect on growth in S. jacobaea as measured by leaf dry mass 7 weeks after treatment. S. sclerotiorum reduced the plant dry mass similarly regardless of wounding; the average reduction was 39%. The lack of an interaction contrasts with the result with C. arvense in the first experiment and suggests that the leaf and petiole cuticle may not have been a significant barrier to infection in this species.

AcknowledgementsThe authors wish to thank Dave Saville for helpful advice with statistical analyses and Christine Galbraith, for assistance in the field studies.

ReferencesDillard HR & Cobb AC, 1995. Relationship between leaf injury and colonization of cabbage

by Sclerotinia sclerotiorum. Crop Protection 14, 677-682.Green S, Gaunt RE, Harvey IC & Bourdôt GW, 1998. Histopathology of Ranunculus acris

infected by a mycoherbicide, Sclerotinia sclerotiorum. Australasian Plant Pathology 27, 73-79.

Hurrell GA, Bourdôt GW & Saville DJ, 2001. Effect of application time on the efficacy of Sclerotinia sclerotiorum as a mycoherbicide for Cirsium arvense control in pasture. Biocontrol Science and Technology 11, 317 -330.

Lumsden RD & Dow RL, 1973. Histopathology of Sclerotinia sclerotiorum infection in bean. Phytopathology 63, 708-715.

Porter DM & Powell NL, 1978. Sclerotinia blight development in peanut vines injured by tractor tires. Peanut Science 5, 87-90.

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Management of Sclerotinia rot in Indian mustard - An Integrated Approach

Saroj Singh

National Centre for Integrated Pest Management Pusa Campus, New Delhi, India –100012

AbstractIn recent past, Sclerotinia rot (Sclerotinia sclerotiorum L. De Bary) is emerging as a new threat to Indian mustard. It has made its appearance in four states of India from mild to moderate form causing yield loss up to 30-45%. An IPM module was made for the management of the disease. Timely sowing (last week of October -early November), cultural practices like field sanitation, burning of stubbles and deep ploughing followed by crop rotation with wet culture rice and seed treatment with benomyl (0.1%) followed by spray of 0.2% benomyl were proved to be effective in managing the disease. In India, there is no resistant variety available to manage this disease, so use of apetalous varieties for the development of disease resistant varieties can play an important role in disease management of the disease in future.

IntroductionOilseed Brassica, commonly referred to as rapeseed-mustard in India, comes next in importance to groundnut. It is one of the most important winter-season (rabi) oilseed crops of India. A number of pests attack the crop and cause enormous losses. The major diseases that cause severe damage to mustard are Alternaria blight (Alternaria brassicae (Berk.) Sacc. & A. brassicicola (Schw.) Wiltshire), white rust (Albugo candida (Pers. ex Fr.) Kuntz., downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr., powdery mildew (Erysiphe cruciferarum Opiz ex Junell) and Phyllody which could cause yield losses ranging from 10-75% depending upon favourable weather condition (Degenhardt et al. 1974; Saharan 1992 & 1998). Sclerotinia rot is emerging as a new threat to rapeseed – mustard and causes yield losses upto 30-45. The disease is of economic importance as yield losses reflect on yield reductions per infected plant and the percentage of infected plants in the crop.

Materials and MethodsAll the disease management practices from presowing to harvesting like host plant resistance, cultural, biological, chemical and mechanical were pooled into a single approach to synthesize an eco-friendly and bio-intensive IDM module for the management of the sclerotinia rot in mustard. This IPM module then validated in farmers' field at village Bhora Khurd (District Gurgaon : Haryana State) during 2000-2001.

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IDM ModuleCrop stage Management practicesPre Sowing Proper field sanitation, removal of debris from

previous crop, summer ploughing of the fields to kill the spores/ residual population of insect pests should be done.

Sowing Use of disease tolerant varieties, selection of sclerotia free seeds. Planting between 15-25 October optimum, early sowing should be avoided. Seed treatment with benomyl @ 0.1%.Planting on raised beds recommended, avoidance of narrow spacing, avoidance of heavy seed rate.

Seedling and vegetative stage Practices for reducing moisture retention in the canopy and promoting aeration to be adopted.Irrigation timing also play an important role hence minimum timely irrigation to be given.

Flowering stage Spraying the crop with fungicides such as benomyl or thiophanate methyl during flowering. Protection is necessary because of the petals play a critical role in infection, fungicide application to be done when most of the plants have reached 20-30% bloom.

Results and DiscussionIt was interesting to note that IDM plots were found less infested with sclerotinia rot besides the crop being in good condition. The disease was observed from 5-10% in IDM plots whereas in control plot it was 20-25%. Higher yield was obtained in IDM plots than corresponding control practices. Thus IDM practices are found to be better and eco-friendly.

Symptoms of the disease appeared from early to late season in the field and dense canopy provided better conditions for the disease development. It was observed that irrigation timing also play an important role in disease development. Developmental traits could also be useful in disease management potential benefits of apetalous cultivars (for example oilseed brassicas) recommended for use in disease management as apetalous cultivars yield more due to more light penetration in plant canopy and avoidance of Sclerotinia disease (Morrall et al. (1991).

Since sclerotia remain viable in the soil for at least 3 years infected fields should be planted with non susceptible crops such as cereals for at least 3 years before susceptible crops are grown again. Rotation with wet rice is possible under Indian conditions in the management of the disease (Kolte, 1985).

ReferencesKolte, S.J.(1985) Disease of annual Edible Oilseed Crops Vol. II-III, CRC Press. Boca Raton,

Florida, USA.Morrall, R.A.A. et al., (1991) Forecasting Sclerotinia stem rot of spring rapeseed by petal

testing In: Mc Gregor, DI (ed) Proc. 8th International Rapeseed Congress, Saskatoon, Canada, pp 483-488.

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Comparative antagonistic activity of Trichoderma harzianum, T. viride and Epicoccum purpurescens against Sclerotinia sclerotiorum causing white rot of brinjal.

Rama S. Singh and Jaspal Kaur

Department of Plant Pathology, Punjab Agricultural University, Ludhiana-141 001, India

AbstractTrichoderma harzianum (Th38) exhibited mycoparasitisim, while Epicoccum purpurescens (Ep5) showed antibiosis against Sclerotinia sclerotiorum in dual culture. T. viride (Tv34), first showed antibiosis, however on extended period of incubation parasitised pathogen colony. The non-volatile metabolites present in culture filtrate were found inhibitory for fungal growth of pathogen. The maximum, 50.9-68.3% growth inhibition was recorded at 50% culture filtrate (CF) concentration of E. purpurescens. The same CF concentration of T. viride and T. harzianum showed 41.4-66.7 and 32.6-41.1 % inhibition respectively.

IntroductionSclerotinia sclerotiorum is a soil borne plant pathogen and commonly cause disease in agricultural and horticultural crops. Sometime significant damage is reported in certain vegetables including brinjal (Bag, 2000). The disease is difficult to manage due to its soil borne nature and wide host range of the pathogen. Several species of Trichoderma have been reported as potential antagonist to reduce a number of soil borne plant pathogens (Papavizas, 1985). E. purpurescens also inhibit the infection of various soil borne and foliar pathogens (Singh, 1985). In general these antagonists act as mycoparasites, antibiosis, competition or sometimes combination of more than one mechanism to reduce infection and loss due to pathogen. In present study, the attempts have been made to find out the extent of variation in inhibition due to their non-volatile metabolites with three fungal antagonists.

Materials and MethodsThe three selected fungal antagonists i.e. T. harzianum (Th38), T. viride (Tv34) and E. purpurescens (Ep5) were selected to observe their antagonistic potential against S. scleroiorum causing white rot of brinjal. Primarily the antagonists were tested in dual culture technique. Further the efficacy of non-volatile metabolites present in the culture filtrate were studied by Poisoned Food technique. The culture filtrate of antagonists were incorporated in growth medium at 0, 10, 20, 30, 40, and 50% concentrations and colony growth inhibition of pathogen was recorded after 48,72,96, and 120 hours of incubation.

Results and DiscussionIn dual culture it was observed that T. harzianum showed mycoparasitism and overgrew on the S. sclerotiorum colonies within 4 days of incubation. T. viride first formed small, 1-2 mm zone of inhibition within 2 days of incubation. However, on extended period of incubation, 10-20% pathogen colony was covered and lysed by the growth of T. viride. Epurpurescens showed exclusively antibiosis by forming 3-5 mm zone of inhibition. Although T. harzianum showed mycoparasitism in dual culture, however, its culture filtrate incorporation in growth medium also inhibited colony growth of S. sclerotiorum. The 14.3% growth inhibition of pathogen was observed at 10% concentration of culture filtrate after 48 hours of incubation. The growth inhibition increase gradually with increased of culture filtrate

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concentration in growth medium and at 50% dilution of culture filtrate, the 57.1 % growth inhibition was

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observed. After 120 hours of incubation, only 10.9,13.0,19.6,26.1 & 32.6% growth inhibition recorded at 10,20,30,40 & 50% concentration of culture filtrate in growth medium (Table 1). The culture filtrate of T. viride gave higher percentage of growth inhibition as compared to T harzianum. The 26.7,33.3,50.0,46.7 & 67.7% colony growth inhibition of S. sclerotiorum was recorded at 10, 20, 30, 40 and 50% culture filtrate concentration. Finally, after 120 hours of incubation, 6.91- 41.4°/o inhibition was estimated at 10-50% dilution of culture filtrate concentration. Among the three antagonists the Epurpurescens showed maximum inhibition of mycelial growth at all level of culture filtrate incorporation in growth medium. There was 24.4% mycelial growth inhibition at 10% culture filtrate concentration, which gradually increased to 68.3% at 50% concentration of culture filtrate after 48 hours of incubation. The extent of inhibition was declined due to increase of incubation period and it was estimated that 16.9-47.9% at 10-50% concentration of culture filtrate after 120 hours of incubation.

Table 1: Effect of culture filtrate of selected fungal antagonists on colony growth inhibition (%) of Sclerotinia sclerotiorum causing stem rot of brinjal at variable incubation period.

CFConc.(%)

T. harzianum (Th38) T. viride (Tv34) E. purpurescens (Ep5)

Incubation Period (hours)

48 72 96 120 48 72 96 120 48 72 96 120

0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

10 14.3 17.9 12.0 10.9 26.7 19.2 11.4 6.9 24.4 20.0 12.3 16.9

20 21.4 23.1 16.0 13.0 33.3 23.1 17.1 13.9 29.3 26.0 14.0 23.9

30 28.6 28.2 20.0 19.6 40.0 30.8 20.0 20.9 43.9 30.0 24.6 25.4

40 42.9 30.8 26.1 24.0 46.7 33.3 28.0 30.2 43.9 32.0 29.8 32.4

50 57.1 41.1 36.0 32.6 66.7 53.0 40.0 41.1 68.3 56.0 50.9 47.9

Fig. 1 showed comparative inhibition of S. sclerotiorum colony with all 3 antagonists at 50% concentration of culture filtrate in growth medium and it was evident that maximum inhibition recorded due to culture filtrate of E. purpurescens followed by that of T. viride. T. harzianum showed minimum inhibition of pathogen growth.

Fig.1: Comparison of growth inhibition of S. sclerotium by 50% concentration of culture filtrate of three fungal antagonists.

ReferencesBag TK, 2000. Status ofvegetcrble diseases caused by Sclerotinia sclerotiorum in different

land use systems of Arunachal Pradesh. Environment and Ecology, 18, 88-91.Papavizas GC, 1985. Trichoderma and Gliocladium: biology, ecology and potential for the

biocontrol. Annual Review of Phytopathology, 23, 23-54.

57.166.7 68.3

41.1

53 56

36 4050.9

32.641.4

47.9

01020304050607080

T. harzianum T. viride E. purpurescens

Gro

wth

inhi

bitio

n (%

)

48 hours 72 hours 96 hours 120 hours

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Singh RS, 1985. Use of Epicoccum purpurescens as an antagonist against Macrophomina phaseolina and Co!letotrichum capsici. Indian E. purpurescens (Ep5) 38: 258-262.

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Evaluation of selected Trichoderma isoaltes against Sclerotinia sclerotiorum causing white rot of Brassica napus L.

A.Srinivasan, I.S.Kang, Rama S.Singh and Jaspal Kaur

Department of Plant Pathology, Punjab Agricultural University, Ludhiana-141001, INDIA

AbstractThe selected isolates of T harzianum (Th38) showed mycoparasitism, however, T. viride (Tv34) exhibited antibiosis and formed zone of inhibition in vitro evaluation. The culture filtrate of both isolates significantly reduced the colony growth of S. sclerotiorum on PDA medium. Under green house conditions the seed and soil inoculation of antagonists increased germination of Brassica napus and reduced disease incidence significantly than uninoculated control. Both the selected antagonists gave 100% inhibition of sclerotial germination of pathogen after 60 days of treatment.

IntroductionSclerotinia sclerotiorum, a soil borne plant pathogen is cosmopolitan in nature and causing white rot of a number of cultivated plants. Brassica napus is most susceptible and sometimes huge loss may occur due to S. sclerotiorum (Scarisbrick & Daniels, 1986). The pathogen is difficult to manage due to its soil borne nature and wide host range. Trichoderma spp. has been found a potential antagonist and minimises pathogen inoculum, hence reduce disease intensity in crop production (Papavizas, 1985; Chet, 1986). The present study has been taken up to evaluate two potential selected isolates, i.e. T. harzianum (Th38) and T viride (Tv34) for their biocontrol efficacy against white rot of B. napus.

Materials and MethodsT.harzianum (Th38) and T viride (Tv34) were already present in the biocontrol lab, Department of Plant Pathology, Punjab Agricultural University, Ludhiana. S. sclerotiorum was isolated from the diseased samples of B. napus. In vitro evaluation of antagonists was done by dual culture method, as well as by poisoned food technique. For green house experiments, sclerotia were inoculated in pot soil before sowing. The antagonists were inoculated as seed and soil treatments with charcoal based formulation. The percentage of seed germination and incidence of disease were recorded during the crop season.

Results and DiscussionIn dual culture T. viride showed 2-3 mm zone of inhibition against S. sclerotiorum. T harzianum showed mycoparasitism and covered 100% colony growth of pathogen within four days of inoculation. The non-volatile metabolites of Trichoderma spp. present in culture filtrate reduced significantly the colony growth of S. sclerotiorum. Various effects of both antagonists are summarised in Table 1. The 50% concentration of culture filtrate of T viride in growth medium inhibited 51.97, 69.48, 73.75 and 75.00 % colony growth of pathogen after 48, 72, 96, and 120 hours of incubation. The culture filtrate of T. harzianum also showed significant inhibition for the colony growth of pathogen. The corresponding figure for the T harzianum was record 48.68, 66,50, 62.94 and 65.33% after 48, 72, 96, and 120

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hours of incubation. It was evident that the culture filtrate of T. viride was more inhibitory as compared to T. harzianum. The presence of culture filtrate of both the antagonists also inhibited sclerotia formation on agar medium as compared to control. There were 60.74 and 48.21 % inhibition of sclerotial production due to presence of 50% culture filtrate of T. viride and T. harzianum respectively. In green house experiment, the seed treatment with T. viride and T. harzianum enhanced 53-68% and 46-70.4% seed germination over control respectively. Similarly the soil inoculation of the two antagonists gave 46.8-71.48% and 50.1-70.44% increased germination over control. The seed treatment with T. viride gave 44.29-63.57% disease inhibition while the T. harzianum seed treatment provided 57,15-74.29% inhibition of disease development. The soil application of antagonists also gave similar results. There was 57.15-80.71% as well as 65.71-91.42% disease inhibition due to soil application of T. viride and T. harzianum respectively. Although the both the antagonists gave significant protection against the infection of S. sclerotiorum, however, it was evident that the application of T. harzianum gave better protection against white rot of B. napus. The efficacy of Trichoderma spp. has also been recognised against S. sclerotiorum causing disease in rape seed and mustard crop (Singh, 1998)

Table 1: Effect of selected isolates of T.viride and T.harzianum on S. sclerotiorum causing white rot of B.napus

Observations Effects of antagonist

T. viride T. harzianumColony growth inhibition* 51.97-75.0 48.68-65.33Sclerotia production * 60.74 48.21Increased germination ofB. napus due to:

(a) Seed treatment 53.0-68.01 46.80-70.44(b) Soil treatment 46.8-71.48 50.12-70.44

Disease inhibition (%)(a) Seed treatment 44.29-63.57 57.15-74.29(b) Soil treatment 57.15-80.71 65.17-91.42

*At 50% culture filtrate concentration in growth medium

ReferencesChet, 1, 1986.Trichoderma - application, mode of action, and potential as biocontrol agent

of soil borne plant pathogenic fungi. IN Chet, 1.(Ed.) Innovative approaches to plant disease control. John Wiley and Sons, New York, pp. 137-160.

Papavizas, G.C. 1985. Trichoderma and Gliocladium: biology, ecology and potential for biocontrol. Annual Review of Phytopatholgy 23, 23-54.

Scarisbrick, D.H. and Daniels, R.W. 1986. Oilseed Rape. Pp 309. Collins Professional and Technical Books. William Collins Sons and Co. Ltd. London.

Singh, Y. 1998. Biological control of sclerotinia rot of rapeseed and mustard caused by Sclerotinia scleroitorum. Plant Disease Research 13, 144-46.

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Biological control of Sclerotinia diseases of vegetables using Coniothyrium minitans A69

A. Stewarta, N. Rabeendrana, I. J. Porterb, T. M. Launonenc and J. Huntd

aSoil, Plant & Ecological Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand; bInstitute for Horticultural Development, Agriculture Victoria, 621 Burwood Hwy, Knoxfield, 3180, Victoria, Australia; cDepartment of Botany, School of Science and Technology, Latrobe University, Bundoora, 3083, Victoria, Australia; and d Agrimm Technologies Ltd, P.O. Box 13245, Christchurch, New Zealand

AbstractConiothyrium minitans A69 has given effective and reproducible control (60-85%) of Sclerotinia diseases of lettuce, cabbage and bean. Best results were obtained when the biocontrol fungus was incorporated into the transplant potting mix (106 spores/g potting medium) and/or applied as a soil amendment at the time of planting (106 spores/g soil). Disease control was equal to or better than the standard fungicide (carbendazim) treatment.

IntroductionConiothyrium minitans is a specialised mycoparasite of Sclerotinia spp. and has been reported to control Sclerotinia disease in both glasshouse and field trials (Budge et al., 1995). A New Zealand isolate of C. minitans A69 (Jones & Stewart, 2000) was shown to have good activity against Sclerotinia diseases, in particular, excellent control was achieved against S. minor on lettuce. In this paper, we describe the results of further field evaluations of C. minitans A69 for control of Sclerotinia diseases of lettuce, cabbage and bean.

Materials and MethodsResearch field trials 1998-2000 - Six trials were conducted to assess the effectiveness of C. minitans A69 in controlling S. minor disease of lettuce and S. sclerotiorum disease of cabbage and bean. There were four treatments; untreated control, standard chemical treatment (carbendazim soil drench at transplanting plus two foliar sprays), C. minitans A69 applied in the transplant potting mix (106 spores/g substrate), C. minitans A69 in maize perlite substrate (106 cfu/g) incoporated into the soil at planting (to 5 cm depth) at a rate of 40kg/ha. Disease was assessed at weekly intervals throughout the growing season and yield data taken at harvest. Data was analysed by ANOVA and differences between treatments compared using Fishers LSD (P=0.05).Commercial trials 2000-2001 - Two field trials evaluated C. minitans A69 for control of S. minor lettuce drop using commercial formulations prepared by Agrimm Technologies Ltd. C. minitans A69 was applied as a combined transplant potting mix treatment and soil incorporation treatment at planting. Disease comparisons were made with an untreated control and standard fungicide (carbendazim) treatment.

Results and DiscussionA summary of the results of the six field trials is given in Table 1. Incorporation of C. minitans A69 into the transplant potting mix was an effective method of delivering the biocontrol agent into the root region of the transplant seedlings prior to planting out in the

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field. Better disease control was achieved using this method (60-85%) compared to the soil incorporation method (40-60%). However, the soil incorporation method still provided excellent control (75%) of Sclerotinia disease in the direct seeded bean trial. Soil monitoring studies showed that best disease control was achieved when the population levels of C. minitans A69 were maintained at 105-106 cfu/g soil.

Table 1: Effect of Coniothyrium minitans A69 on Sclerotinia disease of cabbage, bean and lettuce.

% Sclerotinia diseaseTreatment Cabbage Bean Lettuce

Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2Untreated 49.2 a1 35.0 a 23.8 a 49.9 a 77.2 a 49.2 aFungicide - - 12.8 b 5.0 c 40.0 bc 28.4 bcC. min t/p 8.1 c 15.1 b - - 26.1 c 23.8 cC. min soil 20.3 b 18.2 b 5.3 c 20.5 b 46.2 b 36.2 b1 values followed by the same letter within a column are not significantly different at P<0.05

Under commercial growing conditions, a combined transplant and soil incorporation treatment gave a high level of control of Sclerotinia lettuce drop (Table 2). This control was significantly better than the standard fungicide and resulted in a significant increase in yield. Even when disease levels were low and no biocontrol effects could be detected, the C. minitans treatment resulted in a higher proportion of grade 1 lettuce. Low disease levels were also experienced in lettuce trials conducted in Victoria, Australia. However, similar trends were observed with the transplant application of C. minitans giving approx. 50% control.

Table 2: Effect of Coniothyrium minitans A69 on Sclerotinia infection and yield of lettuce. Trial 1 Trial 2

Treatment % Disease Yield kg/plot % Disease % Grade 1Untreated 28.5 a1 43.4 a 3.7 a 25Fungicide 15.2 b 45.7 a 1.0 a 55C. min (t/p+soil) 6.5 c 60.3 b 1.4 a 751 values followed by the same letter within a column are not significantly different at P<0.05

Our research has shown that C. minitans A69 can provide effective control of Sclerotinia diseases on a range of vegetable crops. Further commercial testing is currently in progress at different sites in NZ and Australia.

AcknowledgementsResearch supported by grants from the NZ Foundation for Research Science and Technology, the New Zealand Vegetable and Potato Growers Federation and Horticulture Australia Ltd.

ReferencesBudge SP, McQuilken MP, Fenlon JS, Whipps JM, 1995. Use of Coniothyrium minitans and

Gliocladium virens for biological control of Sclerotinia sclerotiorum in glasshouse lettuce. Biological Control 5, 513-522.

Jones EE, Stewart A, 2000. Selection of mycoparasites of sclerotia of Sclerotinia sclerotiorum isolated from New Zealand soils. New Zealand Journal of Crop and Horticultural Science 28, 105-114.

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Control of sclerotinia stem rot of canola by aerial application of Coniothyrium minitans

Guoqing Li1; Shanjun Wei1; Daohong Jiang1 and H. C. Huang2

1Department of Plant Protection, Huazhong Agricultural University, Wuhan, China, 430070;2 Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada

AbstractIndoor and filed experiments were carried out to study the control of sclerotinia rot of canola caused by Sclerotinia sclerotiorum (Ss) by aerial applications of Coniothyrium minitans (Cm) in central China. In one indoor experiment, canola petals were inoculated with ascospores o Ss (5.4´103 spores/petal) alone or Ss plus Cm containing 38,3.8´102, 3.8´103 or 3.8´104

pycnidiospores/petal. The petals were placed on detached canola leaves, 1 petal/leaf, and incubated at 20°C for 6 days. Results showed that compared to the control, treatment of Cm significantly (p<0.05) reduced the lesion size by 66.6% and 89.7% at the spore concentrations of 3.8´103 and 3.8´10 4, respectively. In another experiment, canola leaves were sprayed with water or a spore suspension of Cm at 5.0´103, 5.0´104, 5.0´105, 5.0´106 and 5.0´107

pycnidiospores/ml, 8ml/leaf, and each leaf was inoculated with a petal containing Ss at 5.8´103 spores/petal. Results showed that a significant (p<0.05) reduction of lesion size by Cm treatments at 5.0´105 pycnidiospores/ml or higher. Microscopic examinations indicated that Cm could not inhibit germination of ascospore of Ss on canola petals during the first 48 hrs of incubation. But it caused aggregation of cytoplasma and lysis of cell wall of Ss. This suggests that the cell wall degrading enzymes of Cm may be important in suppression of hyphal growth and infection of Ss on canola petals. Field experiments were conducted in 1996-1997 and 1998-1999 by spraying water or Cm (5.9´108 spores/m2) on canola plants three times at 10-day intervals, starting at the early bloom stage (20-30% plants flowered). Result showed that compared to the control, the disease severity was reduced by 75.0% in 1996-1997 and 47.8% in 1998-1999 for the Cm-treatment. Sclerotia formed on diseased plants were infected by Cm and the infection rate was high (>67.9%) in Cm-treated plots, but was low (<6.8%) in control plots. This study suggests that aerial application of C. minitans was effective in reducing severity of sclerotinia rot of canola as well as the survival of sclerotia produced on diseased plants.

P3.14

Suppression of apothecial formation in Sclerotinia sclerotiorum by bacteria

Xinmei Feng and Christian Thaning

Plant Pathology & Biocontrol Unit, SLU, P. O. Box 7035, SE 750 07 Uppsala, Sweden.

AbstractBacteria isolated from lab-produced sclerotia baits, plant roots and field-collected sclerotia and apothecia after surface-sterilization were tested for their effects on apothecial formation in Sclerotinia sclerotiorum in a bioassay. Out of 235 isolates from inside of field-collected sclerotia and apothecia, seven completely suppressed carpogenic germination of sclerotia. Nine out of 300 isolates from sclerotia baits and plant roots also significantly suppressed apothecial formation, one of them, a Serratia plymuthica isolate, A 153, not only completely suppressed apothecial formation in the bioassay, but also showed strong inhibition of carpogenic germination in field experiments when applied to the soil surface one week before sowing oil-seed rape as model plant.

IntroductionThe widely distributed, soil-borne plant pathogen S. sclerotiorum is economically important in many dicotelydonous crops. Its wide host range in combination with its persistent resting structures, the sclerotia, makes it difficult to control. In attempts to develop biocontrol regimes, it has been repeatedly shown that many fungi parasitize the sclerotia and thus strongly influenced their viability (Turner, 1975; Dos Santos, 1982; Phillips, 1989; Adams, 1990; Whipps, 1990), but the role of bacteria in this aspect is not well understood. The bacteria might not only break down the sclerotia but also interfere with their carpogenic germination (Thaning, 2000). In this study a number of bacteria were screened for suppressive ability to the apothecial development.

Materials and methodsSclerotia used in this experiment were produced by growing on autoclaved wheat, and preconditioned until producing stipes before being used in biotests (Thaning, 2000). For testing bacterial affects on apothecial formation, ten even sized (5 to 8 mm) preconditioned sclerotia were placed on the top of one cm depth of a non-sterile commercial peat sand mixture (Hasselfors AB, Sweden; 80 % peat and 20 % sand) in a petri dish (9 cm in diameter), covered with an additional half centimeter of the same soil. Five ml of two-day-old bacterial cultures (in TSA medium) were evenly applied over the soil surface of each petri-dish. For controls, the same volume of sterile tap water was used. The water potential of the soil was finally adjusted to about -0.1 bars, and the petri dishes were sealed with parafilm and placed at a distance of 40 cm under fluorescent tubes (True-Lite 58 W, Rätt Ljus AB, Bromma, Sweden and Duro-Test International Inc., Fairfield, NJ, USA) at a temperature of 18°C and a photoperiod of 16 hours with 50 µE of light m -2s-1. The results were estimated by counting the number of stipes and apothecia periodically. The 235 bacterial isolates from surface-sterilized field-collected sclerotia and apothecia, 280 isolates from roots of cultivated and wild plants, and 20 isolates from lab-produced sclerotial baits that had been buried in both cultivated and non-cultivated soils were tested.Field experiments were carried out in randomized block design. Each plot was artificially infested with S. sclerotiorum by adding 200 sclerotia /m2 and mixing thoroughly to a depth of

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5 cm one month before sowing oilseed rape. Three treatment were included: A, no bacteria control; B, bacteria applied one week before sowing oilseed rape; and C, bacteria applied when first apothecium was observed. Application of bacteria was 1x107 cells/cm2 soil surface. Bacterial effect was read as number of apothecia produced per plot.

Results and discussionOut of 235 bacterial strains, 7 completely suppressed the formation of apothecia, 128 showed significant inhibition and 1 promoted the formation of apothecia. Nine out of 300 bacterial strains also showed suppression of apothecial production to different extents, one of them, A 153 identified as S. plymuthica, was able to completely suppress apothecial formation in bioassay. In field experiments, application of S. plymuthica, A153, one week before sowing plants, significantly reduced the production of apothecia, while bacterial application six weeks after sowing was not effective. The results give evidence that bacteria with ability to inhibit carpogenic germination of sclerotia largely existed in soil and inside sclerotia and most of these bacteria form endospore, it is in agreement with the findings of Zazzerini (1987) and Abd-Elrazik (1985). To our opinion, the reason can be that they can survive in unfavorable condition as sclerotia. It was observed in field experiment the suppression was dissimilar when bacteria were applied at different time, therefore sclerotia in different stages should be tested in bioassay and also more detail experiments should be carried out to find out the right application time of bacteria in field.

AcknowledgementsThis study was supported by the Foundation for Strategic Environmental Research (MISTRA). We thank Mrs. M. Johansson for supplying part bacterial isolates and Mrs. B-M. Jingström for technical assistance.

ReferencesAbd-Elrazik AA, Ei-Shabrawy AM, Sellam MA, Abd-Elrehim, 1985. Effectiveness of certain

fungi and bacteria associated with sclerotia of Sclerotium cepivorum in upper Egypt soil on controlling white rot of onion. Egyptian Journal of Phytopathology 17 (2), 107-114.

Adams PB, Fravel DR, 1990. Economical biological control of Sclerotinia lettuce Drop by Sporidesmium sclerotivorum. Phytopathology 80 (10), 1120-1124.

Dos Santos AF, Dhingra OD, 1982. Pathogenicity of Trichoderma spp. On the sclerotia of Sclerotinia sclerotiorum . Canadian Journal of Botony 60, 472-475.

Phillips AJL, 1989. Fungi associated with sclerotia of Sclerotinia sclerotiorum in South Africa and their effects on the pathogen. Phytophylactica 21, 135-139.

Thaning C, 2000. Ways of managing sclerotinia sclerotiorum inoculum (Ph.D thesis).Turner GJ, Tribe HT, 1975. Preliminary field plot trials on control of Sclerotinia trifoliorum

by Coniothyrium minitans. Plant Pathology 24, 109-113.Whipps JM, Budge SP, 1990. Screening for sclerotial mycoparasites of Sclerotinia

sclerotiorum. Mycological Research 94, 697-700Zazzerini A, Tosi L, 1987. Antsgonistic effects of Bacillus spp. On Sclerotinia sclerotiorum

sclerotia. Phytopathology Medit 26, 185-187.

4. RESISTANCE

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Potential new sources of resistance to white mould in the Phaseolus core collections K. F. Graftona, J. B. Rasmussenb, J. R. Steadmanc and C. Donohueb.

aPlant Sci. Dept.; bPlant Pathology Dept., North Dakota State University, Fargo, ND 58105; cPlant Pathology Dept., University of Nebraska, Lincoln, NE 68583.

AbstractWhite mold, caused by Sclerotinia sclerotiorum, is a serious disease on common bean (Phaseolus vulgaris L.). Sources of genetic resistance are limited, which has hampered development of resistant cultivars. Using two greenhouse screening tests, we evaluated the two core collections of the USDA Phaseolus Plant Introductions to identify new sources of resistance.

IntroductionWhite mold is a serious disease on common bean in temperate regions of the world. Schwartz & Steadman (1989) indicated that Nebraska dry bean crop losses averaged 30%, with individual field losses as high as 92%, because of white mould. Control of white mould is difficult, with some growers relying on timely application of benzimidazole fungicides. Efficacy of these fungicides may be hampered by wet weather, which would favor development and spread of the disease. Genetic resistance and disease avoidance due to plant structure have both been identified as possible mechanisms to reduce white mould damage in dry bean. Miklas & Grafton (1992) reported that partial resistance to white mould is probably controlled by several genes. Complete resistance is unknown and the dearth of unique sources of resistance has hampered developing resistant cultivars. Miklas et al. (1998) evaluated a portion (89 of 182 entries) of the Central and South American (CASA) core of the USDA Phaseolus Plant Introduction collection in an attempt to identify new sources of putative resistance. We expanded their initial evaluation to include all entries of both the Mexico (MEX) and the CASA cores and used both the straw test and the detached leaf assay.

Materials and MethodsThe two core collections were evaluated separately in Fargo, ND, and in Lincoln, NE, with different assays performed at each location. At Fargo, PI’s comprising each of the core collections were evaluated for reaction to white mold with the straw test (Petzoldt & Dickson, 1996). Ten plants, each representing a replicate in an RCBD, of each PI were grown in a greenhouse at 22-34oC with a 14-hr photoperiod. Plants were inoculated 28 d post planting using agar plugs of S. sclerotiorum mycelium. Plants were evaluated as described by Petzoldt & Dickson (1996). The cores were grown in a greenhouse in Lincoln for evaluation using the detached leaf assay as outlined by Steadman et al. (1997). The youngest, fully expanded trifoliolate leaf of each plant was excised, placed in rubber-stoppered tubes filled with sterile distilled water, and inoculated using one agar plug from the advancing mycelial margin of a culture of S. sclerotiorum placed near the center of the middle leaflet. Inoculated leaves were placed in aluminum pans lined with wet paper toweling and covered with plastic food wrap to

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create a humid environment and incubated at 22oC for 48 hr. Lesion size (cm2) was calculated. `Othello’ pinto was used as a susceptible check in all tests.

Results and DiscussionBoth core collections, when tested using the straw test, had normal frequency distributions. A high number of lines scored <5, indicating resistance; however, the relatively warm temperatures in the greenhouse during these tests were sometimes above the maximum limit for disease development (300 C). Othello pinto exhibits plant death in a normal straw test, but it exhibited a less susceptible reaction in these tests, suggesting the relatively warm environment hampered disease development. Also, since PI lines are heterogeneous, there was variability within many PI lines for white mold reaction. Eight lines were selected using results of the straw test, with comparative data from the detached leaf test. In all cases, these PI lines had scores indicating a high level of resistance, with the disease progressing to, but not beyond, the first node. However, these lines scored similar to, or only slightly more resistant than, Othello when tested in the detached leaf assay. These discrepancies may occur because the two methods may be testing different components (leaves vs. stems) of physiological resistance to white mould. Also, eight PI lines from the MEX core were selected on the basis of performance in the detached leaf test. Again, scores were much lower than the susceptible check; in the cases of PI 318695 and PI 319683, scores were lower than the check in both tests. Further evaluation of these putative resistant lines is planned.The best lines from the CASA had straw test scores that were lower than scores for the same lines in the Miklas et al, (1999) test, possibly because of the warmer temperatures in the greenhouse, or possibly due to S. sclerotiorum isolate differences. Although Othello scored as highly susceptible, the warmer temperatures may have retarded disease development in lines with partial resistance. We were able to identify several lines that were classified as resistant in white mold tests. These lines may be used as parents in breeding for improved partial resistance to this important pathogen. Work to verify these reactions is continuing.The value of the core collection is that it offers the possibility of testing the variability that exists in the entire PI collection while keeping entry numbers to a manageable level. Evaluating the two core collections may provide some insight as to the existence of new sources of resistance to white mold. As we identify and verify new sources of resistance, we can then expand the search to PI lines that were collected in the same geographical region.

ReferencesMiklas PN, Delorme R, Hannon R, Dickson MH, 1999. Using a subsample of the core

collection to identify new sources of resistance to white mold in common bean. Crop Sci. 39,569-573.

Miklas PN, Grafton KF, 1992. Inheritance of partial resistance to white mold in inbred populations of dry bean. Crop Sci. 32, 943-948.

Petzoldt, R., and M. H. Dickson. 1996. Straw test for resistance to white mold in beans. Annu. Rep. Bean Improv. Coop. 39, 142-143.

Schwartz H F, Steadman JR, 1989. White mold. In HH Schwartz & M Pastor-Corrales (eds.) Bean Production Problems in the Tropics, 2nd Ed. CIAT, Cali, Colombia.

Steadman, JR, Powers K, Higgins B, 1997. Screening common bean for white mold resistance using detached leaves. Annu. Rep. Bean Improv. Coop. 40, 140-141.

P4.2

Light sensitivity of quantitative resistance in soybean to white mold caused by Sclerotinia sclerotiorum.

B. W. Pennypacker

Department of Agronomy, The Pennsylvania State University, University Park, PA. 16802, U. S. A.

AbstractGreenhouse studies demonstrated light sensitivity and a need for photosynthate in white mold resistance. Field experiments tested the effect of row spacing and planting density on white mold in soybean. Low-density soybeans had more leaves, greater photosynthetic capacity, and fewer main stem lesions. Field and greenhouse results agree.

IntroductionSclerotinia stem rot of soybean (Glycine max) is caused by Sclerotinia sclerotiorum. White mold, as it is commonly known, is a serious threat to soybean production in the northern U.S. when the weather is cool and wet. Fungicides are frequently used to control white mold in high value crops. Unfortunately, fungicides are not cost-effective on soybean. In agronomic crops such as soybeans, resistant cultivars are the primary means of controlling plant diseases. White mold resistance in soybean is quantitative (Kim & Diers, 2000) and thus is sensitive to environmental factors. Unfortunately, when environmental factors such as temperature and moisture favor a severe outbreak of white mold, quantitative resistance is overcome. In an effort to better understand the physiological mechanisms of quantitative resistance to Sclerotinia sclerotiorum, greenhouse light experiments were conducted (Pennypacker & Risius,1999).

Those studies found that quantitative resistance to white mold in soybean is sensitive to the photon flux density (PFD) of photosynthetically active radiation (PAR). Greater PFD is accompanied by a higher expression of resistance, whereas plants under low PFD are more susceptible to white mold (Pennypacker & Risius 1999). Photosynthesis is directly affected by PAR; the higher the PFD of PAR, the greater the rate of carbon assimilation and photosynthate production. The relationship between PFD and expression of resistance to S. sclerotiorum indicates that photosynthate is required for expression of quantitative resistance. Environmental factors that reduce photosynthesis may affect expression of quantitative resistance (Pennypacker, 2000). Agronomic management practices are being examined for their potential to reduce the impact of white mold on soybean yield. This studies objective was to determine whether altering whole plant carbon assimilation by manipulating leaf number per plant through row spacing and planting density effects the incidence and severity of white mold under field conditions.

Materials and MethodsField experiments were conducted in 1999 and 2000 to test the effect of row width (35, 53 and 71 cm) and planting density (370,370 and 185,185 plants/ha) on the incidence and severity of white mold in soybean. Each experiment was a split-plot randomized complete block with a factorial treatment arrangement and 5 replications. The experiments were planted with a precision planter to produce the desired planting density. Final row spacings were achieved

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by hoeing after plant emergence. In late July, healthy plants were harvested from each treatment, separated into leaves and stems, and dry weights were determined. In late August and early September disease incidence and location of the white mold lesions on the plants were recorded. Statistical analyses included an ANOVA of the disease rating data with rep*density as the error term for plant density. Only replications with white mold were used in the data analysis. Regression analysis was used to determine whether there was a relationship between healthy leaf dry weight in late July and incidence or position of white mold lesions.

Results and DiscussionRegression analysis of healthy leaf dry weight against incidence and location of white mold lesions in the 1999 experiment found a significant (P=0.003) negative relationship (r2 = 0.59). The incidence of white mold decreased as the leaf weight of the plants increased. The experiment was repeated in 2000 with similar results. In 2000, there was a significant (P=0.03) negative relationship (r2 = 0.59) between healthy leaf weight and the location of white mold lesions on the plant. Lethal main stem lesions were more prevalent when leaf weight per plant was low, which occurs when plants are grown in narrow rows or at high plant densities. Plants grown under low plant density have greater leaf weight, more metabolic machinery, and a higher photosynthetic capacity per plant (Charles-Edwards, 1986). The field experiments indicate that soybean plants with greater photosynthetic capacity are more likely to express quantitative resistance to white mold. These results also provide some insight into why quantitative resistance fails under heavy white mold pressure. Environmental conditions that favor white mold often coincide with significant cloud cover and reduced PFD, which in turn reduces the expression of quantitative resistance. The field results agree with the greenhouse experiments (Pennypacker & Risius, 1999) that demonstrated the light sensitivity of quantitative resistance to white mold.

AcknowledgementsThis research was supported by grants from The Pennsylvania and Northeast Soybean Promotion Boards. Special thanks to Ken Eck and Bill Wheler, for allowing this research to be conducted on their farms and to Dr. Marvin Risius, Maaike Broos, Rob Dickerson, Buck Fetzer, Wuxing Li, Brent Spangler, Kate Spangler and Ashley Spotts for invaluable assistance.

ReferencesCharles-Edwards DA, 1986. Modeling Plant Growth and Development. Academic Press, NYKim H S, Diers B W, 2000. Inheritance of partial resistance to sclerotinia stem rot in

soybean. Crop Science 40, 55-61.Pennypacker BW, 2000. Differential impact of carbon assimilation o the expression of

quantitative and qualitative resistance in alfalfa (Medicago sativa). Physiological and Molecular Plant Pathology 57, 87-93.

Pennypacker BW, Risius M L, 1999. Environmental sensitivity of soybean cultivar response to Sclerotinia sclerotiorum. Phytopathology 89, 618-622.

P4.3

Sclerotinia minor from peanut: Isolate aggressiveness and host resistance on detached leaves

B.B. Shew and J.E. Hollowell

Department of Plant Pathology, North Carolina State University, Box 7616, Raleigh, NC USA 27695-7616

AbstractA detached leaf method was developed for inoculation of peanut (Arachis hypogaea) with Sclerotinia minor. Peanut lines differed in resistance and isolates in aggressiveness, as measured by lesion length. No isolate x host specificity was found. The relationship between oxalic acid production in vitro and isolate aggressiveness was evaluated. IntroductionSclerotinia blight, caused by S. minor, is a serious disease of peanut. The fungus infects leaves, stems, pods and pegs when cool, moist conditions persist within the canopy. Field selection does not distinguish between disease resistance and disease avoidance, which is based on reduced plant contact with infested soil and a more open canopy. Unfortunately, characters contributing to avoidance (sparse foliage, low pod numbers, and upright growth habit) may reduce yield potential. Rapid, simple, and reliable methods are needed to identify disease resistance, as distinguished from avoidance. We adapted detached leaf inoculation (Pratt, 1996; Steadman et al., 1997) to evaluate resistance to Sclerotinia blight in peanut, characterise isolate aggressiveness, and check for isolate x host specificity. The relationship between oxalic acid production and isolate aggressiveness also was examined.Materials and MethodsAll experiments were conducted twice and data pooled for analysis. Resistance was evaluated on 12 entries. Germplasm lines NC-GP WS 12 and NC-GP WS 15 were derived from a cross with A. cardenasii. The cultivars Tamrun 98, VA 93B, VA 98R and Perry have field resistance to Sclerotinia blight and NC 7 is susceptible. Advanced breeding lines N91026E, N96009C, N92056C, N96074L, and N96076L were included. After plants were grown in the greenhouse 8 weeks, the second fully expanded leaf was detached from the main stem. A 19 x 16 x 4 cm plastic box was filled with 275 cm3 sand, water was added, and a wire screen was placed on the sand. Ten leaflets per entry were arranged, adaxial sides up, in each box. Sclerotinia minor was cultured on potato dextrose agar, and a plug 4 mm diam was placed in the centre of each leaflet, mycelium side down. Lesion lengths were measured after 2 and 3 days of incubation in the dark at 20°C. Four randomised complete blocks were used in the experiment.Forty-eight isolates of S. minor were collected from peanut and evaluated for aggressiveness on detached leaflets of NC 7. Isolates were inoculated on a single leaflet per replicate and isolates were arranged in ten randomised complete blocks.Based on the results of the resistance and aggressiveness trials, five peanut lines representing a range of resistance to S. minor (NC-GP WS12, N92056C, N91026E, Perry, NC 7) were grown for 9 weeks in the greenhouse and field. A standard isolate (58), two aggressive isolates (13, 20), one intermediate isolate (22), and two unaggressive isolates (24, 42) were individually inoculated on leaves detached from each line and source (field or greenhouse). The experimental design was a split-split plot with isolates as whole plots, source as subplots, and 4 blocks (replications).

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Oxalic acid production was quantified in vitro for nine isolates grown for 2 and 3 days in 25 ml potato dextrose broth. Controls were broth without cultures. Mycelium was removed from the cultures by filtration, dried at 35 C for 48 h, and weighed. Culture filtrates were assayed with a commercial kit consisting of an oxalate oxidase and colour indicators for the conversion of oxalate to hydrogen peroxide (Sigma). Colour intensities were measured in a spectrophotometer at 590 nm and standard curves were used to calculate concentrations in mmol/L/mg of mycelium. Each isolate was replicated twice.Results and DiscussionDetached leaf inoculations consistently resulted in infections and gave results within 3 days. Significant differences were found among entries, with mean lesion length ranging from 11 mm on NC-GP WS 12 to 24 mm on Perry. The mean lesion length across all entries was 19 mm. The longest lesions were found on Perry, Tamrun 98 (23 mm) and VA 98R (23 mm), indicating that field resistance in these cultivars may be related to disease avoidance. Resistance to Sclerotinia blight previously was found with detached stem inoculations (Melouk et al, 1992; Chappell et al, 1995). The detached leaf method was simple, rapid, used little plant material, and gave consistent results.The 48 isolates differed significantly in aggressiveness. Three days after inoculation, mean lesion length was15 mm. The most frequently observed (n = 19) mean lesion lengths were 15-18 mm. Isolate 13 was most aggressive, with a mean lesion length of 24 mm. Four isolates produced lesions <3 mm long on average and isolate 24 was least aggressive, with a mean lesion length of 2 mm. Leaf source effects were not consistent, but NC-GP WS 12 was ranked as resistant regardless of leaf source or isolate. Isolate and line main and interaction effects were significant. The interaction was attributed to the inability of unaggressive isolates (e.g., 24 and 42) to discriminate among lines and not to host x isolate specificity.The isolates tested differed significantly in oxalic acid production. Isolate aggressiveness as measured by lesion length was correlated with mycelial dry weight (r > .96) and mmol/L oxalate, but not with mmol oxalate/L/mg of mycelium.AcknowledgementsWe thank Dr. Marc Cubeta and Mr. Brian Cody for their advice and Ms. Monica Smith for technical assistance. Dr. Thomas Isleib kindly provided peanut breeding lines and North Carolina State University Field Faculty assisted in isolate collection. ReferencesChappell GF II, Shew BB, Ferguson JM, Beute, MK, 1995. Mechanisms of resistance to

Sclerotinia minor in selected peanut genotypes. Crop Science 35, 692-696.Melouk HA, Akeem CN, Bowen C, 1992. A detached shoot technique to evaluate the reaction of

peanut genotypes to Sclerotinia minor. Peanut Science 19, 58-62.Pratt RG, 1996. Screening for resistance to Sclerotinia trifoliorum in alfalfa by inoculation of

excised leaf tissue. Phytopathology 86, 923-928.Steadman JR, Powers K, Higgins B, 1997. Screening common bean for white mold resistance

using detached leaves. Annual Report of the Bean Improvement Cooperative 40, 140-141.

P4.4

Defense gene expression in Sclerotinia infected susceptible and resistant soybean cultivars.

A. Dorrancea, M. Grahamb and T. Grahamb

a Department of Plant Pathology, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691 USA; bDepartment of Plant Pathology, The Ohio State University, Kottman Hall, 2021 Coffey Rd., Columbus, OH 43210 USA

AbstractDefense gene expression, following infection by Sclerotinia sclerotiorum, of a susceptible (Williams 82) and a partially resistant cultivar (S-1990) are being compared for both secondary product and pathogenesis related protein genes. Initial results indicate a higher expression of certain PR proteins in the resistant compared to the susceptible cultivar.

IntroductionSclerotinia sclerotiorum, which causes stem rot, is an economically important pathogen of soybean in Ohio. Yield losses ranging from 5 to 25% have been recorded at periodic intervals since the early 1980's. This soybean disease is best managed by planting cultivars with resistance. Resistance to S. sclerotiorum is a type of partial resistance, in that fewer plants develop Sclerotinia stem rot and stem lesions are smaller compared to more susceptible cultivars. Biochemical mechanisms of partial resistance to S. sclerotiorum are unknown at this time. However, fairly extensive phenylpropanoid (Graham, 1994) and some limited PR gene responses (Tkeuchi et al, 1990; Quotob et al, 2000) have been examined in soybean’s interactions with Phytophthora sojae. The objective of this study was to compare defense gene expression in lines differing in partial resistance to Sclerotinia. Both constitutive and post-infection expression of genes for certain PR-proteins were examined.

Materials and Methods Growth chamber assay. Twelve seeds of soybean cultivars to be assayed for defense gene expression were planted in vermiculite in 11.5 cm pots. Plants were placed in a greenhouse and were inoculated when the unifoliates could be seen but were not expanded, usually 10 to 12 days after planting. A hole, 3 mm was cut in the cotyledon with a paper hole punch. Inoculum consisted of a single Sclerotinia colonized oat kernel which was placed in the hole, one cotyledon per plant was inoculated and all of the plants in a pot were inoculated Following inoculation plants were placed in a mist chamber for 48 hrs at 20oC.

RNA isolation. Plants were removed from the mist chamber and brought to the lab, where tissue, 2 to 4 mm wide, was cut from the edge of the lesion on the cotyledon, placed in a sterile eppendorf tube and immediately frozen in liquid nitrogen. Plant tissues were stored at -80OC until RNA extraction. RNA was extracted using Trizol® Reagent according to manufacturers instructions (Life Technologies, Rockville, MD).

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Northern blots. Northerns were carried out using standard protocols. Oligonucleotide primer pairs (20 mers) for each gene analyzed were designed with information from the soybean EST database. PCR or RT-PCR products were gel purified and labelled with 32P by random priming (Life Technologies) and used as hybridization probes.

Results and DiscussionProbes were developed for several PR protein genes, including PR1a, the elicitor-releasing β-1,3-glucanase (GLU, PR2, Tkeuchi et al, 1990), a win-like protein (WLP, PR4) and the Kunitz trypsin inhibitor (KTI, PR6). A probe for the phenylpropanoid enzyme chalcone synthase (CHS) was also used for comparison. The PR genes were all highly expressed after Sclerotinia infection and thus may be involved in defense to this pathogen. The induction levels were very similar among the 15 cultivars tested for KTI and WLP, whereas expression of PR1a and GLU were highly variable in the different cultivars. While there was a low level of constitutive expression of WLP and GLU in some cultivars, there was no baseline expression of PR1a or KTI. KTI was induced in some wounded controls, while constitutive GLU expression was suppressed in wounded tissues. In some experiments, PR1a and KTI expression were induced to much higher levels in S-1990 than in Williams 82, whereas there was no such difference in CHS mRNA levels. However, this differential expression was not seen in all experiments. We are currently looking into the potential source of this variation, which could be a reflection of the differential sensitivity in responsiveness of these genes. Thus, while intriguing variations in expression of certain genes were seen among lines, the correlation of any given response to partial resistance remains unclear at present. Because we expect partial resistance to be multigenic, this is not surprising and it may be combinations of differences in gene expression that are important.

Acknowledgments We wish to thank Mr. Jerome Weidner (Columbus) and Mr. Rob Smith, an undergraduate intern from Otterbein College, Westerville, OH for technical assistance. Financial support was provided in part by the Ohio Agricultural Research and Development Center, by grants to AD and TG from the Ohio Soybean Council and by a seed grant to MG from the Ohio Plant Biotechnology Consortium.

ReferencesGraham TL. 1994. Cellular biochemistry of phenylpropanoid responses of soybean to

infection by Phytophthora sojae. In: Handbook of Phytoalexin Metabolism and Action. M. Daniel and R. P.Purkayastha, eds., pp. 85-116. Marcel Dekker Inc. N.Y.

Qutob D, Hraber PT, Sobral BWS, Gijzen M, 2000. Comparative analysis of expressed sequences in Phytophthora sojae. Plant Physiol. 12, 243-253.

Tkeuchi Y, Yoshikawa M, Takeba G, Tanaka K, Shibata, D, Horino O, 1990. Molecular cloning and ethylene induction of messenger RNA encoding a phytoalexin elicitor-releasing factor, beta-1,3-endoglucanase, in soybean. Plant Physiol. 93, 673-682.

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Lactofen induces multiple defense mechanisms in soybean

T. Grahamb, A. Dorrancea, S. Landinib and M. Grahamb

a Department of Plant Pathology, The Ohio State University, 1680 Madison Ave., Wooster, OH 44691 USA; and b Department of Plant Pathology, The Ohio State University, Kottman Hall, 2021 Coffey Rd., Columbus, OH 43210 USA

AbstractDefense expression, following treatment with lactofen (Cobra®) was analysed both by metabolic profiling and northern analysis. Both secondary product and pathogenesis related protein (PR) genes were examined. Multiple modes of action were uncovered and will be discussed in relation to local and systemic protection against Sclerotinia.

IntroductionSclerotinia stem rot of soybean is an economically important disease in Ohio. Recently it has been reported that application of the herbicide Cobra® (lactofen) to field grown soybeans at the R1 stage can lead to partial protection against S. sclerotiorum. While the precise mechanisms of lactofen protection against S. sclerotiorum are unknown, it has been assumed that it involves the induction of host defense mechanisms. Consistent with this, earlier reports showed that lactofen treated field soybeans had higher levels of the soybean phytoalexin, glyceollin (Dann et al., 1999). In this study we have examined the effects of lactofen on soybean defense responses in several well established laboratory bioassays as well as on field grown soybeans. We employed metabolic profiling to examine the induction of a wide range of phenylpropanoid defense responses and Northern analyses for genes involved in the phenylpropanoid pathways and for a couple of PR proteins. The effects of lactofen are compared to those for jasmonic acid and 1-aminocyclopropane carboxylic acid (ACC). Results suggest that a complex, multi-component defense response is triggered by lactofen.

Materials and Methods Plant material and cotyledon assays. For metabolite profiling, the cut cotyledon assay was performed (cultivar Williams) as described elsewhere to examine proximal and distal cell responses (Graham & Graham, 1991). For Northern analyses, a third transverse section (1 mm thick) through the cotyledon was used to sample cells at a point much further (1 cm) from the point of treatment. In the field, Williams 82 soybeans were treated with 6 oz/acre lactofen at the R1 stage.

HPLC and Northern analyses. HPLC metabolic profiles were performed as described elsewhere (Graham, 1991). All phenylpropanoids were identified and quantified by comparison to purified standards. RNA samples were harvested and frozen immediately in liquid nitrogen and stored at -80oC. RNA was isolated by a LiCl precipitation method. Northerns were carried out using standard protocols. Oligonucleotide primer pairs (20 mers) for each gene analyzed were designed with information from the soybean EST database. PCR or RT-PCR products were gel purified and labeled with 32P by random priming (Life Technologies) and used as hybridization probes.

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Results and DiscussionHPLC metabolic profiles were examined for cotyledon tissues treated with lactofen in the presence or absence of the glucan elicitor from P. sojae. The glucan elicitor was used because it is one of the most characterized fungal general resistance elicitors and has been very thoroughly studied in the soybean system for its effects on phenylpropanoid defense pathways (Graham & Graham, 1999). Lactofen alone caused a very massive induction of isoflavone aglycones, predominantly the 5-deoxyisoflavones, daidzein and formononetin. The glucan alone caused large accumulations of the isoflavone conjugates of daidzein and genistein, and some glyceollin. The combination of lactofen and glucan induced a composite response, but with much higher (5X) levels of glyceollin. Taken together these results suggest that lactofen has two separate and highly complementary effects on phenylpropanoid defenses. First of all, it is a very potent inducer (“loader”) of 5-deoxyisoflavone pools, and secondly, it greatly enhances the capacity of soybean cells to utilize isoflavone precursors for the accumulation glyceollin in response to the glucan elicitor (elicitation competency). Lactofen causes a similar massive increase in isoflavone aglycones in leaves of field soybeans at the R1 stage, except that a slightly different profile of isoflavones is induced, with genistein predominant. Consistent with its induction of isoflavones, lactofen induces mRNA for chalcone synthase and isoflavone synthase in cotyledon tissues. Induction is seen not only in local (proximal and distal) cells, but also at the farthest end of the cotyledon from the point of treatment. Lactofen also induced proximal, but not distal accumulation of mRNA for PR1a. It had no effect on expression of the Kunitz trypsin inhibitor gene (a PR6).

Acknowledgments Financial support was provided in part by matched funds to TG from the Ohio Agricultural Research and Development Center (OARDC) and Valent Technologies and by grants to TG and AD from the Ohio Soybean Council.

ReferencesDann, EK, Diers, BW, Hammerschmidt, R, 1999. Suppression of Sclerotinia stem rot of

soybean by lactofen herbicide treatment. Phytopathology 89, 598-602 Graham, TL, 1991. A rapid, high resolution HPLC profiling procedure for plant and

microbial aromatic secondary metabolites. Plant Physiol. 95, 584-593. Graham, TL, Graham, MY, 1991. Glyceollin elicitors induced major but distinctly different

shifts in isoflavonoid metabolism in proximal and distal soybean cell populations. Mol. Plant Microbe Interact. 4, 60-68.

Graham, TL, Graham, MY, 1999. Role of hypersensitive cell death in conditioning elicitation competency and defense potentiation. Physiol. Molec. Plant Pathol. 55, 13-20.

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Evaluation and breeding soybeans for resistance to Sclerotinia stem rot

T.D. Vuonga, L. Kulla, D.D. Hoffmana, B.W. Diersa, W.L. Pedersona, L. Aydogdua, R.L. Nelsona,b, and G.L. Hartmana,b

aDept. of Crop Sciences, University of Illinois; bUSDA-ARS, 1101W. Peabody Dr., Urbana, Illinois 61801, USA

AbstractA cooperative research program at the University of Illinois has actively been investigating various aspects of Sclerotinia stem rot of soybeans. The goal of the research program is to find sources of resistance, study the mechanisms and inheritance of resistance, and evaluate isolates for their pathogenic and genetic variability.

IntroductionSclerotinia stem rot (SSR) of soybean, caused by Sclerotinia sclerotiorum, is one of the major diseases in the north central states of the U.S. (Hartman et al., 1999). Efforts to identify resistant sources of germplasm have been of interest since most current soybean varieties lack the desired level of resistance. Under field conditions, some varieties may be rated as partially resistance due to physiological and escape mechanisms (Bolland & Hall, 1988). In the greenhouse, disease reactions are the result of physiological resistance (Nelson et al., 1991).

The goal of our cooperative research program is to find sources of resistance, study the mechanisms and inheritance of resistance, and evaluate isolates for their pathogenic and genetic variability. The specific objectives are to (i) study genetic control of SSR resistance of plant introductions; (ii) investigate effectiveness of single plant selection and develop breeding germplasm for SSR resistance; (iii) determine whether shoots or roots are associated with SSR resistance and evaluate intact stem inoculation method for soybean and other crops; and (iv) study genetic variability of S. sclerotiorum isolates using randomly amplified polymorphic DNA.

Materials and MethodsGenetics. Stem lesion lengths on F2 plants derived from crosses among susceptible (Asgrow 2242 and Merit) and resistant genotypes (PI194.634, PI194.639, and NKS19-90) were analyzed. Crosses of NKS19-90 and other partial resistant PIs (PI358.318A, PI391.589B, and PI561.353) were evaluated in the field to determine inheritance of field resistance. Selections. A total of 10 F2 and backcross populations derived from the crosses of PIs (567.650B, 567.158, 507.353, 504.489, 257.435, 548.379, 578.496, 196.151) and commercial cultivars (Pana, Savoy, and Dwight) were made. In early generations, single plants were selected for different resistance levels based on field-inoculated evaluations. Selection procedures were repeated for F3 and F4 progeny. Promising selected F2:4 lines were evaluated under field conditions. Simple sequence repeat analyses were employed for identification of linked molecular markers. Grafts and stem inoculation. Self and reciprocal grafts among NKS19-90, Asgrow 2242, and Williams 82, were made using single- and double-scion grafting techniques. Grafted plants were evaluated for SSR resistance based upon percentage of plant survival at 5 days after inoculation. For the intact stem inoculation method, main stems of 5–6 wk seedlings of

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soybean, dry bean, and sunflower were severed above either the third or fourth node. Mycelial plugs were placed directly on the cut stem followed by 2 days of incubation at near 100% humidity and 20oC. Stem lesion lengths were measured and used for statistical analysis. Genetic variability. From an initial screening of 220 RAPD primers for molecular polymorphisms, 32 primers were selected. Sixty S. sclerotiorum isolates from Argentina, the U.S., and different crops were subjected to RAPD analyses. Dissimilarity and clustering analysis using SAS programs were conducted for the evaluation of genetic variability of these isolates.

Results and DiscussionGenetics. The stem lesion lengths of all F2 populations had a normal distribution indicating that resistance is multigenic. These greenhouse results agreed with field evaluations, which had an h2

b estimate of 0.37. From the field and greenhouse studies, it was concluded that inheritance of SSR resistance is multigenic. Selections. Selection for SSR resistance was investigated by classifying inoculated F2 and F3 plants. Single plant selection reduced the size of breeding populations and identified 90% of the most resistant families. In the population derived from the Williams 82 x NKS19-90 cross, two simple sequence repeat markers, Satt129 and Satt163, were identified with field resistance and seed yield. These markers can be used for marker-assisted breeding programs. Grafts and inoculation method. In all grafting combinations, a resistant or susceptible response was consistently associated with resistant or susceptible shoots regardless of the rootstocks. The results indicate that soybean resistance to S. sclerotiorum is controlled by shoots and not roots. The grafting techniques provided a better understanding of the SSR resistance mechanism in soybean.

Response of resistant and susceptible genotypes to S. sclerotiorum in different crops were consistently differentiated by an intact stem inoculation method. The increase in stem lesion lengths in greenhouse evaluations was positively correlated with disease severe index (DSI) of field evaluations indicating the reliability and usability of the inoculation method. Genetic variability. Cluster analysis based on RAPDs revealed genetic variability among isolates from geographic locations and different crops. At least four clusters were identified for each location and diverse crops. Of these, highly divergent clusters containing one isolate were also determined. The results indicate that RAPD is a very discriminative and an efficient method for differentiating and studying genetic diversity of S. sclerotiorum isolates.

AcknowledgementsThe authors wish to thank the Illinois Soybean Check-off Board and the North Central Soybean Research Program for providing funds for this research, and the Office of Research-International Activities, College of ACES, University of Illinois, for travel grant.

ReferencesBolland, GJ and Hall, R, 1988. Epidemiology of Sclerotinia stem rot of soybean in Ontario.

Plant Dis. 78, 1241-1245.Hartman, GL, Sinclair, JB, and Rupe, JC, 1999. Compendium of Soybean Diseases (4th ed).

The American Phytopathological Society. Nelson, BD, Helmes, TC, and Olson, MA, 1991. Comparison of laboratory and field

evaluation of resistance in soybean to Sclerotinia sclerotiorum. Plant Dis. 75, 662-665.

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Tracing the origin of QTLs for resistance to Sclerotinia sclerotiorum in soybean ancestral lines

V. Arahanaa, G. Graefa, J. Spechta and J. Steadmanb

aDept. Agronomy and Horticulture and bDept. Plant Pathology, University of Nebraska, Lincoln, NE 68583-0915

AbstractEighteen Simple Sequence Repeat (SSR) markers, putatively linked to quantitative trait loci (QTLs) for sclerotinia stem rot resistance, were used to screen 44 soybean lines in a pedigree analysis. A marker-phenotype (detached leaf assay) association was expected for those lines carrying a particular QTL allele. We verified previously identified QTLs and showed that QTL alleles with even relatively small effects on phenotype can be traced through ancestral pedigrees or future crosses to facilitate the development of resistant cultivars.

IntroductionIdentifying resistance to Sclerotinia sclerotiorum in soybean has proven difficult. Despite multiple screening studies, only partial resistance to S. sclerotiorum in soybean has been described (Kim et al., 1999, Boland & Hall, 1987), and it is regarded as multilocus and complex (Kim and Diers, 2000). Using molecular markers, putative QTLs for resistance have been reported (Kim & Diers, 2000, Arahana et al., 2001). Tracing the origin of a putative QTL back to the ancestral lines with molecular markers could be interpreted as one form of QTL verification. Ancestors pass on blocks of their own genome to derived cultivars, which can be traced with molecular markers. A marker-phenotype combination is expected to be found in the ancestral lines as well as in the derived cultivar depending on their linkage association. A cultivar that does not receive both a marker allele and a phenotype from the same parent is considered to be a recombinant cultivar (Lorenzen et al., 1995). The objective of this study was to verify QTLs identified by Arahana et al. (2001) and identify their origin by tracing the marker-phenotype combination in soybean ancestral lines.

Materials and MethodsForty-five soybean cutivars, constituting the pedigrees of ‘Corsoy 79’, ‘Vinton 81’, ‘Dassel’, ‘NKS 1990’, and ‘DSR 173’ were evaluated. Plants of all 45 cultivars were grown in the greenhouse on two planting dates. Each 12-inch pot contained four plants of a given cultivar. Experimental design for the detached leaf assay in the laboratory was an alpha lattice with four replications per planting date. In each replication the 45 lines were randomized to 12 pans, four leaves per pan. Pans were considered incomplete blocks. One replicate consisted of one leaf from a single plant of each line. At the V4 developmental stage, the most recently developed fully expanded leaf was removed from the plants and inoculated using an agar plug with isolate 143 of S. sclerotiorum following the protocol for a detached leaf assay described by Arahana et al. (2001). Lesion area was recorded after 48h incubation. Nineteen SSR markers linked to QTLs for resistance were used to trace the origin of the respective marker allele in ancestral soybean cultivars. DNA was extracted from leaves of each cultivar following the mini-extraction CTAB procedure (Based on Saghai-Maroof et al., 1984). DNA amplification used a PTC-100 thermocycler as described by Arahana et al. (2001).

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LSMEANS for lesion area were calculated for each cultivar using the PROC MIXED procedure of SAS with PAN(REP) declared a random effect. For each SSR marker, association between a favorable marker allele and decreased lesion size was determined by calculating single degree-of-freedom contrasts comparing cultivars carrying the favorable allele with those carrying a different allele. The ancestral origin of a QTL was deduced by following the favorable allele of the respective SSR marker linked to it and its association with a decreased lesion size phenotype.

Results and DiscussionWe verified the QTLs on Linkage Groups A2 (Satt424), F (Satt114), and O (Satt243-Sat_109, and Satt478) identified previously, and traced their probable origin through analysis of ancestral lines. Most of the QTLs accounted for 3% to 5% of the variation for lesion size in the QTL study by Arahana et al. (2001), while the QTL identified by markers Sat_109 and Satt243 on LG O accounted for 8% of the variation for the trait. Even with relatively small effects, these QTLs were traced through some pedigrees to identify possible sources for the resistance alleles. Factors that confounded our ability to infer possible donor genotypes from this pedigree analysis include heterogeneity in the original accessions, recombination between marker and QTL alleles, and lack of information due to missing breeding lines in some pedigrees. The results show that QTL alleles with relatively small effects on phenotype can be traced through ancestral pedigrees or future crosses to facilitate development of resistant cultivars. We are currently incorporating these QTL alleles into high-yielding soybean cultivars. The putative QTL donor genotypes Mandarin, A.K. Harrow, Adams, PI 257435 and Capital should be included in regional disease nurseries to evaluate their resistance to S. sclerotiorum.

AcknowledgementsWe thank Becky Higgins & Kris Powers for help phenotypic screening cultivars; Perry Cregan for SSR primers; Novartis Seeds Inc., Dairyland Seeds Company, Inc., & Randy Nelson for cultivar & seed lines. Partial support was provided by grants from the Nebraska Soybean Board.

ReferencesArahana VS, Graef GL, Specht JE, Steadman JR, Eskridge KM, 2001. Identification of

QTLs for resistance to Sclerotinia sclerotiorum in soybean. Crop Sci. 41, 180-188.Boland GJ, Hall R, 1987. Evaluating soybean cutivars for resistance to Sclerotinia

sclerotiorum under field conditions. Plant Dis. 71, 934-936.Kim HS, Diers BW, 2000. Inheritance of partial resistance to sclerotinia stem rot in soybean.

Crop Sci. 40, 55-61.Kim HS, Sneller CH, Diers BW, 1999. Evaluation of soybean cultivars for resistance to

sclerotinia stem rot in field environments. Crop Sci. 39, 64-68.Lorenzen LL, Boutin S, Young N, Specht JE, Shoemaker RC, 1995. Soybean pedigree

analysis using map-based molecular markers: Crop Sci. 35, 1326-1336.Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW, 1984. Ribosomal DNA spacer

length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA, 91, 5466-5470.

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Pyramiding transgenes with QTLs to enhance resistance to Sclerotinia sclerotiorum in soybean

V. Arahanaa, G. Graefa, T. Clementea, J.R. Steadmanb, J. Spechta, A. Mitrab and T. Buhra

aDept. Agronomy and Horticulture and bDept. Plant Pathology, University of Nebraska, Lincoln, NE 68583-0915

AbstractAn oxalyl-CoA decarboxylase (oxc) gene clone from Oxalobacter formigens was integrated into the genome of the soybean cultivar Thorne through Agrobacterium-mediated transformation. We transferred the oxc gene into partially resistant cultivars NKS1990 and Vinton 81, in which we previously identified QTLs for resistance to S. sclerotiorum, via backcrossing. SSR markers and glufosinate detect QTLs and the oxc gene; the detached leaf assay evaluates resistance. IntroductionComplete genetic resistance to Sclerotinia sclerotiorum in soybean in unknown (Kim et al., 1999, Boland & Hall, 1987). Partial resistance is considered multilocus and complex (Kim & Diers, 2000). Quantitative trait loci (QTLs) for resistance have been reported in soybean (Kim and Diers, 2000; Arahana et al., 2001). However, the resistance offered by the few QTL is insufficient to completely protect plants from fungus attack since each QTL accounts only for 5% to 10% of the phenotypic variation. S. sclerotiorum produces oxalic acid which plays a significant role in fungal pathogenicity (Godoy et al., 1990). Dickman & Mitra (1992) proposed a biological control strategy based on catabolism of oxalate in the host plant. Transformed oilseed rape, tobacco, and tomato with oxalate oxidase or oxalate decarboxylase genes had improved resistance to S. sclerotiorum (Thompson et al., 1995; Kesarwani et al., 2000). We transformed tobacco and soybean with the oxc gene from the bacterium Oxalobacter formigenes under control of the CaMV35s promoter. Although transgenic tobacco displayed improved resistance to the pathogen in some detached leaf assays, the results were inconclusive. Similar results were obtained for transformed soybeans. The objective of this study was to investigate the effect of combining endogenous physiological resistance conferred by QTL with exogenous resistance conferred by the oxc gene from the bacterium O. formigenes. Materials and MethodsSoybean cultivars NKS1990 and Vinton 81 are known to have QTL alleles for resistance to the pathogen (Arahana et al., 2001). NKS1990 has a QTL allele on LG F, linked to marker Satt114; the QTL in Vinton 81 is on LG O, flanked by markers Satt243 and Sat_109. The source of exogenous resistance was two trangenic events (285-11A, 285-11B) incorporating the oxc gene into the soybean cultivar Thorne by Agrobacterium-mediated transformation. The transgene construct (pPTN178) also contained the bar gene conferring glufosinate resistance as a selectable marker. Both genes were under the control of the CaMV 35s promoter, followed by tobacco etch virus translational leader (TEV) and ending with the CaMV T35 terminator. Northern hybridization detected mRNA-transcripts for the oxc gene in these two events. Resistance to S. sclerotiorum was evaluated in the transgenic events and the QTL-containing cultivars using the detached leaf assay in 20 replications of a randomized complete block design (RCBD). At the V4 developmental stage, the most recently developed

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leaf was removed from the plants and inoculated using an agar plug of S. sclerotiorum. Lesion size was measured after 48 h incubation (Arahana et al., 2001). Each block was represented by a pan with five leaves per pan, one from each of the transgenic events, one from each of the QTL-containing lines and one from Thorne. Transgenic plants and F1 plants from crosses involving transgenics were evaluated for glufosinate resistance at the V3 stage by swabbing a solution (100 mg/mL) of the herbicide Liberty® onto one of the unifoliolate leaves. The herbicide-susceptible plants were discarded. Hybridity of F1 plants was also assessed by PCR using SSR markers Sat_109, Satt114 and Satt243. Parental, F1, and control plants were evaluated for reaction to S. sclerotiorum using the detached leaf assay. Leaves from plants involved in each cross were randomly assigned to four pans in a RCBD. Each pan constituted a block. Each pan contained three leaves, one from the F1 plant, and one for each of the parents. A negative control cross between non-transgenic Thorne and the QTL-containing cultivars was also included to measure the Thorne background effect. Four replications in each of two different runs were used for these tests. Analysis of variance and LSMEANS were generated using the SAS PROC MIXED procedure applied to F1 plants and parental lines for a RCBD. Pan and run were considered random effects. Single-degree-of-freedom contrasts between F1 plants and each of their respective parents were also performed.Results and DiscussionTransgenic events 285-11A and 285-11B had significantly smaller lesion sizes than Thorne and NKS1990. Lesion size for Vinton 81 was also smaller than Thorne and NKS 1990, and similar to the two transgenic events. In the analysis of parental and F1 plants, there was a significant run x line interaction, and there was no advantage of the transgenic line or the F1 containing the transgene-QTL combination compared with the QTL-containing cultivar. Because there is no significant advantage to using the transgene, approaches to combine and maximize different sources of endogenous resistance are recommended. AcknowledgementsWe thank B. Higgins and K. Powers technical assistance, and Nebraska Soybean Board funding. ReferencesArahana VS, Graef GL, Specht JE, Steadman JR, Eskridge KM, 2001. Identification of QTLs

for resistance to Sclerotinia sclerotiorum in soybean. Crop. Sci. 41, 180-188.Boland GJ, Hall R, 1987. Evaluating soybean cultivars for resistance to Sclerotinia

sclerotiorum under field conditions. Plant Dis. 71, 934-936.Dickman MB, Mitra A, 1992. Arabidopsis thaliana as a model for studying Sclerotinia

sclerotiorum pathogenesis. Physiol. Mol. Plant Pathol. 41, 255-263.Godoy G, Steadman JR, Dickman MB, Dam R, 1990. Use of mutants to demonstrate the role

of oxalic acid in pathogenesis of Sclerotinia sclerotiorum in Phaseolus vulgaris. Physiol. Mol. Plant Pathol. 37, 179-191.

Kesarwani M, Azam M, Natarajan K, Mehta A, Datta A, 2000. Oxalate decarboxylase from Collybia velutipes: Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. J. Biol. Chem. 275,7230-7238.

Kim HS, Diers BW, 2000. Genetic analysis of partial resistance to sclerotinia stem rot in soybean. Crop. Sci. 40, 55-61.

Kim HS, Sneller CH, Diers BW 1999. Evaluation of soybean cultivars for resistance to sclerotinia stem rot in field environments. Crop. Sci. 39, 64-68.

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Use of herbicide induced resistance to control soybean Sclerotinia stem rot

X.B. Yang and P. Lundeen

Department of Plant Pathology, Iowa State University, Ames, USA.

AbstractFrom 1997 to 2000, field experiments were conducted to determine the efficacy of resistance induced by herbicides (lactofen and Action) for integrated disease and weed management. Application of these herbicides consistently reduced Sclerotinia stem rot (SSR). Control results were equal to fungicides registered for Sclerotinia stem rot control. Yield benefits from induced resistance were evident when SSR was severe. Use of risk prediction models should enhance return of this control measure.

IntroductionSystematically induced resistance is a physiological immune response triggered by pathogen infection or by synthetic or natural activators (Kessmann et al., 1994). SAR responses are not pathogen specific, can be effective to reduce infection by a broad range of pathogens, and have been found in many crops, including bean and soybean (Kessmann et al., 1994; Sticher et al., 1997). These immune resistance responses are systemic within the plant. Because SAR will provide a stable and broad-spectrum disease control, implementation of SAR provides a novel alternative to disease prevention and management. Applications of several synthetic compounds have been reported to induce SAR. Lactofen when applied to soybean causes localized necrosis, a symptom morphologically similar to those caused by fungi, and induces resistance to the disease. The objective of this study was to determine the effectiveness and consistency of systemic acquired resistance induced by synthetic activators in reducing soybean infections by Sclerotinia sclerotiorum at experimental plots and production fields.

Materials and MethodsThe study was conducted in 1997-2000 growing seasons in Iowa in fields with a history of Sclerotinia stem rot. The fields used for experiments had been in a corn-soybean rotation with corn the previous crop. Two separate experiments were conducted in two different scales: 1) an experiment with randomized completed block design to determine the effects of application date and rate on disease reduction, 2) a replicated experiment with application for production conditions (except for 1999). At each location-year, a white mold susceptible variety (Ag 2242 for 1997 and 1998 and LG 6293 for 1999 and 2000) was used each year and was planted in May. Chemical and hand weeding was applied for weed control during the growing season.

Synthetic chemicals tested were Cobra (lactofen), Action, and Actigard. The first two chemicals are used in weed control in soybeans. Treatments of application rate and application times varied over years. In each year Benlate or Topsin, fungicides registered for controlling Sclerotinia stem rot, were used as controls. Disease severity data were taken in late August, and plants of central two rows of the plots were harvested for yield. In 2000, leaf spot severity was also rated.

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Large scale application experiments were also conducted in corn-soybean rotation fields in collaboration with soybean producers to examine lactofen’s effectiveness. At each location and year, replicated plots at least 0.3 ha in size were established for each treatment. Varieties used in the experiments were susceptible to the disease. The SAR induced chemicals were applied with conventional farm equipment at soybean flowering time. Disease severity data were taken in late season, and the treatments were harvested for yield.

Results and DiscussionApplications of lactofen resulted in small brown lesions on leaves. Actigard did not produce leaf lesions but leaves appeared pale after application in 1998. Compared with non-spray control, the synthetic compounds reduced disease severity and increased yields, although yield was not significant, especially in fields where disease severity was high. Results from multiple year-location experiments in Iowa showed that applications of both Action or Cobra reduced severity of Sclerotinia stem rot, an indication of induced resistance by these synthetic compounds. Their control results were equivalent to fungicides. The results were consistent over years and locations. In 2000, SSR was light but was less in treated compared with control. Leaf spot was also reduced with in plots treated with lactofen and action.

At production scale, application of lactofen has consistently reduced disease severity. In 1997, spray and non-spray had severity of 6% and 67%, respectively. The yields were 2.69 and 3.50 tons/ha for spray and non sprayed. In 1998, the disease was light in Iowa and out of four experiments, one had severity 40% in non-sprayed plots compared with 10% in sprayed plots. Yields were 4.12 vas 4.51 tons/ha for non-spray and spray treatments.

ReferencesKessmann, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., Ward, E., Uknes, S., and

Ryals, J. 1994. Induction of systemic acquired disease resistance in plants by chemicals. Annu. Rev. Phytopathol. 32, 439-59.

Sticher, L., Mauch-Mani, B., and Metraux, J.P. 1997. Systemic acquired resistance. Annu. Rev. Phytopathol. 35, 235-70.

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Inheritance and mechanisms of resistance to Sclerotinia sclerotiorum and in vitro mutagenesis via microspore culture in Brassica napus

S-Y Liua, Z.Y. Xua, J.K. Zhanga, H.Z. Wanga Y.J. Huanga and L.Y. Heb

aOil Crops Research Institute of CAAS, Wuhan 430062; bInstitute of plant Protection of CAAS, Beijing 100094.

IntroductionStem rot (Sclerotinia sclerotiorum) a main disease on oilseed rape (Brassica ssp) has become a limited factor of rapeseed production in China. To promote resistant breeding and effective control the following objectives were studied: (1) Understand inheritance of resistance; (2) Exploit resistant mechanisms to oxalic acid, a toxin produced by the fungus as a key factor of the pathogenesis; (3) Develop resistant lines by microspore culture and in vitro mutagensis.

Materials and methods1. Inheritance of resistance to S. sclentiorum and its toxin In view of varietal performance of resistance, a diallele mating design following Griffing's method four with six parents was employed for genetic analysis. Crossing parents of resistance and susceptibility were bred during last ten years. Methods of resistance evaluation included greenhouse disease nursery, seedlings fed oxalate (OA) through roots, detached leaves fed OA through petioles and inoculation of detached leaves with mycelia. Significant variances were detected for disease index (disease severity) or for lesion size, and the variance components indicated a preponderance of both additive and dominant effects throughout four methods of disease evaluation. In disease nursery evaluation, the additive variance was larger than the dominant variance and they contributed a rate of 40.3% and 34.7% to the total, respectively. Contrarily, the additive variance was lower than the dominant variance and they accounted for 23.6%-24.4% and 32.6%-34.0% of the total, respectively, in both evaluation methods using OA. Significant heritabilities of resistance to both the fungus infection and OA toxicity were estimated for disease index or lesion size by four evaluation methods, indicating that selection for resistance would be generally effective. Genetic effects in resistant and susceptible parents were different. Resistant parents generally showed negative additive effect. Of them, line ZhongR-783-3 had significant negative additive effect and significant negative dominant effect in crosses with other parents for disease index, indicating it is an elite parent for breeding. Additive effects in susceptible parents were generally positive, suggesting use of susceptible parents should be avoided for breeding. Dominant effects in crosses between resistant and susceptible parents were either negative or positive depending on specific cross and thus resistance of a specific cross or hybrid should be evaluated by experiments.2. Inheritance of resistance to disease spreadExperiments with mycelium inoculation of detached leaves showed that lesion size (Y) increased with time (x) in form of equation Y=a xb . Lesion spread of susceptible varieties was significantly faster than that of resistant ones. Analysis of conditional variance further conformed that the existing variation of resistance between varieties was controlled by heritable factors, which had different genetic effects between resistance and susceptible varieties as described above. This analysis method is more sensitivity to detect heritability, for example, heritability increased by 40 percent after roots treated with OA when compared with that estimated by the general variance analysis. It could reveal dynamic gene change effects.3. Mechanisms of resistance to oxalic acid in B. napusRape detached leaves and seedlings were fed 14C-oxalate through petioles and roots, respectively. The organic substances in leaves were extracted by acetic acid-ethanol solution and were fractionated by chemical and chromatographic methods. It was found from autoradiographic profile of seedlings

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and detached leaves that radioactivity in resistant varieties to S. sclerotiorum was mainly confined to major veins of leaves and stems, whereas in susceptible varieties radioactivity was distributed rather uniformly throughout the seedlings. Therefore, resistant varieties limited the transportation of OA into interveins. The results of detecting both dry leaves and the extracts from fresh leaves revealed consistently difference of the absorbed amount of OA by leaves as determined by radioactivity between resistance and susceptible varieties. The amount in susceptible variety 84039 is four folds higher than that in resistant one ZhongR-783. The difference was also found between basal leave and the upper leaves, i.e. radioactivity in the former is 72.0-77.4 percent of the latter. More than 57% of OA absorbed was metabolized into other organic substances, and the higher rate of its metabolism was found in resistant varieties than that in susceptible ones, revealing a mechanism of detoxification in seedlings. Radioactivity detected in soluble OA was rather small (about 1.2% of the total), but the activity in the susceptible variety increased by 76.3 percent when compared with that in resistant one. Radioactivity detected in insoluble OA ranged from 112581 to 300675 cpm/gF.W. between varieties, accounting for 28% of the total. The insoluble OA content of the resistant variety decreased by 28.4%-56.2% when compared with that of susceptible one. Deposit of insoluble oxalate would partially explain mechanism of pathogenesis of OA. In the acetic acid-ethanol fraction, radioactive neutral substances (mainly sugars) accounted for 43.2%-81.1%. There were no relation between resistance and rates of anmino, other organic acid and sugars.4. In vitro mutagenesis and selection for resistance by microspore culture in B. napus.Microspore culture was carried out following the Manual for Microspore Culture Technique for Brassica napus. The buds of the donor plants came from selfed lines, open pollinated population (OPP), F1 and F2 generations of crosses. Results showed that embryo yield is different between differently pollinated populations. The frequency of embryogenesis of crosses and OPP was higher than that of selfed lines in microspore culture. The highest yield per bud reached more than one hundred embryos. There was no relation between embryo yield and resistance. For in vitro mutagenesis including application of chemical EMS (Ethyl methane sulfonate) and gamma rays, haploid callus were induced from plantlets developed from embryos. In vitro screening of mutants was undertaken using OA added into media as selection pressure. Experiments showed that the mortality rate of the callus increased when OA concentration increased. An obvious difference was found between the callus from resistance and susceptible donor varieties when OA concentration rose up to near half lethal dose of 3mmol/L. During the second cycle screening with higher concentration of OA, a similar case was observed. There was an accumulated effect of OA toxicity observed to callus, but the growth rate of living callus under selection pressure on media was not affected by OA. The mortality rate of haploid callus on media containing EMS increased with EMS concentration, and there was no survival when EMS concentration reached 0.3 percent. EMS also reduced survival rate and the growth rate of the living callus during subsequent culturing on media containing OA. A similar trend was observed after callus were radiated with gamma rays with half lethal dose of 1000 roentgen. However, screening by using OA promoted plantlet regeneration from the callus when repeating transfer and multiplication of the callus not treated with OA reduced plantlet regeneration. On the basis of the above studies, a procedure for in vitro mutagenesis for resistance to S. Sclerotiorum and its toxin has successfully be established by using haploid callus derived from microspore culture. As the product and final purpose of the procedure, two DH lines of resistant mutants M083 and M004 have been developed. The resistant evaluation indicated that disease index of M083 and M004 decreased by 40% and 34.2%, respectively, when compared with those of original populations of 083 and 004 as donors. In a field identification of resistance, disease index of Zhongyou 821, a most resistant variety, was 17.1%, but that of M083 and M004 was only 3.9% and 5.2%, respectively. Furthermore, the two DH lines own good agronomic characters, for example, high yield per plant, heavy weight per 1000 seeds and good plant type (shape).

P4.11

QTL Analysis of Resistance to White Mould in Common Bean

J. D. Kelly a and J.M. Kolkmanb

aDepartment of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA; andbDepartment of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

AbstractWhite mould (Sclerotinia sclerotiorum) is a destructive yield-limiting disease of common bean (Phaseolus vulgaris L.). Molecular markers, identified using selective multivariate genotyping of DNA bulks, were linked to QTL for resistance to S. sclerotiorum. Individual markers accounted for 17% of disease variation, and 37% of yield variation under white mould pressure.

IntroductionWhite mould is a destructive fungal disease that seriously limits bean production in temperate regions (Steadman, 1983). Epidemics of white mould in common bean occur during seasons of high yield potential. Total seed yield is reduced due to lower number of seeds produced per plant, reduced number of pods per plant and smaller seed size (Kerr et al., 1978). In a recent survey of Michigan dry bean producers, 64% of the respondents indicated that white mould was the number one disease problem. Breeding for resistance in bean offers a stable, long-term strategy to reduce yield loss to white mould in common bean. The objectives of this study were to i) study the inheritance of resistance to white mould, and ii) identify markers linked to quantitative trait loci (QTL) conferring resistance to white mould in bean, using selective multivariate genotyping based on single and multiple phenotypic traits.

Materials and MethodsThree populations were developed and rated for resistance, and agronomic avoidance traits at two locations in Michigan over three years (1996-98). The first population consisted of 27 resistant and/or susceptible elite lines or cultivars. The second population (BN) was comprised of 98 F3:6 families derived from a cross between Bunsi, an indeterminate (Type II) resistant cultivar with an open porous canopy, and Newport, a determinate (Type I) susceptible cultivar. The third population (HN) was a 28-entry recombinant inbred line (RIL) population, derived from the cross between the resistant indeterminate cultivar, Huron, and Newport. RIL populations were developed by single seed descent and no selection for agronomic traits was made during generation advance. Plots were rated for disease shortly before harvest, when the majority of plants had reached physiological maturity. Thirty plants per plot were each given a rating from 0 to 4, where 0 = no disease present, 1 = 1 to 25% of the plant with white mould symptoms, 2 = 26 to 50% of the plant with white mould symptoms, 3 = 51 to 75% of the plant with white mould symptoms, and 4 = 76 to 100% of the plant with white mould symptoms (Hall & Phillips, 1996). Disease incidence (DI) was calculated for each plot as (the number of plants infected)/30 plants, based as a percentage. Disease severity index (DSI) was calculated for each plot on a percentage basis as: 100[ (rating of each plant)/4(30)]. Populations were evaluated for physiological resistance in the greenhouse using the oxalate assay (Kolkman & Kelly, 2000).

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Selective genotyping, using both single traits and multiple traits, was used to create DNA bulks, and identify significant markers for the BN population. Three DNA bulks were established for extreme phenotypes of DSI, DI, and oxalate. The multivariate bulks were comprised of lines that were either resistant and high-yielding, or susceptible and low-yielding, within a fixed flowering range from 40 to 45 days. The four sets of DNA bulks were screened with the RAPD and AFLP primer pair combinations in order to identify markers linked to the resistance phenotype. Primers that were polymorphic in the bulks were then tested for polymorphism in the population. Significant markers were identified via analysis of variance and correlation analysis to indicate linkage between markers, as well as the confirmation of linkage to resistance or agronomic traits. Linkage and linkage order of markers were determined with MAPMAKER/EXP, using the Kosambi mapping function, a minimum LOD score of 3.0 and a maximum recombination frequency of 0.30.

Results and DiscussionHeritability estimates were moderate for DSI (0.49) and DI (0.42), in the BN population, and were higher for DSI (0.82) and DI (0.76) in the HN population (Kolkman & Kelly, 2001). Agronomic avoidance traits that correlated to DSI and DI in the BN and HN populations were not significant in the first population. Markers linked to QTL for resistance to S. sclerotiorum were identified in each of the DNA bulked screening methods, and were consistent across field environments and populations. In the BN population, the most significant marker on linkage group B2 of the bean core map was associated with DSI (12%) and DI (13%), while individual markers on linkage group B7 were associated with DSI (17%), DI (13%), and yield (37%). Located on B7 was a unique locus for determinate growth habit in navy bean that was associated with susceptibility to white mould. Other QTL for white mould resistance have been reported on B7 (Miklas et al. 2001; Park et al., 2001). In the HN population, one marker on B2 was also associated with DSI (40.3%) and DI (35.4%), while one marker on B7 was associated with resistance to oxalate (24.3%) and yield (47.0%). Markers located near the PvPr-2, and ChS genes on B2 have previously been associated with QTL for resistance to common bacterial blight and Fusarium root rot. Linkage group B2 may have a cluster of defence-related genes that have broad application across pathogens. Markers identified in this study will be used in marker-aided breeding for resistance to white mould in common bean.

References Hall R., L.G. Phillips LG. 1996. Evaluation of parameters to assess resistance of white bean to

white mold. Annu. Rep. Bean Improv. Coop. 39,306-307.Kerr ED, Steadman JR, Nelson, LA. 1978. Estimation of white mold disease reduction of yield

and yield components of dry edible beans. Crop Sci. 18,275-279.Kolkman JM, Kelly JD, 2000. An indirect test using oxalate to determine physiological resistance

to white mold in common bean. Crop Sci. 40, 281-285.Kolkman JM, Kelly JD, 2001. Analysis of agronomic factor affecting inheritance of resistance to

white mold in common bean. Crop Sci. 41, (accepted).Miklas, P.N., R. Delorme, W.C. Johnson, and P. Gepts. 2001. QTL conditioning physiological

resistance and avoidance to white mold in dry bean. Crop Sci. 41,309-315.Park, S.O., D.P. Coyne, J.R. Steadman, and P.W. Skroch. 2001. Mapping of QTL for resistance

to white mold disease in common bean. Crop Sci. 41, (in press).Steadman J. 1983. White mold- a serious yield-limiting disease of bean. Plant Dis. 67, 346-350.

5. PATHOLOGY AND EPIDEMIOLOGY

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Sclerotinia sclerotiorum - A new threat to mustard cultivation in Rajasthan, India

A. Shivpuri, A.K. Bhargava and H.P. Chhipa

Department of Plant Pathology, Rajasthan Agricultural University, A.R.S, Durgapura, Jaipur - 302018

AbstractIndian mustard (Brassica juncea) is an important oilseed crop of the state of Rajasthan, India. Currently stem rot disease caused by Sclerotinia sclerotiorum (Lib). de Bary has become an important problem in the state, appearing endemically in the fields causing considerable yield loss. Integrated approaches like use of chemicals, pant extracts and varietal resistance have given encouraging results for the management of the disease.

IntroductionIn India among the oilseed crops mustard occupies the second largest area after groundnut, i.e., 6.10 million hectares with productivity of 940 kgs/ha. Rajasthan state alone contributes about 38 per cent to the total production of mustard of the country. The estimated yield loss caused due to different diseases ranges from 13-70 per cent in rapeseed and mustard (Bulletin - Vision 2020,NRCM, Bharatpur, India. Stem rot of mustard induced by Sclerotinia sclerotiorum has set its foot in the state infecting a wide range of dicotyledonous plant species including rapeseed/ mustard. Most of the cultivated varieties of Brassica juncea (T-59, RH-30, Pusa bold and Bio 902) were found susceptible to this pathogen (Shivpuri et al., 1997 a). Horning (1983) and Liu (1991) have reported more than 50 per cent yield loss in Germany and China respectively. In India, seed yield loss has been estimated upto 90 per cent when plants are infected at early stage of flowering (Shivpuri et. al., 2000). Henderson (1962) reported that ascospores were the most important source of infectionas compared to mycelium and germinating sclerotia. He also suggested that sclerotia were important source of inoculum. The infection on the plants is chiefly caused by ascospores. Williams and Stelfox (1980) working on this disease, trapped ascospores from 20 cms to 117 cms above the soil surface in rapeseed field.

Materials and MethodsIn a field experiment five chemicals, viz. Carbendazim 50 WP (0.1 %), Mancozeb 75% WP (0.2%), Thiophanate methyl (0.2%), Neem oil (0.3%) and a growth hormone Triacontanol (0.05%) were sprayed twice (60 & 75 DAS) on Brassica juncea cv. T-59.

In a lab experiment leaf extracts (aqueous suspension) of 18 plant species were incorporated into Czapek's agar medium before autoclaving and assessed for growth and sclerotial production of the pathogen. Besides 70 Brassica were evaluated under artificial soil-inoculation conditions for their resistance to stem rot.

ResultsCarbendazim 50 WP (0.1%) sprayed twice at 60 and 75 DAS was comparatively superior in reducing the stem rot and significantly increasing the seed yield. Effectiveness of

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Carbindazim against sclerotial rot of rapeseed has also been reported (Shen, 1992). Of the 18 botanicals, species of viz., Withamia somnifera, Vinca rosea, Ocimum sanctum, Dhatura stramonium, Azadirachta indica and Polialthia longifolia had almost inhibited the growth and sclerotia production in vitro. However some of the plant species stimulated the sclerotial production. Thus it indicates that some species secrete stimulants to S. sclerotiorum and others, inhibitory substances to it (Shivpuri et al., 1997b). Of the 70 genotypes tested, ZYR-6, Cutlass and PCR-10 (B. juncea), Westar (B. napus), Parkland, Tobin, Torch and Candle (B. campestris) were found resistant against stem rot disease.

AcknowledgementAuthors are thankful to Indian Council of Agricultural Research (ICAR), New Delhi for granting the project, Rajasthan Agricultural University Plant Pathology Department, A.R.S., Durgapura for providing facilities for work and to Dr. R.B.L. Gupta, Associate Professor, Plant Pathology for helpful suggestions during experimentation.

ReferencesHenderson RM, (1962) Some aspects of the lifecycle of the plant pathogen Sclerotinia

sclerotiorum in Western Australia, J. Royal Soc. W. Aust., 45, 133-135.Horning H, (1983). Weitere Untersuchungen Und Eskenntnisse Zur Krankhaften Abreife

RAAPS, 5 Jg. (I): 24-29.Liu CQ, (1991). Study on tolerance on Sclerotinia sclerotiorum and the inheritance

properties in Brassica napus. Scientia Agricultura Sinica, 24, 43-49Shivpuri A, Chhipa HP, Gupta RBL, Sharma KN (1997). Field evaluation of rapeseed

mustard genotypes against white rust, stem rot and powdery mildew. Annals of Arid Zone, 36, 387-389

Shivpuri A, Sharma OP and Jhamariya, S.L. (1997b) Fungitoxic properties of 10 plant extracts against five pathogenic fungi. Indian J. Mycol. Pl. Pathol, 27, 29-31.

Shivpuri A, Sharma KB, Chhipa HP (2000) Some studies on stem rot disease of mustard in Rajasthan.Indian J. Mycol. Pl. Pathol. 30, 268.

Williams JR, Stelfox, D (1980). Dispersal of ascospores of Sclerotinia sclerotiorum in relation to Sclerotinia stem rot of rapeseed. Plant Disease Repr.,63, 395399.

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Adaptation and importance of Sclerotinia sclerotiorum in North Dakota

Berlin D. Nelson

Department of Plant Pathology, North Dakota State University, Fargo, ND 58105, USA

AbstractSclerotinia sclerotiorum has become the major pathogen of row crops in North Dakota. The acreage of susceptible crops has expanded dramatically and all growing areas are infested. Losses from Sclerotinia diseases occur each year. The pathogen is well adapted to the agro-ecosystem in ND, is a serious threat to row crop production and adequate controls are lacking.

Adaptation and Importance of S. sclerotiorum in North DakotaSclerotinia sclerotiorum is considered the most important row crop pathogen in North Dakota. It causes important diseases known as white mold, Sclerotinia wilt or stalk rot, and Sclerotinia head rot on a wide variety of broadleaf crops. It commonly damages dry beans, sunflowers, soybeans and canola. Other susceptible crops are field pea, potato, mustard, safflower, lentils, borage, crambe, buckwheat, chickpea, lupine, faba bean and numerous vegetables. Some of these crops, such potato are rarely damaged by S. sclerotiorum, while others are highly susceptible. Numerous weeds such as marsh elder, lambsquarters, pigweed, Canada thistle, sow thistle, and wild mustard are also hosts and play a role in disease cycles. With the expansion of row crops and close rotations of these crops, Sclerotinia will continue to increase in importance. High levels of host resistance are lacking and cultural controls and prediction systems are inadequate; thus the control of Sclerotinia diseases is a serious challenge. The two other species S. trifoliorum and S. minor are not reported in North Dakota.

Although S. sclerotiorum was reported in North Dakota in the 1930's, it was not considered an important pathogen until the 1970's when white mold of dry beans and Sclerotinia wilt of sunflower began appearing as serious diseases in the eastern portion of North Dakota, primarily in the Red River Valley. It was during that decade that both state and USDA plant pathologists began intensive research on biology and control of S. sclerotiorum in the state. The pathogen was so serious on sunflowers in the Red River Valley during the 1980's, that many growers with infested fields were forced to abandon sunflowers or rotate to less susceptible crops. White mold continues to be the most important disease of dry bean and sunflower.

North Dakota agriculture was traditionally based on cereals, but during the 1960's to 1970's the acreage of susceptible row crops, especially dry bean and sunflower began to expand. Low prices for cereals and an emphasis on crop diversity, resulted in growers including susceptible row crops in their rotations. In 1960, the acreage of susceptible broadleaf row crops was about 300,000 acres. In 2000 it was 6.1 million acres and is expected to continue increasing, especially for soybean and canola. These broadleaf crops are expanding into the western portions of the state. In the 1990's, a combination of low cereal prices and a devastating epidemic of Fusarium head blight of wheat and barley put additional pressure on growers to plant Sclerotinia susceptible crops such as canola and soybean, especially in northeastern North Dakota.

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During rapid expansion of the sunflower acreage in the late 1970's and 1980's, the distribution of, and increase in inoculum densities of, S. sclerotiorum were greatly enhanced. This pathogen infects sunflowers through the roots causing Sclerotinia wilt. The disease occurs every year sunflowers are planted on infested soils and low inoculum densities result in high levels of Sclerotinia wilt (Holley et al., 1986). Also, during wet summers, Sclerotinia head rot was common. Head rot was especially important because it allowed the fungus to move from field to field via ascospores. These factors allowed the fungus to build up sclerotia populations over a wide acreage. Over the past 30 years, S. sclerotiorum has spread from the eastern half to the western portion of the state. It is now found in almost all counties, although the incidence is low in the far western counties where dryer conditions limit row crop acreage. The most heavily infested areas are the Red River Valley and the northeast quarter of the state.

Yield losses due to Sclerotinia have not been adequately measured in all susceptible crops. The 1999 epidemic of Sclerotinia head rot of sunflower was estimated to cost the industry around 100 million dollars over the North Dakota, South Dakota and Minnesota region. In canola, S. sclerotiorum was estimated to cause losses of 18 million dollars in 2000 in North Dakota (Lamey et al., 2001). In addition, the cost of controlling Sclerotinia is high. Sclerotinia affects management decisions such as which crops to rotate in fields, the length of rotations, and which fungicides to use and how and when to use them. For example, some sunflower seed companies decided that hybrid seeds should not be produced in this region because of head rot epidemics.

Besides the wide acreage of susceptible crops there are other important factors behind the success of Sclerotinia in ND. Cold soil temperatures for half the year limit the microbial activity that can reduce survival of sclerotia. Research in ND has shown that populations of sclerotia decline about 20% per year (Nelson, 1987), thus fields with high inoculum densities require numerous years in rotations to non susceptible crops to reduce levels of the pathogen. Also, compared to other states, the lower precipitation (averages 43 to 30 cm) favors drier soils and thus longer survival of sclerotia. Furthermore, precipitation is common in July and August when susceptible plants are flowering and canopies are closed, two conditions favorable for disease development. Apothecia are commonly produced in these months and ascospore inoculum can be widespread (Nelson, 1985).

ReferencesHolley, R.C., Nelson, B. 1986. Effect of plant population and inoculum density on

incidence of Sclerotinia wilt of sunflower. Phytopathology 76, 71-74. Lamey, A., Knodel, J., McKay, K., Endres, G., Fore, Z., Andol, K., and Draper, M. 2001.

2000 Canola Disease Survey. NDSU Extension Service, Report 63, Jan. 2001. Fargo, ND.Nelson, B. 1985. Ecology of Sclerotinia sclerotiorum in North Dakota. Proceedings of the

North Dakota Academy of Science 29,33.Nelson, B. 1987. Effect of crop rotation on survival of Sclerotinia sclerotiorum in soil, p6-7.

In: Proceedings Sunflower Researchers Workshop, Aberdeen, S.D. Dec. 10, 1986. 15pp.

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Environmental conditions influencing apothecial production and lettuce infection by Sclerotinia sclerotiorum in field conditions

C. S. Younga, J. A. Smitha, M. Watlingb, J.P. Clarksonc and J. M. Whippsc.

aADAS Consulting Limited, ‘Woodthorne’, Wergs Road, Wolverhampton, WV6 8TQ, UK;bADAS Consulting Limited, Terrington-St-Clement, King’s Lynn, Norfolk, PE34 4PW, UK;cHorticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.

Abstract Paper mulch stimulated germination of sclerotia of Sclerotinia sclerotiorum, but a soil fungicide inhibited germination. Batches of sclerotia, buried every fortnight during the growing season, germinated at the same times, indicating a strong relationship with environmental conditions. A simple soil temperature and water potential model for germination of sclerotia is being developed from laboratory and field data.

IntroductionSclerotinia sclerotiorum is often found at low incidence in iceberg field lettuce, but in recent years losses in some plantings have been greater than 50%. There is only one foliar applied fungicide available and there is little information on which to base decisions for optimum spray timing. The objectives of this project were to determine the key environmental factors in the field that influence apothecial production and disease, and to investigate the effects of soil mulch and fungicides.

Materials and MethodsIn 1999, at sites in Cheshire and Norfolk, plots of iceberg lettuce were planted in April, June and August, with 60 sclerotia of a local isolate buried on the same day as lettuce planting in each of the appropriate plots. The sclerotia were produced in culture and conditioned at 4oC. Treatments included paper mulch, soil fungicide (calcium cyanamide, ‘Perlka’) and foliar fungicide (iprodione, ‘Rovral Flo’). In 2000, naturally occurring disease was monitored weekly in each planting of a growers crop on a high risk peat soil in Cheshire. In Norfolk, 10 crops of lettuce were planted fortnightly throughout the 2000 season and were inoculated at three different growth stages with ascospore suspensions, and were monitored for sclerotinia disease. At both the Cheshire and Norfolk sites, sclerotia of local isolates (produced as above) were buried fortnightly from early April (10 burials in total), and were assessed for germination every week until November. At all sites, environmental conditions were monitored using a Delta T logger, with probes to record air and soil temperatures, relative humidity, rainfall, surface wetness, soil moisture and soil water tension.

Results and DiscussionAt both sites in 1999, paper mulch resulted in increased germination levels compared to non-mulched plots, e.g., in Cheshire, germination was 48% under mulch and 20% without mulch.

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Germination under mulch resulted in the formation of stipes, but not apothecia. Soil temperatures were generally, but not always, increased under mulch by up to 1oC. Soil fungicide prevented the germination of nearly all sclerotia, whereas foliar fungicides did not affect germination. The levels of disease were low overall in plots and an effect of foliar fungicide on disease could not be detected.

In 2000, the final disease incidence in the field lettuce in Cheshire was greatest in crops planted in May, reaching a maximum of 31% disease. The first disease was detected 5-7 weeks after planting. Disease incidence was <2% in crops planted from June onwards. In Norfolk, 2000, disease in plants inoculated with ascospores was generally low with several plantings having no disease. There were two exceptions: 57% infection of lettuce planted on 17 April, (inoculated after two weeks growth) and 40% infection of lettuce planted on 15 May (inoculated after 4 weeks growth). There was no consistent relationship between growth stage of lettuce at inoculation and final disease levels. The weather at both sites was particularly wet in April, May and September to November. The Cheshire site had rainfall on most days from the end of July through August, but the Norfolk site was drier than average during June to August. In the fortnightly burials of sclerotia in Cheshire, 2000, the maximum % germination was greatest (95%) in the first burial. In successive burials, the maximum germination declined and in the later burials from July onwards, the maximum % germination reached was 10% or less. The time from burial to first appearance of apothecia ranged from 6 to 14 weeks. In Norfolk, 2000, the maximum % germination (45%, in first burial) was much lower than in Cheshire. The sclerotia also took longer to germinate, but the times to first germination decreased with successive burials (24, 22, 20, 19, 17, and 16 weeks for burials 1- 6, respectively). Peaks of germination for separate burials clearly coincided in time, indicating a strong relationship with soil environmental conditions. Using a similar approach to a temperature/water potential model developed for germination of Botrytis sclerotia (Clarkson et al., 2000), a preliminary model was derived from laboratory data, based on the time to appearance of apothecia at different temperatures. When the model was applied to Cheshire field data, an upper threshold temperature of approximately 25°C and a minimum water potential value of 6-20KPa were found to give the best prediction of observed apothecial appearance in the field. These values are in the same range as other studies investigating factors affecting carpogenic germination of S. sclerotiorum (Phillips, 1987). Further analysis is in progress to refine the model and determine the conditions necessary for apothecial production for both Cheshire and Norfolk isolates.

AcknowledgementsThis work was funded by the UK Ministry of Agriculture, Fisheries and Food.

ReferencesClarkson JP, Kennedy R, Phelps K, 2000. The effect of temperature and water potential on the

production of conidia by sclerotia of Botrytis squamosa. Plant Pathology 49, 119-128Phillips AJL, 1987. Carpogenic germination of sclerotia of Sclerotinia sclerotiorum: a review.

Phytophylactica 19, 279-283.

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Infection of oilseed rape (Brassica napus) by petals containing ascospores of Sclerotinia sclerotiorum

A. McCartney, A. Heran and Q Li

Plant Pathogen Interactions, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK.

AbstractIn oilseed rape crops infection by Sclerotinia sclerotiorum is usually via ascospore-bearing petals. At petal-fall ascospore-infected petals stick to leaves initiating infections that can develop into stem rot lesions. The effects of ascospore numbers and environmental conditions on infection of oilseed rape leaves by ascospore-bearing petals are reported.

IntroductionStem rot, caused by Sclerotinia sclerotiorum, is an economically damaging disease of rapeseed crops in many parts of the World. The disease is monocyclic and symptoms are usually only apparent four to six weeks after initial infection, when significant damage has already been done to the crop. In oilseed rape the main route of infection is via ascospores deposited on oilseed rape petals. The petals act as a substrate for ascospore germination, and infection occurs when ascospore-bearing petals are deposited on leaves. We report the results of studies on the effects of ascospore numbers and environmental conditions needed for infection of oilseed rape leaves by petals.

Materials and MethodsSclerotia of S. sclerotiorum, produced by the wheat-Perlite method (Sansford & Coley-Smith, 1992), were placed in Magenta vessels containing sterilised coarse grade Perlite moistened with distilled water. A 37mm cellulose filter holder was fixed to the lid of each vessel. The cultures were kept at 15°C until the appearance of stipes, after which they were placed under near-UV radiation to encourage apothecia growth (Mylchreest & Wheeler, 1987). Ascospores released from the apothecia were harvested onto the cellulose filters. Petals were inoculated by placing them in sterile aqueous suspensions of ascospores. Plants were inoculated by placing petals containing ascospores on leaf surfaces. Experiments were done in a CE chamber where the temperature, humidity and lighting conditions could be controlled. Most experiments were done with the spring type cultivar Rebel and the same S. sclerotiorum isolate. Lesion size was assessed using the length plus width as a lesion index (LI). Experiments were done on the time needed for ascospore germination and infection of petals; the effect of ascospore and petal number on infection; the requirements of water for infection; the effect of petal age on infection; and humidity requirements for infection.

Results and DiscussionAscospores germinated on petals within a few hours of deposition, providing the petals were wet, and the petals could infect plants for at least 24h. The effectiveness of infection, as determined

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by lesion size, depended on the ascospore numbers on the leaf, up to about 100 ascospores per leaf, above this, infection appeared to be independent of ascospore numbers (Fig. 1). Similarly infection efficiency increased with the number of petals up to about 4 per leaf. It appeared to make little difference whether the petals were overlapping, touching or spaced out on the leaf. Petal age influenced the effectiveness of infection, “old” petals, that is ones from flowers that had partially formed pods, produced larger and less variable lesions than younger petals.Lesions were produced at all test temperatures (between 15 and 30°C) when the humidity was kept continuously close to saturation. Lesions were initiated in 2-4 days at temperatures between 20 and 25°C. But it took longer to initiate lesions at both 15 and 30ºC. Free water was not needed for infection, provided that humidity was high (>c100%). However, lesion sizes were two or three times larger when water was present. Between 24 and 48h of continuous high humidity (c100%) were needed to initiate lesion formation from ascospores on petals placed on leaves. However, lesions size, assessed 6 days after inoculation, increased significantly as the period of continuous humidity increased (Fig. 2). Petals which dried and were then re-wetted were still be able to cause infections: in one experiment lesions formed even when ascospore-

bearing petals were dried only 4h after being placed on the leaf and re-wetted 24h later. How long they can remain dry before re-wetting and still infect is not know.Humidity and leaf wetness clearly plays an important role in oilseed rape infection via petals. Knowledge of crop microclimate and the effects of intermittent wetting and dying of petals on infection are needed to understand and assess the risk of stem development in rapeseed crops. AcknowledgementsThis work was supported by a grant from the European Union (No. ERBIC18CT970173).ReferencesMylchreest M.J. & Wheeler B.E.J. (1987) A method for inducing apothecia from sclerotia of

Sclerotinia sclerotiorum. Plant Pathology 36, 16-20.Sansford C.E. & Coley-Smith J.R. (1992) Production and germination of Sclerotia of Sclerotinia

sclerotiorum, Plant Pathology 41, 154-156.

0

10

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1 10 100 1000 10000

Ascospores/plant

Les

ion

Inde

x

OverlappingTouchingSpaced

0

5

10

15

0 12 24 36 48 60 72 84 96 108

Time at 100% RH (h)

Les

ion

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x

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PAUC61

COCRI821

COCRI681

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P5.5

Petal fall, petal retention and petal duration in Oilseed Rape crops

A McCartneya, A Herana, Q Lib and J Freemana


AbstractOilseed rape petals bearing ascospores of Sclerotinia sclerotiorum play an important role in initiating stem rot disease in oilseed rape crops. Patterns of flowering, petal fall and petal retention measured in oilseed rape crops in the UK and china are reported.

IntroductionSclerotinia sclerotiorum is the causal pathogen of stem rot in oilseed crops and can cause significant economic damage in many parts of the World. One of the principal routes of S. sclerotiorum infection of oilseed rape crops is via ascospore infected petals. Inoculum, in the form of airborne ascospores, is deposited on petals, which in turn are deposited on the crop. The petals act as substrates for ascospore germination and growth allowing the fungus to penetrate and infect leaves, which can lead to damaging stem lesions. Thus patterns of petal fall and the duration of petals on leaves play an important role in the initiation of stem-rot epidemics. We report results of studies of the patterns of petal fall and duration made in the UK and China.

Materials and MethodsFour sets of measurements were made. (a) The rate of flowering was measured by counting the number of fully open flowers on a random sample of flower heads. The number of flower heads per unit area was also measured. (b) The rate of petal fall was measured by placing plastic trays at three different heights in the crop to collect fallen petals. (c) Petal deposition on leaves was estimated by randomly sampling leaves from three layers in the crop (0-25 cm, 25-50 cm, 50-75 cm) and counting the number of petals sticking to each leaf. (d) The duration of petals on leaves was estimated by randomly marking plants and tagging individual leaves at different heights. The number and position of petals sticking to the leaves were recorded every two to three days and the duration of each petal on the leaf was determined from these measurements. Measurements were done, using the same methodology, over 3 consecutive seasons at IACR-Rothamsted in the UK and 2 seasons at Anhui Academy of Agricultural Science (AAAS) in China.

Results and DiscussionThere were broad similarities in patterns of petal fall and petal retention at both sites. The rates of petal fall broadly reflected the pattern of flowering, except that the pattern was delayed by

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about 6-12 days, and the duration of petal fall was less (Fig. 1). The number of petals deposited on leaves at the two sites on most years tended to reach a maximum of between 10 and 15 per leaf, except in 2000 at AAAS when the maximum did not exceed about 7. At IACR fewer petals

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tended to be deposited on leaves lower in the canopy. While at AAAS, in 2000, fewer petals were deposited on the upper layer of the crop than on the middle and lower layers, but in 1999 petal deposit on all layers was similar. At both sites, generally, more than half of the petals did not persist on leaves for more than 5 days, and few lasted more than 10 days (Table 1). Petal duration on leaves tended to be shorter in 2000 at both sites than in previous years. At AAAS this difference (and the lower number of petals on leaves) could have been cause by the hot dry weather during flowering preventing petals from sticking to leaves on the upper parts of the canopy. While at IACR the shorter duration may have been due to a relatively wet season washing petals from leaves. These results suggest that weather may play a role in the duration of fallen petals on leaves, and consequently on the risk of the development of epidemics.

Table 1. Duration of petals on leaves in different layer of the crop. Values averaged over all the years of the study.

Height % of fallen petals on leaf for=2 days 3-5 days 6-10 days >10 days

UK China UK China UK China UK China<40 cm 35.0 38.5 26.7 30.0 32.3 16.5 6.0 15

40-60 cm 37.8 33 24.1 34 20.8 17.5 17.3 15.5>65 cm 28.8 32.5 31.7 38.5 15.0 14.8 24.5 14.2

Patterns of petal fall and petal retention on leaves appear to be broadly similar for European and Chinese crops. Roughly one third of petals deposited on leaves do not survive more than 2 days and a further third last less than 6 days. As it probably takes more than two days to infect leaves (under ideal conditions) it is likely than about half the ascospore-bearing petals will not persist long enough to cause infections. Petals lasted slightly longer on the lower and middle parts of the canopy than in the upper part, but the numbers of petals deposited on leaves in the lower part of the canopy was about half of that in the upper and mid-parts. Although conditions in the lower part of the crop may be more favourable for infection, the difference in the number of petals deposited probably means that the leaves in the mid-canopy are most at risk of infection. The pattern of petal fall and petal deposit on leaves suggests that the crop is most vulnerable to infection towards the end of flowering, about 25 days after the beginning of flowering in the UK.

AcknowledgementsThis work was supported by a grant from the European Union (No. ERBIC18CT970173).

P5.6

Temporal and spatial distribution of ascospores of Sclerotinia sclerotiorum around tobacco greenhouses in North Carolina

H.M. Hartzog, T.A. Melton and H.D. Shew

Department of Plant Pathology, NC State University, Raleigh, NC 27695-7616, USA.

AbstractAscospores numbers of Sclerotinia sclerotiorum were monitored monthly over a 21/2 year period to determine when inoculum was present around tobacco greenhouses. Ascospore production occurred between February and May, with no activity during summer and fall. Data are being used to design new inoculum management strategies for S. sclerotiorum.

IntroductionDisease management for tobacco seedling production in greenhouses is based on providing environmental conditions that favor plant health but limit disease development. Because the environmental parameters required for optimal seedling production also favor development of disease caused by Sclerotinia sclerotiorum, and no chemicals are currently labeled for control of the disease in the greenhouse, emphasis has been placed on inoculum management. Ascospores of the pathogen are the primary inoculum for the disease and enter greenhouses through open side vents (Gutierrez & Shew, 1998). Disease develops once a dense canopy is formed and wounding occurs (Gutierrez & Shew, 2000).Ascospores of S. sclerotiorum can be trapped and quantified with a semi-selective agar medium (Gutierrez & Shew, 1998). Trapping between 9 a.m. and noon, the peak ascospore release times, can pinpoint inoculum sources and show trends in activity around houses. Environmental factors that induce apothecia in culture have been identified, but little is understood about conditions necessary for ascospore production around tobacco greenhouses. The objectives of this study were to determine the temporal and spatial distribution of ascospores around greenhouses during the tobacco transplant production season and throughout the year and to attempt to correlate environmental factors with ascospore production.

Materials and MethodsThree greenhouses in central North Carolina were chosen to monitor ascospores numbers of S. sclerotiorum. Trapping was performed three to five times per week during the spring transplant production period (February to April of 1999, 2000 and 2001) and then less frequently throughout the rest of the year. Petri dishes containing a semi-selective agar medium (Gutierrez & Shew, 1998) were arranged around the periphery of each greenhouse and opened for one hour between 9:30 and 11:30 a.m. Exposed dishes then were incubated in the dark for 72 hours before colony counts were made. Representative colonies were plated onto new medium and identification of S. sclerotiorum was confirmed. Weather data loggers (HOBO series, Spectrum Technologies) were placed at two of the greenhouse locations and hourly measurements of

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temperature, relative humidity, and rainfall were recorded in 2000 and 2001. Disease assessment were made in houses to determine when first foci of disease developed.

Results and DiscussionPeak ascospore release occurred in March in each year of the study, with significant numbers of ascospores also trapped in late February and most of April. These dates represent the period of rising temperatures after winter and during tobacco seedling production. Few or no ascospores were trapped at other times of the year, indicating that the isolates of S. sclerotiorum that infect tobacco release ascospores only in the Spring of the year. Other isolates of S. sclerotiorum in North Carolina release ascospores predominantly in the fall to early winter (Hudyncia, 2000).

Trapping results confirm previous observations (Gutierrez & Shew, 1998) that tobacco isolates of S. sclerotiorum have a steep dispersal gradient from source apothecia. Localized areas of inoculum were detected around greenhouses A and B, whereas inoculum distribution was uniform around greenhouse C. These clusters of inoculum were observed over the three spring trapping seasons. The reoccurrence of localized areas of inoculum around certain sites suggests the fungus is cycling on alternative hosts around greenhouses, probably weed hosts, with little decline in fecundity. Trapping during the spring production season can be used as a tool to locate inoculum sources, which may allow for improved strategies for inoculum management such as directed fungicide sprays or biocontrol

Rainfall events were significantly correlated with ascospore release at only one location, and ambient temperatures were not correlated with ascospore numbers over the three spring-trapping periods, Therefore, precise forecasting of collar rot occurrence based on environmental parameters is not possible at present.

ReferencesGutierrez WA, Shew HD, 1998. Identification and quantification of ascospores as the primary

inoculum for collar rot of greenhouse produced tobacco seedlings. Plant Dis. 82, 485-490. Gutierrez WA, Shew HD, 2000. Factors that affect development of Sclerotinia collar rot on

tobacco seedlings grown in greenhouses. Plant Dis. 84:1076-1080.Hudyncia J, 2000. Examination of the ecology and reproductive biology of Sclerotinia

sclerotiorum on cabbage and tobacco. MS Thesis, NC State Univ.

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Susceptibility of spring oilseed rape to sclerotinia stem rot in Poland and the Czech Republic

Jedryczka M.a, Dakowska S.a and Plachka E.b

a Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, Polandb OSEVA PRO, Institute of Oil Crops, Purkynova 6, 746-01 Opava, the Czech Republic

AbstractFourty-one cultivars of spring oilseed rape of different origin (Australia, Canada and several countries in Europe) were inoculated in field (Poland and the Czech Republic) and glasshouse conditions. For artificial infection 2 isolates of Sclerotinia sclerotiorum and 3 methods were used: inoculation with wheat grains, agar discs and wooden cloth pins. Cultivars differed from tolerant to very susceptible.IntroductionIn Poland and the Czech Republic the prevailing form of oilseed rape is the winter type (Brassica napus subs. oleifera forma biennis). However, for the last few years the spring form has been also cultivated on some areas. The increase of spring rape cultivation is the result of harsh winters in 1995 and 1996, which severely damaged winter types of rapeseed. The spectrum of the pathogens attacking both types of oilseed rape is the same, but the severity of particular diseases is different. Recent observations of plants in natural conditions have shown that one of the most frequent diseases is dark spot caused by fungi from the genus Alternaria (A. brassicae, A. brassicicola) (Sadowski et al., 2001). Plant damage can be also caused by the attack of the fungus S. sclerotiorum, resulting in stem rot. The disease was observed on spring rape in Canada (Morrall & Dueck, 1982), China, many countries in Europe, including Poland, the Czech Republic and Russia (Jedryczka et al., 2001). The main source of inoculum are the ascospores, which are produced in apothecia and released in the spring. At this time the plants of spring rape are young and vulnerable to the disease. So far, in Poland and the Czech Republic, severe damage of plants was observed in the adult stage of plant development, but the occurrence of strong disease symptoms in the rosette stage was already reported in the USA by Phillips (1999).The aim of this work was to evaluate the susceptibility of numerous cultivars of spring oilseed rape to S. sclerotiorum. To achieve this goal similar experiments with artificial inoculation of spring rapeseed plants were done in Poland and the Czech Republic. The cultivars represented broad diversity in respect to their geographical origin and the genetic background. Materials and MethodsPlants: 41 cultivars of spring oilseed rape originating from Poland, Germany, Sweden, Denmark, France, Russia, Canada and Australia. Fungi: 2 isolates of S. sclerotiorum (isolate Sc23 from winter oilseed rape, isolate B1 from sunflower). For each cultivar, 50 plants were inoculated (2 reps per 25 plants) in the field and 48 plants in the glasshouse (4 pots with 6 plants in 2 reps). Inoculation techniques: 1) Wheat grains overgrown with mycelium of a particular isolate were attached to the stem, 15-25 cm from the ground level. Stems were scratched in the place of inoculation. The grain was supplied with the moist cotton wool and attached to the stem with aluminium foil. 2) Plants were inoculated with wooden cloth pins overgrown with the mycelium.

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The mycelium was grown on liquid Czapek-Dox medium for 10 days, in static culture. 3) Mycelium was subcultured on Potato Sucrose Agar medium for 10 days and than 5 mm agar discs were attached to the wounded stem. Aluminium foil stripes protected inoculum from drying and falling off the plants. Evaluation of symptoms: In field conditions the symptoms were evaluated 2, 3 and 4 weeks after inoculation, in glasshouse the disease symptoms were scored after 1 and 2 weeks. The disease parameters were as follows: 1) length of stem discolouration, 2) scoring according to 0-9 scale, where 0 was no symptoms and 9 was the dead plant. Location of field experiments: 1) Cerekwica (ca. 20 km from Poznan), Poland - experiment field of the Institute of Plant Genetics PAS, 2) suburb of Opava - experiment field of the Institute of Oil Crops, the Czech Republic.Results and DiscussionFrom three methods of inoculation used, infection with wheat grains, proposed by Lewartowska et al. (1994), was the most effective in the field. Inoculation with agar discs was the best for glasshouse conditions. Thicker plant stems and changing weather conditions in the field required inoculation with material allowing to better keep the moisture in order to prevent the inoculum from drying too quickly after inoculation. However, the symptoms of such inoculation method used in the glasshouse were too quick and strong, so a less invasive inoculation with agar discs is recommended. Inoculation with wooden pins was the most unreliable in respect to repetitiveness of results. Very hot weather after inoculation, caused fast drying of mycelium on wooden cloth pins, which were not covered with aluminium foil. There was high statistical correlation between both parameters of evaluation (length of stem discolouration and bonitation score). Both parameters can be used in order to describe the symptoms of infection. The results obtained in all times of evaluation were highly correlated, which suggests a high flexibility in choosing the time of disease scoring. The best time for symptom evaluation is between 2 and 3 weeks after inoculation and depends on temperature and humidity in the field and glasshouse. The most tolerant lines were Polish cv./lines Margo, POHO 01, MAH 599 & MAH 1400 (ZDHiAR-Malyszyn), cv. Narenda & Dunkeld from Australia, cv. Licosmos & Lisonne from Germany (DSV), cv. Sponsor from Sweden (Svalof) and Star from Denmark (DLF). Most cultivars from Russia (Slawuticz, Rubiez, Dubrawinskij, Rytm, Lira, Ratnik) & Canada (Profit, Quantum & Westar) were highly infected by S. sclerotiorum. The difference between tolerant and susceptible lines was equal to 2 scores of the 0-9 scale or 4 cm length of stem discolouration.ReferencesJedryczka M, Nikonorenkov AV, Levitin M, Gasich E, Lewartowska E, Portenko L, 2001. Spectrum and

severity of fungal diseases on spring oilseed rape in Russia. IOBC/wprs Bulletin (in press)Lewartowska E., Jedryczka M, Frencel I, 1994. The methods of winter oilseed rape (Brassica napus L.)

resistance evaluation against Sclerotinia sclerotiorum (Lib.) de Bary. Plant Science (Sofia) 31 (7), 252-254Morrall RAA, Dueck J, 1982. Epidemiology of sclerotinia stem rot of rapeseed in Saskatchewan. Canadian

Journal of Plant Pathology 4, 161-168.Phillips DV, 1999. Clonality in Sclerotinia sclerotiorum isolates producing different symptoms on canola

across southern USA. 10th International Rapeseed Congress, 26-29.09.1999, Canberra, Australia. Proceedings on CD-Rom, session C05

Sadowski Cz, Dakowska S, Lukanowski A, Jedryczka M, 2001. Occurrence of fungal diseases on spring rape in Poland. IOBC/wprs Bulletin (in press).

P5.8

Factors affecting on the production of ascospores of Sclerotinia sclerotiorum (Lib) De Bary and their role in infection of sunflower.

Rajender Singh and N.N.Tripathi

Department of Plant Pathology, CCS HAU-Hisar-125004-India

AbstractSclerotinia sclerotiorum causes Sclerotinia-rot of sunflower. Mature sclerotia produced apothecia in laboratory condition in 19 days at 15±5°C. Germination of ascospores was recorded between 9-30°c, highest being 64.66% at 21°C. Maximum head-rot was recorded after 72hr wetting period, when inoculated with 2x103 ascospores/ml. No infection was obtained on uninjured plant parts except leaf blade petiole junction after inoculation.

IntroductionInfection of above ground parts of sunflower incited by airborne ascospores released from apothecium of carpogenically germinated sclerotium of Sclerotinia sclerotiorum has been reported (Young & Morris, 1927). A successful infection by ascospores required a film of water on host surface (Purdy, 1958). The present systematic studies have been iniatiated to find out role of physical factors on ascospores germination and infection of aerial parts.

Materials and MethodsApothecia were produced by the modified method of Bedi (1956) by placing sclerotia at 2 cm depth in sandy soil under 40-watt tubelight in laboratory conditions (15±5°c). Standardised ascospores suspensions were put on sterilised glass slide in moist chamber and finally kept at different temperature to find out optimum condition for germination.Ascospores suspension having 1 x 103 ascospores/ml (Hawthome & Jarvis, 1973) was used to inoculate at bud initiation stage with sterilised hypodermic syringe with ten replication of each treatment along with control. Symptoms were recorded with the help of 0-5 scale (Yang & Thomas, 1980) for head-rot measurement. Detached heads were inoculated with 5 ml ascospores suspensions on receptacle side and pedicel submerged in water to see the effect of wetness period. The inoculated part was covered with moist cotton swab which were removed after an interval of 6 hr. Ascospores suspension was inoculated on the stem, leaf blade, petiole and leaf blade-petiole junction at 10,12,14 and 17th leaf position to find out successful infection. Sclerotia were placed in the vicinity of root zone as proposed by Huang & Kozub (1990) to determine systemic infection.

Results and DiscussionApothecia were produced in 19 days. Ascospores germination was initiated at 9°c, highest being at 21°c (64.66%) followed by 18°C. Sedum & Brown (1987) also noted best ascospores germination at 20 ± 1 °C. Maximum head-rot (43.33%) was recorded at 2 ml ascospores suspension (Table-2). Infection did not initiate on uninjured plant parts. It seems that injury was a prerequisite for head infection. Sedum & Brown (1987) also opined the similar view. Rotting of head initiated after 36 hr of wetting period, which attained

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maximum after 72hr (Table-3). While working on head-rot, Lamarque (1983) corroborated the present findings. Infection was not recorded on any of the plant parts where ascospores suspension was placed except leaf blade-petiole junction. First lesion developed after 6 days on leaf blade petiole junction irrespective of leaf position. After 25 days, disease progressed on stem upto 36 cms irrespective of petiole thickness, stem girth and plant height. It may be due to

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availability of more moisture and exudation of nutrients at leaf blade-petiole junction, which may be responsible for ascospores germination and further infection.Light brown lesion girdled entire stem at collar region in 10 days followed by white mycelial growth on infected stem. The disease progressed very fast and finally plant dried off in 28 days irrespective of crop stage. The heads of such plants, which were covered with cloth bags, were not found infected, though drying was most visible. It is, therefore, concluded that infection could not reach systemically from root to head of sunflower. Ascospores have been recorded as major source of infection in present investigation, which get positive support of Young & Morris (1927).

Table 1. Effect of temperature on ascospores germination of Sclerotinia sclerotiorum.Temperature (°c) 9 12 15 18 21 24 27 30Per cent germination 25.00 28.57 40.00 61.53 64.66 58.28 55.55 32.28

(30.00) (32.30)* (39.23) (51.33) (53.32) (55.55) (48.13) (34.60)C.D at 5% 1.72.

Table 2. Effect of ascospores concentration on development of Sclerotinia head-rot.Inoculum (x103/ml) 0.5 1.00 1.5 2.00 ControlDisease intensity (%) 21.33 28.57 38.33 43.33 0.00

(32.31)* (38.31) (38.25) (41.15)C.D at 5% 1.72 *Fig in parentheses are angular transformed value.

Table 3. Effect of wetness period on the development of Sclerotinia head-rot.Wetness period (hr) 6 12 24 30 36 42 48 54 60 72Rotting after days

Initiation 0 0 0 0 0 6 5 3 3 2 Completion 0 0 0 0 0 0 12 10 8 8

Rotting index 0 0 0 0 0 3.3 4.1 4.6 4.6 5.1

ReferencesBedi KS, 1956. A simple method for production of apothecia of Sclerotinia sclerotiorum (lib)

De Bary. Indian Phytopathology 9, 39-43.Lamarque C, 1983. Climatic condition necessary for sunflower infection by Sclerotinia

sclerotiorum : Forecasting of local epidemic. EPPO Bulletin. 13, 75-78.Hawthorne BT, Jarvis WR, 1973. Differential activity of fungicides in various stage in the

life cycle of Sclerotinia spp. New Zealand Journal of Agriculture Research 16, 551-557.Huang HC, Kozub, GC 1990. Cyclic occurrence of Sclerotinia wilt in western Canada. Plant

Disease 74, 766-770.Purdy LH,1958. Some factors affecting penetration and infection by Sclerotinia sclerotiorum.

Phythopathology 48, 605-609.Sedum FS , Brown JF, 1987. Infection of sunflower leaves by ascospores of Sclerotinia

sclerotiorum. Annals of Applied Biology 110, 275-285.Yang SM, Thomas CA, 1980. An effective technique for inoculating glasshouse grown

sunflower species of Rhizopus. Annals of Phytopathological Society of Japan 46, 553-555.

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Young PA, Morris HE, 1927. Sclerotinia wilt of sunflower Bulletin 208, University of Montana Agricultural Experimental Station, Montana.







Delegate List

Title Forenames Surname Company Country Email

Prof Nazira Aitkhozhina Insitute of Microbiology and Virology Kazakstan [email protected]

Dr Paul Beales Central Science Laboratory UK [email protected] Olivier Belet Natural Resources Institute UK [email protected]

Sharon Billings Syngenta USA [email protected]

Dr Greg J Boland University of Guelph Canada [email protected] Graeme W Bourdôt Agresearch Ltd New Zealand [email protected]

Mr Gary Bradbury Fisher Foods Limited UK [email protected]

Christopher Bruel Universite Claude Bernard France [email protected]

Miss Odile Carisse Agriculture and Agri-food Canada Canada [email protected] Kelly Chenault USDA – ARS USA [email protected]

Dr John M Clarkson Horticulture Research International UK [email protected] Gordon Couper Aberdeen University UK [email protected] Dermot P Coyne University of Nebraska USA [email protected] Michel De Lange Sygenta Seeds BV Netherlands [email protected]

Yves Decroos BIPA Belgium [email protected]

Dr Joachim Degener Aventis CropScience NV Belgium [email protected]

Dr Anne Dorrance The Ohio State University USA [email protected]

Sandra Beatriz Durman University of Buenos Aires Argentina [email protected]

Dr Jamshid Fatehi Swedish University of Agricultural Sciences Sweden [email protected]

Dr Xinmei Feng Swedish University of Agricultural Sciences Sweden [email protected]

Dr A Ruth Finlay Fargro Ltd UK [email protected]

Ing Agr Vivienne Gepp Facultad de Agronomia Uruguay [email protected] Peter Gladders ADAS UK [email protected] George Graef University of Nebraska USA [email protected]

Prof Kenneth F Grafton North Dakota State University USA [email protected]

Dr Thomas Gulya US Department of Agriculture USA [email protected]

Dr Volker Hahn State Plant Breeding Institute Germany [email protected]

Dr Nigel Hardwick CSL UK [email protected]

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Dr Mark J Hocart SAC UK [email protected]

Dr Stephen Hoyte Hortresearch New Zealand [email protected]

Dr Henry Huang Agriculture and Agri-food Canada Canada [email protected]

Mrs Yong-Ju Huang IACR Rothamsted UK [email protected]

Mr Kelvin Hughes Central Science Laboratory UK [email protected]

Mr Geoff A Hurrell Agresearch Ltd New Zealand [email protected]

Dr Malgorzata Jedryczka Insitute of Plant Genetics Pas Poland [email protected]

Mr Clinton Jurke Advanta Canada Inc Canada [email protected]

Prof James D Kelly Michigan State University USA [email protected]

Prof Linda M Kohn University of Toronto Canada [email protected]

Mr Steven Koike University of California USA [email protected]

Mrs Cezarina Kora University of Guelph Ontario [email protected]

Dr Joseph M Krupinsky USDA-ARS USA [email protected]

Linda Sue Kull University of Illinois USA [email protected]

Dr Charles Lane Central Science Laboratory UK [email protected]

Dr Thomas A Lee Jr Texas Agricultural Extension Service USA [email protected]

Dr Roseann Leiner UAF AFES Palmer Research Center USA [email protected]

Dr Jean Liu Aventis CropScience Canada [email protected]

Dr Peter Lüth Prophyta Germany [email protected]

Dr Christian Martin Agriphyto France [email protected]

Dr Charlie A Martinson Iowa State University USA [email protected]

Dr Michael E Matheron University of Arizona USA [email protected]

Dr Alastair McCartney IACR Rothamsted UK [email protected]

Dr Mark J McQuilken Scottish Agricultural College UK [email protected]

Dr Hassan A Melouk USDA-ARS, PSWCRL USA [email protected]

Zeljko Micic State Plant Breeding Institute Germany [email protected]

Dr Phil Miklas USDA/ARS USA [email protected]

A Moret University of Barcelona Spain [email protected]

Prof Berlin D Nelson North Dakota State University USA [email protected]

Sclerotinia 2001195


Dr Albert O Paulus University of California USA [email protected]

Dr Dan Phillips University of Georgia USA [email protected]

Dr Ian Porter Agriculture Victoria Australia [email protected]

Dr Khalid Y Rashid Agriculture and Agri-food Canada Canada [email protected]

Dr S Roger Rimmer Agriculture and Agri-food Canada Canada [email protected]

María Alejandra Rodriguez University of Buenos Aires Argentina [email protected]

Dr Barbara Shew North Carolina State University USA [email protected]

Prof David Shew North Carolina State University USA [email protected]

Dr Asha Shivpuri Rajasthan Agricultural University India [email protected]

Dr Daina Simmonds Agriculture and Agri-Food Canada Canada [email protected]

Dr Rama Singh Punjab Agricultural University India [email protected]

Miss Julie Smith ADAS Wolverhampton UK [email protected]

Mr Brent Spangler The Pennsylvania State University USA [email protected]

Mrs Kate Spangler The Pennsylvania State University USA [email protected]

Prof James R Steadman University of Nebraska USA [email protected]

Prof Alison Stewart Lincoln University New Zealand [email protected]

Prof Dr A v Tiedemann Universität Rostock Germany [email protected]

Miss Vicky Toussaint Agriculture and Agri-food Canada Canada [email protected]

Mr M Turner Oxford Plant Sciences Ltd UK [email protected]

Dr Eva Twengström Swedish University of Agricultural Sciences Sweden [email protected]

Dr Rogério Faria Vieira EPAMIG Brazil [email protected]

Dr Tri D Vuong University of Illinois USA [email protected]

Prof John M Whipps HRI UK [email protected]

Dr Paul Wood University of Bristol UK [email protected]

Dr Chunren Wu Limagrain Canada Seeds Inc Canada [email protected]

Prof X B Yang Iowa State University USA [email protected]

Dr Caroline Young ADAS UK [email protected]

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Outline Programme

Day Coach 08.15 09.00-11.00 Coffee 11.30-13.00 Lunch 14.00-15.30 Tea 16.00-17.30 Coach 17.45 19.30

Sunday 8 Registration Dinner & Welcome receptionUniversity

Monday 9 Session I Session II Session III Session IV

Biology, taxonomy &

molecular biology

Biology, taxonomy &

molecular biology

Posters Poster discussion DinnerUniversity

Tuesday 10 Session V Session VI Session VII

Control Control Coach trip around North Yorkshire Dinner York

Wednesday 11 Session VIII Session IX Session X Session XI

Resistance Pathology & epidemiology

Epidemiology ISPP Sclerotinia Committee

DinnerUniversity

Thursday 12 Departure

Sclerotinia 2001

1 · Web view07.30 Breakfast 08.15 Coaches to Central Science Laboratory Session I Biology,...

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Transcript of 1 · Web view07.30 Breakfast 08.15 Coaches to Central Science Laboratory Session I Biology,...