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IOBC / WPRS

Working group „Integrated Protection of Fruit Crops” Sub group “Pome Fruit Diseases”

Proceedings of the Meetings

at

Lindau (Germany)

31 August – 5 September 2002

and

Piacenza (Italy)

31 August – 3 September 2005

Edited by

Cesare Gessler, Vittorio Rossi and Simona Giosuè

IOBC wprs Bulletin Bulletin OILB srop Vol. 29 (1) 2006

The content of the contributions is in the responsibility of the authors

The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palearctic Regional Section (IOBC/WPRS) Le Bulletin OILB/SROP est publié par l‘Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles, section Regionale Ouest Paléarctique (OILB/SROP) Copyright: IOBC/WPRS 2006 The Publication Commission of the IOBC/WPRS: Horst Bathon Federal Biological Research Center for Agriculture and Forestry (BBA) Institute for Biological Control Heinrichstr. 243 D-64287 Darmstadt (Germany) Tel +49 6151 407-225, Fax +49 6151 407-290 e-mail: [email protected]

Luc Tirry University of Gent Laboratory of Agrozoology Department of Crop Protection Coupure Links 653 B-9000 Gent (Belgium) Tel +32-9-2646152, Fax +32-9-2646239 e-mail: [email protected]

Address General Secretariat: Dr. Philippe C. Nicot INRA – Unité de Pathologie Végétale Domaine St Maurice - B.P. 94 F-84143 Monfavet Cedex France ISBN 92-9067-184-8 http://www.iobc-wprs.org

Pome Fruit Diseases IOBC/wprs Bull. 29(1), 2006

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Preface This Bulletin contains the proceedings of the meetings of the IOBC/WPRS Working Group “Integrated Protection of Fruit Crops” subgroup “Pome fruits diseases”. This volume includes the contributions from two workshops: the 6th International Workshop on Pome fruit Diseases held in Lindau, Germany, 31 August – 5 September 2002, and the 7th International Workshop on Orchard Diseases, held in Piacenza, Italy, 31 August – 3 September 2005. The Bulletin contains 41 contributions: 16 from the Lindau meeting and 25 from Piacenza. They concern several aspects of the most important fruit crop diseases, including epidemiology, biological and integrated control. Also diseases of increasing importance in Europe, like brown spot and bark canker on pears, were deeply investigated by several contributions of participants and round tables. In aggregate, 78 scientists from 15 countries in Europe, United States and Canada attended the Workshops. The local organization of the Workshops was handled by Peter Triloff and myself in collaboration with competent co-workers, for Lindau and Piacenza, respectively. Both organizers took care of the scientific planning and also handled the organizing details to guarantee successful meetings. In both occasions excursions were planned to visit pear and apple orchards both conventional and organic, showing different cropping methods, pathological problems, and strategies of fungicide applications. The next Workshop of this subgroup will be organized in Denmark in 2008, probably in October, by Arne Stensvand and two colleagues from Denmark. I believe that this Bulletin will be an useful tool for scientists and technicians as an updating of the knowledge about epidemiology and optimal control strategies for both well known and emerging diseases. Vittorio Rossi Convenor of the subgroup “Pome fruit diseases”

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We thank all the sponsors of the meeting in Lindau (2002):

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Contents Preface........................................................................................................................................ i Sponsors of the Lindau Meeting .............................................................................................. ii List of Participants in Lindau, 2002 ........................................................................................ vii List of Participents in Piacenza, 2005 ...................................................................................... xi Meeting at Lindau (Germany), 2002 Control of apple scab (Venturia inaequalis) in organic apple growing. StopScab: A

Danish research programme for screening substitutes to copper fungicides Marianne Bengtsson, John Hockenhull ........................................................................... 1

Apple scab IPM: preliminary report on the application of a new sampling technique to determine “scab-risk” J. Reardon, L. Berkett, M. Garcia, A. Gotlieb, T. Ashikaga, G. Badger.......................... 5

Spatial distribution of ascospores of Venturia inaequalis within the tree canopy O. Carisse, D. Rolland, J. Charest................................................................................... 9

Sanitation practices to reduce apple scab inoculum in orchards Piet Creemers, Alida Vanmechelen, Kjell Hauke .......................................................... 15

Effect of apple cultivar mixtures on the epidemic of Venturia inaequalis in a treated orchard F. Didelot, L. Brun, S. Clément, L. Parisi ...................................................................... 25

Phytotoxic effect of lime sulphur on apple and pear Bart Heijne, Peter Frans de Jong, Imre Janos Holb ..................................................... 31

Pome fruit storage diseases Joana Henriques ............................................................................................................ 37

Durable disease resistance and high fruit quality, a challenge for apple breeding Markus Kellerhals, Cornelia Sauer, Ernst Höhn, Barbara Guggenbühl, Jürg Frey, Robert Liebhard, Cesare Gessler ......................................................................... 43

Geographical distribution of Venturia inaequalis strains virulent to the Vf gene in Europe L. Parisi, F. Laurens, F. Didelot, K. Evans, C. Fischer, V. Fouillet, F. Gennari, H. Kemp, M. Lateur, A. Patocchi, H. Schouten, C. Tsipouridis..................................... 49

Factors influencing deposition of Venturia inaequalis ascospores on apple trees Vittorio Rossi, Simona Giosuè, Riccardo Bugiani ........................................................ 53

A Chorus tolerant population of Venturia inaequalis found in a South African apple orchard Wolf Schwabe ................................................................................................................ 59

Evaluation of in-vitro grown apple shoot sensitivity to Venturia inaequalis using a detached leaf assay E. Silfverberg-Dilworth, Andrea Patocchi, Cesare Gessler .......................................... 67

An adaptation of the New Hampshire degree-day model to predict ascospore release of Venturia inaequalis in Norway Arne Stensvand, David M. Gadoury, Terje Amundsen, Robert C. Seem ....................... 75

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Meteorological data for warning systems: some views concerning sensors Christer Tornéus ............................................................................................................ 83

The simulation of ascospore release from apple scab: do we use suitable climatic data? Peter Triloff .................................................................................................................... 87

Chemical control of apple powdery mildew (Podosphaera leucotricha): mode of actions Xiangming Xu, Joyce Robinson, Angela Berrie ............................................................. 95

Meeting at Piacenza (Italy), 2005 Biological characteristics of dicarboximide-resistant isolates of Stemphylium vesica-

rium from Italian pear orchards Giulia Alberoni, Marina Collina, Agostino Brunelli .................................................. 109

Control of brown spot of pear in organic pear orchard Loredana Antoniacci, Riccardo Bugiani, Rossana Rossi ........................................... 117

Screening of organically based fungicides for apple scab (Venturia inaequalis) control and a histopathological study of the mode of action of a resistance inducer Marianne Bengtsson, Hans J. Lyngs Jørgensen, Anh Pham, Ednar Wulff, John Hockenhull ................................................................................................................... 123

Development of an integrated pest and disease management system for apples to produce fruit free from pesticide residues – Aspects of disease control Angela Berrie, Jerry Cross........................................................................................... 129

Evaluation of alternative treatments to urea to eliminate leaf litter in organic apple production Angela Berrie, Barbara Ellerker, Karen Lower .......................................................... 139

Heterogeneity in apple scab: implication for management Odile Carisse, C. Meloche, Tristan Jobin, D. Rolland ................................................ 145

Field and in vitro sensitivity of Valsa ceratosperma (Cytospora vitis) to fungicides Marina Collina, Elena Cicognani, Benedetta Galletti, Agostino Brunelli .................. 151

Sensitivity in vitro of Stemphylium vesicarium to fungicides Marina Collina, Giulia Alberoni, Agostino Brunelli .................................................. 155

Relationship between biological agent populations and biocontrol of Monilinia spp in peaches Antonieta De Cal, Inmaculada Larena, Belén Guijarro, Rosario Torres, Mar Liñan, Pietro Domenichini, Alberto Bellini, Xavier Ochoa de Eribe, Josep Usall, Paloma Melgarejo ....................................................................................................... 163

Modelling dynamics of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot of pear Simona Giosuè, Vittorio Rossi, Riccardo Bugiani, Chiara Mazzoni ........................... 169

Fungicide resistance in apple scab in the province of Québec: an overview of the problem and its implications for disease management Tristan Jobin, Odile Carisse ........................................................................................ 177

New strategies to improve the efficacy of BSPcast for control of Stemphylium vesicarium on pear Isidre Llorente, A. Vilardell, Pere Vilardell, Emilio Montesinos ................................ 181

Preliminary studies on biology and epidemiology of Valsa ceratosperma (Cytospora vitis), the causal agent of bark canker on pear in Italy

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Carla Montuschi, Marina Collina, Loredana Antoniacci, Elena Cicognani, Silvia Rimondi, Roberta Trapella, Eva Baruzzi, Chiara Mazzoni, Mirco Iotti, Agostino Brunelli......................................................................................................................... 183

Virulence of Stemphylium vesicarium isolates from pear and other host species Elisabetta Pattori, Vittorio Rossi, Riccardo Bugiani, Simona Giosuè......................... 195

Organic field-testing of compounds to control apple scab (Venturia inaequalis) in combination with alleyway cover crops Hanne Lindhard Pedersen, Lars P. Christensen, Marianne Bengtsson, Klaus Paaske, John Hockenhull ............................................................................................. 207

Evaluating the use of RIMpro and Metos weather stations for control of apple scab (Venturia inaequalis) in Denmark 2002-2005 Hanne Lindhard Pedersen, Karen Linddal Pedersen, Klaus Paaske .......................... 213

Use of bulk ascospore samples for monitoring apple scab fungicide resistance in individual orchards Vincent Philion ............................................................................................................ 219

Temperature and humidity requirements for germination and infection by ascospores of Pleospora allii, the teleomorph of Stemphylium vesicarium Vittorio Rossi, Elisabetta Pattori, Simona Giosuè ...................................................... 223

Equations for the distribution of Venturia inaequalis ascospores versus time during infection periods Vittorio Rossi, Simona Giosuè, Riccardo Bugiani ...................................................... 231

Climatic conditions prior to green tip of apple affect ascospore maturation in Venturia inaequalis Arne Stensvand, Håvard Eikemo, David M. Gadoury, Robert C. Seem ..................... 243

Application of the BSPCast model to control Stemphylium vesicarium in a district of the Emilia-Romagna region Clelia Tosi, Massimo Liboni, Riccardo Bugiani ......................................................... 249

Resistance management in Vf resistant organic apple orchards Marc Trapman ............................................................................................................. 253

Infection risk and biological parameters: automating fungal spore count and leaf growth measurements Stijn Van Laer, Peter Jaeken, Piet Creemers .............................................................. 259

Alternaria alternata, causal agent of dead (dormant) flower bud disease of pear M. Wenneker, L.T. Tjou-Tam-Sin, A.S. van Bruggen, P. Vink .................................... 265

Testing alternative chemicals against apple scab and powdery mildew Xiangming Xu, Joyce Robinson, Angela Berrie .......................................................... 271

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List of Participants, Lindau 2002 Bengtsson, Marianne V............... Plant Pathology Section,Department of Plant Biology

The Royal Veterinary and Agricultural University (KVL) Thorvaldsensvej 40 DK-1871 Frederiksberg C (Denmark) [email protected]

Berkett, Dr. Loraine .................... Department of Plant & Soil Science University of Vermiont, Hills Building 105 Carrigan Drive Burlington, VT 05405 (USA) [email protected]

Berrie, Dr. Angela....................... East Malling Research New Road East Malling ME19 6BJ Kent (United Kingdom) [email protected]

Boshuizen, Adrie......................... Horti Bureau Wageningen P.O.Box 592 NL-6700 AN Wageningen (The Netherlands) [email protected]

Bugiani, Riccardo........................ Servizio Fitosanitario Regione Emilia-Romagna Via di Corticella 133 I-40129 Bologna (Italy) [email protected]

Creemers, Piet ............................. Research Mycology RSF-Royal Research Station of Gorsem De Brede Akker, 13 B 3800 Sint-Truiden (Belgium) [email protected]

Didelot, Frédérique ..................... Unité de Pathologie Végétale I.N.R.A. 42, rue Georges Morel B.P. 57 49071 Beaucouzé (France) [email protected]

Gessler, Dr. Cesare...................... Institute of Plant Sciences Pathology ETH-Zentrum LFW Universitätstr 2 CH 8092 Zürich (Switzerland) [email protected]

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Giosuè, Simona ........................... Istituto di Patologia vegetale Università Cattolica del Sacro Cuore Via Emilia Parmense 84 29100 Piacenza (Italy) [email protected]

Giraud, Michel ............................ Ctifl-Centre de Lanxade BP21 24130 Prigonrieux (France) [email protected]

Haug, Phillip ............................... Säntisstr. 4b 78464 Konstanz (Germany)

Heijne, Dr. Bart........................... Applied Plant Research (PPO-fruit) PO Box 200 NL 6670 AE Zetten (The Netherlands) [email protected]

Henriques, Joana ......................... CEFA - Centro de Estudos de Fitossanidade de Armazenamento Rua Almirante Candido Reis, 8-3º 2500-125 Caldas da Rainha (Portugal) [email protected]

Hockenhull, John ........................ Plant Pathology Section Royal Vet. & Agricultural University Copenhagen (Denmark) [email protected]

Hull, Dr. Jerome.......................... Department of Horticulture. Michigan State University A338 Plant & Soil Sciences Bldg East Lansing Michigan 48824 (USA) [email protected]

Kellerhals, Dr. Markus................ Eidg. Forschungsanstalt Postfach 185 CH-8820 Wädenswil (Switzerland) [email protected]

Köhl, Dr. Jürgen.......................... Plant Research International P.O. Box 16 6700 AA Wageningen (The Netherlands) [email protected]

Kovacs, Dr. Gabriele................... Abteilung Wirksamkeitsprüfung und Phytotoxizität Institut für Pflanzenschutzmittelbewertung & -zulassung Spargelfeldstrasse 191 1226 Wien (Austria) [email protected]

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Liebhard, Robert ......................... Institut of Plant Sciences Pathology ETH-Zentrum LFW Universitätstr 2 8092 Zürich (Switzerland) [email protected]

MacHardy, Bill............................ 34 Woodridge Road Durham NH 03824-3597 (USA) [email protected]

Parisi, Luciana............................. Unité de Pathologie Végétale INRA 42, rue Georges Morel (B.P. 57) 49071 Beaucouzé (France) [email protected]

Patocchi, Andrea ......................... Institute of Plant Sciences Pathology ETH-Zentrum LFW Universitätstr 2 8092 Zürich (Switzerland) [email protected]

Persen, Ulrike.............................. Abteilung Phytopathologie in Raumkulturen Institut für Pflanzengesundheit Spargelfeldstrasse 191 1226 Wien (Austria) [email protected]

Philion, M.Sc. agr. Vincent......... Phytopathologiste, IRDA 3300 Sicotte, St-Hyacinthe, Québec, J2S 7B8 (Canada) [email protected]

Polfliet, Matty ............................. Fruit Consult Intl. Vlinderstraat 26 3500 Hasselt (Belgium) [email protected]

Ramborg, Svend.......................... Raadgivningsudvalget for Frugt og Baer Rugaardsvej 197 DK-5210 Odense NV (Denmark) [email protected]

Reiß, Dr. Karin............................ Syngenta agro Am Technologiepark 1-5 D-63477 Maintal (Germany) [email protected]

Rossi, Vittorio ............................. Istituto di Patologia Vegetale Università Cattolica del Sacro Cuore Via Emilia Parmense 84 I-29100 Piacenza (Italy) [email protected]

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Schloffer, Karl............................. Obstbaufachschule Gleisdorf Pirching 80 A-8200 Gleisdorf (Austria) [email protected]

Schouten, Henk J. ....................... Plant Research International P.O Box 16 NL-6700 AA Wageningen (The Netherlands)

Schwabe, Wolf ............................ Drommedarisweg 24 7130 Somerset West (South Africa) [email protected]

Stensvand, Arne .......................... Norwegian Crop Research Institute Plant Protection Centre Fellesbygget N-1432 Aas (Norway) [email protected]

Stentebjerg-Olesen, Kirsten ........ Fruit and Vegetable Advisory Service Agrovej 1 Oe Toreby DK-4800 Nykoebing F (Denmark)

Tornéus, Christer......................... Swedish Board of Agriculture Box 12 SE-230 53 Alnarp (Sweden) [email protected]

Trapman, Marc............................ Bio Fruit Advies Dorpstraat 32 4111 KT Zoelmond (The Netherlands) [email protected]

Van den Putte, An ....................... Fruitteeltcentrum K.U.Leuven W. DeCroylaan 42 B-2220 Hverlee (Belgium) [email protected]

Triloff, Peter................................ Marktgemeinschaft Bodenseeobst eG Albert-Maier-Str. 6 88045 Friedrichshafen (Germany) [email protected]

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List of Participants, Piacenza 2005 Antoniacci, Loredana .................. Servizio Fitosanitario, Regione Emilia-Romagna

Via Saliceto, 81 40128 Bologna (Italy) [email protected]

Auwerkerken, Annemarie ........... Laboratory for Fruit Breeding and Biotechnology Willem de Croylaan, 42 3001 Heverfee (Belgium) [email protected]

Baruzzi, Eva ................................ Servizio Fitosanitario, Regione Emilia-Romagna Via Saliceto, 81 40128 Bologna (Italy)

Bengtsson, Marianne .................. Department of Plant Biology, KVL (The Royal Veterinary and Agricultural University) Thorvaldsensvej 40 DK-1871, Frederiksberg (Denmark) [email protected]

Berrie, Angela ............................. East Malling Research Institute New Road, East Malling, Kent (UK) [email protected]

Boshuzien, Adrie ........................ Horti Bureau Wageningen P.O. Box 592, 6700 AN Wageningen (The Netherlands) [email protected]

Broggini, Giovanni...................... Swiss Federal Institute of Technology Universitätsstr. 2 CH-8092, Zürich (Switzerland) [email protected]

Bugiani, Riccardo........................ Servizio Fitosanitario, Regione Emilia-Romagna Via Saliceto, 81 40128 Bologna (Italy) [email protected]

Carisse, Odile .............................. Agriculture and Agrifood Canada 430 Gouin St Jean sur Richelieu, J3B3E6 Québec (Canada) [email protected]

Collina, Marina............................ Dipartimento di Protezione e Valorizzazione Agroalimentare Università di Bologna Viale G. Fanin 46 40127 Bologna (Italy) [email protected]

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De Cal Maria, Antonieta ............. INIA Crta. De la Coruna km. 7 28040, Madrid (Spain) [email protected]

de Jong, Peter Frans .................... Applied Plant Reserch-Fruit Research Unit P.O. Box 200 6670 AE, Zetten (The Netherlands) [email protected]

De Wit, Inge ................................ Better3fruit Willem de Croylaan, 42 3001 Heverfee (Belgium) [email protected]

Galli, Paolo.................................. Swiss Federal Institute of Technology Universitätsstr. 2 CH-8092, Zurich (Switzerland) [email protected]

Galliano, Aldo ............................. Creso Via Falicetto, 10 12030 Manta (CN) (Italy) [email protected]

Gessler, Cesare ............................ Swiss Federal Institute of Technology Universitätsstr. 2 CH-8092, Zurich (Switzerland) [email protected]

Giosuè, Simona ........................... Istituto di Entomologia e Patologia vegetale Università Cattolica del S. Cuore Via Emilia Parmense 84 29100 Piacenza (Italy) [email protected]

Heijne, Bart ................................. Applied Plant Research (PPO-fruit) P.O. Box 200 6670 AE, Zetten (The Netherlands) [email protected]

Jijakli, Haissam ........................... Faculté des Sciences Agronomiques de Gembloux Passage de Déportes, 2 5030 Gembloux (Belgium) [email protected]

Khan, Awais ................................ Swiss Federal Institute of Technology Universitätsstr. 2 CH-8092, Zurich (Switzerland) [email protected]

Köhl, Jürgen ................................ Plant Research International P.O. Box 16 6700 AA, Wageningen (The Netherlands) [email protected]

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Liboni, Massimo.......................... OVR-Ortofrutticola Valle del Reno Via Nuova 12/a Corporeno 44040, Ferrara (Italy) [email protected]

Llorente, Isidre ............................ Institue of Food and Agicultural Technology, University of Girona Campus de Montilivi 17071 Girona (Spain) [email protected]

MacHardy, William..................... University of New Hempshire, Dept.of Plant Biology Spaulding Hall 03824, Durham, NH (USA) [email protected]

Marmiroli, Elisa .......................... Istituto di Entomologia e Patologia vegetale Università Cattolica del S. Cuore Via Emilia Parmense 84 29100 Piacenza (Italy)

Melgarejo, Paloma ...................... INIA Crta. De la Coruna km. 7 28040, Madrid (Spain) [email protected]

Montuschi, Carla ......................... Servizio Fitosanitario, Regione Emilia-Romagna Via Saliceto, 81 40128 Bologna (Italy) [email protected]

Natalini, Giovanni ....................... Servizio Fitosanitario Via Fontivegge, 51 06100 Perugia (Italy) [email protected]

Patocchi, Andrea ......................... Swiss Federal Institute of Technology Universitatstrasse 2 CH-8092 Zurich (Switzerland) [email protected]

Pattori, Elisabetta ........................ Istituto di Entomologia e Patologia vegetale Università Cattolica del S. Cuore Via Emilia Parmense 84 29100 Piacenza (Italy) [email protected]

Pedersen, Karen Linddal ............. Fruit and Vegetable Advisory Service Rugaardsvej 197 5210 Odense NV (Denmark) [email protected]

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Philion, Vincent........................... IRDA 3300 Sicotte J2S 7B8, St-Hyacinthe Québec (Canada) [email protected]

Ribbert, Markus........................... Fraunhofer IME Worringerweg 1 52074 Aachen (Germany) [email protected]

Rimondi, Silvia............................ Servizio Fitosanitario, Regione Emilia-Romagna Via Saliceto, 81 40128 Bologna (Italy)

Rossi, Vittorio ............................. Istituto di Entomologia e Patologia vegetale Università Cattolica del S. Cuore Via Emilia Parmense 84 29100 Piacenza (Italy) [email protected]

Stensvand, Arne .......................... The Norwegian Crop Research Institute, Plant Protection Centre, Department of Plant Pathology Høgskoleveien 7 1432 Ås (Norway) [email protected]

Trapman, Marc ............................ Bio Fruit Advies Dorpsstraat, 32 4111 KT, Zoelmond (The Netherlands) [email protected]

Trappella, Roberta....................... Servizio Fitosanitario, Regione Emilia-Romagna Via Saliceto, 81 40128 Bologna (Italy)

Triloff, Peter ................................ Marktgemeinschaft Bodenseeobst Albert Maier Str. 6 8045 Friedrichshafen (Germany) [email protected]

Van Laer, Stjin ............................ PCF-Royal Research Station of Gorsem de brede akker 13 3800 Sint-Truiden (Belgium) [email protected]

Vilardell, Pere/Battlori, Lluis ...... Estación Experimental Fundación Mas Badia Ctra. De la Tallada, s/n 17134 La Tallada, Girona (Spain) [email protected]

Wenneker, Marcel ...................... Applied Plant Research (APR/PPO), section fruit P.O. Box 200 6670 AE, Zetten (The Netherlands) [email protected]


Lindau 2002


pp. 1-3

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Control of apple scab (Venturia inaequalis) in organic apple growing. StopScab: A Danish research programme for screening substitutes to copper fungicides Marianne Bengtsson, John Hockenhull Plant Pathology Section, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. E-mail: [email protected], [email protected] Abstract: The Danish research project StopScab (2002-2004) has recently been initiated to identify and begin to develop some new approaches and materials for the control of apple scab in organic growing. Four linked objectives are included: 1) screening of materials for scab control 2), histopathological investigation of host-pathogen interactions 3) orchard testing of selected control compounds and 4) characterisation of metabolite profiles in apples. Key words: Apple scab, Venturia inaequalis, organic growing, alternative control, screening, plant extracts, induced resistance. Introduction Apple scab, caused by Venturia inaequalis, causes serious losses in quality and yield of organi-cally grown apples in Denmark and elsewhere. Also conventional apple growers in Denmark are facing growing problems with scab. Cultural practices that lessen environmental conditions favourable for infection, sanitation practices that reduce the ascosporic inoculum at source in over-wintering leaves on the orchard floor (Bengtsson, 2001) and utilisation of resistant cultivars are some of the management strategies available for organic growers in Denmark. However, the presence of the new races 6 and 7 of Venturia inaequalis in Denmark (Bengtsson et al., 2000) has limited the use of Vf resistant cultivars. No eradicative or curative fungicides are available for use by organic growers, and while protective, copper-based fungicides are permitted in most European countries, only sulphur is allowed in Denmark and sulphur is not very effective against scab. There is thus a need for innovative research in the area of scab control, particularly in order to develop new approaches and materials that can be used by organic apple growers. The Danish research project StopScab (2002-2004) has recently been initiated to identify and begin to develop some new approaches and materials for the control of apple scab in organic growing. Four linked objectives are included: screening of materials for scab control, histopathological investigation of host-pathogen interactions, orchard testing of selected control compounds and characterisation of metabolite profiles in apples. Screening of candidate materials for scab control Candidate scab control materials are collected, including plant extracts, essential oils, compost tea, biocontrol agents (e.g. Bacillus subtillis, Clonostachys rosea), resistance inducers (e.g. Bion) etc. Reference materials will include sulphur and Cu-fungicides. Candidate materials will first be screened by using an in vitro leaf disc assay (Benaouf & Parisi, 1998). Candidate materials will

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be applied to disinfected apple leaf discs on agar before inoculation with the pathogen and scab symptoms will be assessed after three weeks incubation under controlled conditions. In a second screen selected materials will be applied to apple seedlings ‘Jonagold’ grafted on rootstocks before and after inoculation of the pathogen. Inoculated plants will be observed for development of scab symptoms after 2-3 weeks incubation using the grading scale of Parisi et al. (1993). Histopathological investigations of host-pathogen interactions Where control of scab is found in the greenhouse screen microscopy will be used to determine whether the controlling effect appears to be direct (fungicidal or fungistatic) or whether it appears to involve an activation of host plant resistance mechanisms (induced resistance) as described by (Orthega et al., 1998). Infection courses of the pathogen in the host with and without previous application of an inducer will be made and defence reactions will be studied. Investigations on induced resistance are aiming at finding evidence that natural defence responses in plants are enhanced by application of the material. All materials showing activity will be considered for inclusion in further orchard level tests. Pending additional funding molecular analysis of induced defence responses in apple will also be carried out. Orchard testing of selected control compounds In organically managed orchards at the Research Station Aarslev, Denmark, the most promising anti-fungal materials are to be tested firstly on single trees and next, for the most promising materials, on blocks of trees. For resistance inducing compounds and contact compounds application will be made before apple scab infection periods occur (both primary and secondary infections). Most attention will be given the development of primary apple scab infections. The PC-warning program RIMpro will be used to predict application time in the primary infection period. Assessment of scab infection on leaves after heavy primary scab periods will be carried out. Leaf scab infections in secondary infection periods will be carried out once and scab evaluation on fruits will be carried out at harvest Secondary metabolite characterization in apples An advantage that has been related to induced resistance is the production of defensive compounds, which can increase the quality of foodstuffs (Treutter, 2000). One important group is the phenolics, which may possess human disease-preventive properties (King & Young, 1999). Differences in the aroma profile of apples will affect taste and might thus influence consumer preference. Some volatiles might also be important from a health perspective (Sangwan et al., 1998). The level and composition of volatiles might change during induction of resistance as plants use these compounds to attract or deter insects and animals. The phenolic profile of apples harvested from trees treated with selected control compounds will be determined by HPLC and categorised as free and bound compounds, comprises phenolic acids and flavonoids. Volatile compounds will be measured in harvested apples by dynamic headspace sampling followed by GC and GC-MS. Acknowledgements This project is funded by the Danish Ministry of Food, Agriculture and Fisheries; The Danish Research Centre for Organic Farming: DARCOF II (2002-2005). The project involves Danish research groups at the Plant Pathology Section, The Royal Veterinary and Agricultural University, The Departments of Horticulture and Crop Protection at The Danish Institute of Agricultural Science, at Aarslev and Flakkebjerg respectively.

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References Bénaouf, G. & Parisi, L. 1998: Characterisation of Venturia inaequalis pathogenicity on leaf

discs of apple trees. European Journal of Plant Pathology 104: 785-793. Bengtsson, M. 2001: Biological control of the over-wintering stages of cherry leaf spot

(Blumeriella jaapii) and apple scab (Venturia inaequalis). Ph.D. thesis. The Royal Veteri-nary and Agricultural University, Copenhagen, Denmark.

Bengtsson, M., Lindhard, H. & Grauslund, J. 2000: Occurrence of races of Venturia inaequalis in an apple scab race screening orchard in Denmark. IOBC wprs Bulletin 23 (12): 225-229.

King, A. & Young, G. 1999: Characteristics and occurrence of phenolic phytochemicals. Journal of the American Diet Association 99: 213-218.

Ortega, F., Steiner, U. & Dehne, H.W. 1998: Induced resistance to apple scab: Microscopic studies on the infection cycle of Venturia inaequalis (Cke. Wint). Journal of Phytopathology 146: 399-405.

Parisi, L., Lespinasse, Y., Guillaumes, J. & Krüger, J. 1993: A new race of Venturia inaequalis virulent to apples with resistance due to the Vf gene. Phytopathology 83: 533-537.

Sangwan, N.S., Shanker, S., Sangwan, R.S. & Kumar, S. 1998: Plant-derived products as anti-mutagens. Phytotherapy Research 12: 389-399.

Treutter, D. 2000: Induced resistance in plant pathology – Consequences for the quality of plant foodstuffs? Journal of Applied Botany 74: 1-4.


pp. 5 - 7

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Apple scab IPM: preliminary report on the application of a new sampling technique to determine “scab-risk” J. Reardon, L. Berkett, M. Garcia, A. Gotlieb, T. Ashikaga, G. Badger Departments of Plant & Soil Science and Medical Biostatistics, University of Vermont, Burlington, VT 05405 USA Abstract: To facilitate grower adoption of apple scab management thresholds, a simplified, autumn sequential sampling technique to determine the ‘scab-risk’ of an orchard was proposed in the scientific literature. However, this technique had not been field validated. This research determined that: (i) the sequential sampling technique provided ‘scab-risk’ orchard ratings consistent with the original, non-sequential procedure, at potentially a significant time savings; (ii) similar ‘scab-risk’ ratings were obtained by using different combinations of trees and shoots within an orchard; (iii) following the ‘delayed-spray’ strategy in ‘low-risk’ orchards did not result in significant differences in fruit scab at harvest compared to spraying from the green-tip bud stage. Introduction Most fungicides applied to apple orchards in the Northeast United States are targeted at the management of apple scab. Researchers have developed action thresholds that aid in decision-making on whether early spring fungicide applications could be eliminated without a significant increase in the incidence of fruit scab at harvest. To facilitate grower adoption of these thresholds, a simplified, autumn sequential sampling technique to determine the ‘scab-risk’ of an orchard next spring was proposed in the scientific literature (MacHardy et al., 1999).

However, this technique had not been field validated. The major objectives of this research were to: (i) determine whether the outcome of the sequential sampling technique consistently agreed with the outcome of the original sampling procedure (non-sequential) (MacHardy, 1998) to estimate the ‘scab-risk’ of an orchard; (ii) determine how consistent the sequential sampling technique was in identifying whether an orchard is a ‘low-’ or ‘high-risk’ orchard when different trees and extension shoots were chosen for sampling; and (iii) determine if using the ‘delay-spray’ strategy in orchards identified as ‘low-risk’ by the sequential sampling technique would result in commercially unacceptable levels of scabbed fruit at harvest (i.e., >1%). Materials and methods In the autumn of 1999, 2000 and 2001, orchards were evaluated for foliar scab to determine the ‘scab-risk’ rating using both the sequential sampling technique (MacHardy et al., 1999) and the original procedure (MacHardy, 1998). Data were further analyzed using a simulation program to determine if results would change if different shoots or trees were used (Reardon et al., 2002). In 2000 and 2001, ‘delayed-spray’ experiments were conducted in two of the assessed orchards to evaluate delayed-spray and full-spray treatments. Delayed-spray replicates were to receive no fungicide sprays until after the third primary infection period (but before the fourth) or until the pink stage of bud development, whichever came first, which was the recommended action

6

threshold (MacHardy, 1994). However, in 2001, there were no infection periods until the bloom stage and, thus, the first fungicide was applied during bloom after the infection period. Full-spray replicates received fungicide sprays starting at the green-tip stage of bud development onwards as recommended in the New England Apple Pest Management Guide (Berkett, 2000). Foliar and fruit scab incidence was determined for treatment replicates. Results and discussion Results validated the sequential sampling technique as providing ‘scab-risk’ orchard ratings consistent with the original, non-sequential procedure, at potentially a significant time savings.

The sequential sampling technique, in which only 10 trees were sampled, required an average of 36 min, whereas the original procedure in which 60 trees were sampled required an average of 254 min. In 2000 and 2001, there were 20 assessments out of a total of 180 assessments where sampling a single set of 10 trees in the orchard did not result in a 'low-risk' or 'high-risk' rating. In these 20 assessments the number of scabbed leaves was above the threshold for 'low-risk' but below the threshold for 'high-risk', indicating sampling should 'continue'. However, a rating usually could be attained by adding only 1 or 2 additional sets of 10 trees. For all other assessments (i.e., 160 out of 180 assessments), evaluation of any of the 10 tree sets within an orchard provided a ‘low-risk’ or ‘high-risk’ rating consistent with the assessment of any of the other sets of 10 trees and was in agreement with the 'scab-risk' rating produced by the original, non-sequential procedure involving 60 trees. Further analysis using simulated, sequential sampling produced very consistent results within all orchards in both years.

Following the ‘delayed-spray’ strategy in ‘low-risk’ orchards did not result in significant differences in fruit scab at harvest compared to spraying from the green-tip bud stage (i.e., the full-spray treatment). Implementation of the ‘delayed-spray’ strategy in commercial orchards would be beneficial since it would reduce the total fungicide dose applied per orchard and allow for better integration of fungicide and insecticide application at the pink bud stage. Spray integration has many advantages to the grower, including reduced cost of labor, equipment, and materials, and reduced early season travel through the orchard in wet spring conditions. Reduction in applications of some fungicides may also translate into reduced pressure for the development of fungicide resistance. However, prior to implementing the ‘delayed-spray’ strategy, the grower must determine the orchard’s ‘scab-risk’ rating and growers have been reluctant to do this because of the amount of time required to assess the orchard using the original, non-sequential procedure. It is hoped that this research will address the reservations that apple growers may have in assessing the ‘scab-risk’ of their orchard, since the new sequential sampling technique involves less time, and facilitate adoption of the ‘delayed-spray’ strategy in ‘low-risk’ orchards. References Berkett, L.P., ed. 2000: Disease management. In: 2000-2001 New England Apple Pest

Management Guide. G. Koehler, ed. Cooperative Extension Service. Universities of Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island, and Vermont. 154 pp.

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MacHardy, W.E. 1994: A "PAD" action threshold: the key to integrating practices for managing apple scab. In: Integrated Control of Pome Fruit Diseases. Denis J. Butt, ed. Norw. J. Agric. Sci. Suppl. 17: 75-82.

MacHardy, W.E. 1998: Action thresholds for managing apple scab with fungicides and sanitation. Proc. Int. Conf. Integrated Fruit Prod. Acta Hort. 525: 123-131.

MacHardy, W.E., Berkett, L.P., Neefus, C.D., Gotlieb, A.R. & Sutton, D.K. 1999: An autumn foliar scab sequential sampling technique to predict the level of "scab-risk" next spring. Phytopathol. 89: S47.

Reardon, J.E. 2002: Field validation of a new sequential sampling technique for determining ‘risk’ of apple scab in Vermont apple orchards. M.S. Thesis. University of Vermont. Burlington. 91 pp.


pp. 9 - 14

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Spatial distribution of ascospores of Venturia inaequalis within the tree canopy O. Carisse1, D. Rolland1, J. Charest2 1 Agriculture and Agri-Food Canada, Horticultural Research and Development Centre, 430

Gouin, Saint-Jean-sur-Richelieu, Québec, Canada, J3B 3E6 2 MAPAQ, Direction Régionale de la Montérégie, 491-2, rue Sainte-Marie, Marieville,

Québec, Canada, J3M 1M4 Abstract: The amount of airborne Venturia inaequalis ascospores present during an infection period is rarely considered. Airborne ascospore concentration (AAC) can be measured, in real time with spore traps. However, the level of heterogeneity of the AAC within an orchard block makes it unreliable. Moreover, when the spore traps are located near the ground (height of 40 cm), the correlation between AAC and scab development is poor. To optimize the use of AAC as a decision tool, the spatial distribution of V. inaequalis ascospores within tree canopy was studied.

The airborne (AAC) and rainborne (RAC) ascospore concentration were measured in 31 locations within tree canopies (cv McIntosh) and at 6 heights (20 to 275 cm) from the ground with volumetric air samplers, passive traps and funnels devices during 6 major rain events of the spring 2001 to 2002. Scab severity was measured on leaves surrounding the trapping devices. The AAC measured with the volumetric spore traps decreased with increasing height from 20 to 275 cm and the RAC increased with increasing heights. Higher correlations were obtained between scab severity and AAC when the volumetric samplers were located at the tree level (80 to 120 cm) than at the ground level (20 to 40 cm). The experiment was conducted in an orchard block with high inoculum potential. In commercial orchards with lower inoculum, it would be more difficult to measure ascospores at this height due to the reliability of the trap. Nevertheless, the results of this experiment showed that it was possible to measure rainborne ascospore concentration and the AAC are not uniformly distributed within tree canopy. Introduction Apple scab is the most important disease of apple in Canada and causes important economic losses in many apple production areas. Apple scab control depends almost exclusively on frequent use of fungicides. In northeastern America, fungicides are mainly used to control the primary infections and to avoid epidemic build-up caused by secondary infections. Models of ascospore maturation based on degree-day accumulation (Gadoury & MacHardy, 1982, St-Arnaud & Neumann, 1990), are use to predict the beginning and the end of the ascospore ejection season, however, most models are regionally based and generally are not accurate enough to provide the time at which the first and the last fungicide applications should be done. In general, fungicides are applied based on risk of infection that is estimated from weather, amount of susceptible host tissue, and ascospore maturity. The amount of airborne ascospores present during an infection period is rarely considered.

The potential ascospore dose (PAD) is a valuable tool for estimating the amount of inoculum in an orchard and has been shown to significantly improve apple scab management (MacHardy et al., 1993). A complement to PAD measurement would be to assess airborne ascospore concentration (AAC) during the ascospore ejection period in the spring (Philion et al., 1997). Aylor (1998) and Aylor & Kiyomoto (1993) showed that the number of primary

10

apple scab lesions is quantitatively related to the airborne ascospore concentration. Charest (2000) studied the relationship between AAC and lesions development under natural conditions for five cultivars: Empire, McIntosh, Jonagold, Royal Gala, and Spartan. Potted trees were exposed to different airborne ascospore inoculum and the corresponding AAC were measured using spore traps. The number of lesions per tree increased as the AAC increased (R2 = 0.61 and P < 0.0001). However, it was not possible to establish a threshold of AAC for infection because of the large variation observed under low AAC (R2 = 0.16). The use of volumetric spore samplers for apple scab management is based on the hypothesis that the AAC is uniformly distributed within an apple orchard. Charest et al. (2002) clearly showed that the level of heterogeneity of the AAC within an orchard is high making AAC measurements unreliable. In their study Charest et al. (2002) placed the samplers at 40 cm from the ground. In theory (Aylor, 1995), as the height of trapping increases the level of homogeneity should increases and concentrations of ascospores decrease. In practice there is no data on spatial distribution of ascospores within the tree canopy. Therefore, the objective of this study was to identify the pattern of spatial distribution of the ascospores V. inaequalis within the apple tree canopy. Materials and methods The experiment was conducted in an orchard block of McIntosh with a high level of inoculum. The orchard was located at the experimental farm of Agriculture and Agri-Food Canada, Frelighsburg, Quebec. Prior to selected rain events (experimental runs) of the spring of 2001 and 2002, different spore trap devices were installed in the tree. The last experimental run was conducted before the appearance of lesions in order to avoid conidia infections. The airborne ascospore concentrations (AAC) were measured with volumetric samplers (rotary impaction samplers) and microscope slides one greased on the top and one greased on the bottom (Aylor, 1993). Rainborne ascospore concentration (RAC) was measured with funnels. The rotary samplers were placed on a pole near the apple tree at heights of 20, 40, 80, 109, 196 and 257 cm from the ground. The microscope slides and funnels were placed near to each other in the center of the tree at the same heights than the rotary samplers and on two horizontal axes oriented north south and east west at a distance of 75 cm between each device.

The airborne (AAC) and rain borne (RAC) ascospore concentrations were measured during 6 major rain events and scab severity was measured on leaves surrounding the trapping devices.

Within each experimental run, the data were transformed to proportion of maximum AAC or RAC. Different statistical tests were used to estimate the distribution of ascospores within the apple tree. The variance to mean ratio was used to determine the type of distribution pattern (regular, random or clustered). The variance to mean ratio was calculated by dividing the sample variance by the sample mean. When the ratio of s2 / x is < 1, =1, or >1, the pattern of distribution is considered regular, random, or aggregated respectively (Campbell & Madden, 1990). The Poisson probability distribution was used to determine if the spatial pattern of distribution was random or non-random (11). In other words, when the frequency distribution of ACC or RAC corresponded to the expected frequency distribution of Poisson as determined with a Chi square test, the spatial distribution was considered to be random. The negative binomial probability distribution was used to test a departure from randomness or aggregation (Campbell & Madden, 1990; Scherrer, 1984).

11

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Figure 1. Vertical profiles of concentrations of Venturia inaequalis ascospores measured with

volumetric samplers (A), microscope slides (B) and funnels (C).

12

Results and discussion Only the data from 2002 are presented (3 experimental runs). The AAC measured with the volumetric spore traps and the microscope slides decreased with increasing height from 20 to 275 cm and the RAC increased with increasing heights (Figure 1).

Overall, similar effect of heights was observed when the microscope slides were greased on the top and the bottom but more ascospores were captured when the slides were greased on the top. The effect of trapping heights on AAC observed was similar to results reported by Aylor (1995) except that in Aylor’s study only 6% of the AAC was trapped at 3 m compared to 10 to 45% in our study at 2.57 m. To our knowledge this is the first report of rainborne ascospore measurement. The diminution of RAC with increasing height could be explained by the interception of rain by the apple leaves (Aylor & Sutton, 1992).

Different horizontal profiles of concentrations of ascospores were observed when the ascospores were collected with the microscope slides (AAC) and with the funnels (RAC) (Figures 2).

Rel

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Figure 2. Horizontal profiles of concentrations of Venturia inaequalis ascospores measured with microscope slides (A) and funnels (B).

The variance to mean ratios for most of the AAC and RAC sampling dates were < or

equal to 1, indicating a regular or random pattern of distribution. The frequencies of the different classes of AAC and RAC followed the Poisson pattern of distribution (P < 0.01). This indicates that the AAC and RAC were randomly distributed within the tree canopy. Moreover, the classes of the AAC and RAC did not follow the negative binomial probability distribution (P < 0.01), indicating that the ascospores concentrations were not aggregated.

13

Best correlation between ascospore concentration and scab severity was obtained with the rain borne ascospores measurements. Higher correlations were obtained between scab severity and AAC when the volumetric samplers were located at the tree level (80 and 120 cm) than at the ground level (20 or 40 cm). Overall, similar infection efficiencies were observed (5 to 10%) than those reported by Aylor & Anagnostakis (1991) who observed infection efficiency varying from 5 to 14 % on potted trees.

It is generally admitted that the severity of primary infections depends directly on the AAC surrounding susceptible apple leaves. However, the amount of ascospores in the air and ascospore deposition can only be estimated using mathematical equations (Aylor 1998; Aylor & Kiyomoto, 1993). Direct measurements of wet and dry deposition combined with these models could increase our capacity to both understand and manage apple scab more efficiently

The experiment was conducted in an orchard block with a high level of inoculum. In commercial orchards with lower inoculum, it would be more difficult to measure ascospores at this height due to the capacity of the trap to measure low amount of ascospores. Nevertheless, the results of this experiment showed that it was possible to measure rainborne ascospore concentrations and that AAC are not uniformly distributed within tree canopy. References Aylor, D.E. 1993: Relative collection efficiency of Rotorod and Burkard spore samplers for

airborne Venturia inaequalis ascospores. Phytopathology 83: 1116-1119. Aylor, D.E. 1995: Vertical variation of aerial concentration of Venturia inaequalis ascospores

in an apple orchard. Phytopathology 85: 175-181. Aylor, D.E. 1998: The aerobiology of apple scab. Plant Dis. 82: 838-849. Aylor, D.E. & Anagnostakis, S.L. 1991: Active discharge distance of ascospores of Venturia

inaequalis. Phytopathology 81: 548-551. Aylor, D.E. & Kiyomoto, R.K. 1993: Relationship between aerial concentration of Venturia

inaequalis ascospores and development of apple scab. Agriculture and Forest Meteorology 63: 133-147.

Aylor, D.E. & Sutton, T. 1992: Release of Venturia inaequalis ascospores during unsteady rain: relationship to spore transport and deposition. Phytopathology 82: 532-540.

Campbell, C.L. & Madden, L.V. 1990: Introduction to Plant Disease Epidemiology. Toronto: John Wiley and Sons.

Charest, J. 2000: Spatial distribution and dose-disease relationship of airborne ascospores of Venturia inaequalis on apple. M.Sc. Thesis, McGill University.

Charest, J., Debwney, M., Paulitz, T., Philion,V. & Carisse, O. 2002: Spatial Distribution of Venturia inaequalis Airborne Ascospores Under Commercial Orchard Conditions. Phytopathology 92: 769-779.

Gadoury, D.M. & MacHardy, W.E. 1982: A model to estimate the maturity of ascospores of Venturia inaequalis. Phytopathology 72: 901-904.

MacHardy, W.E., Gadoury, D.M. & Rosenberger, D.A. 1993: Delaying the onset of fungicide programs for control of apple scab in orchards with low potential ascospore dose of Venturia inaequalis. Plant Dis. 77: 372-375.

Philion, V., Carisse, O., Garcin, A. & Vanesson, S. 1997: Monitoring airborne ascospore of Venturia inaequalis in the control of apple scab. IOBC/WPRS Bulletin 20 (9): 180-184.

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St-Arnaud, M. & Neumann, P. 1990: Évaluation au Québec d'un modèle de prédiction de la fin de la période annuelle d'éjection des ascospores du Venturia inaequalis. Phytoprotection 71: 17-23.

Scherrer, B. 1984: Biostatistique. Gaëtan Morin. Boucherville, Québec.


pp. 15 - 23

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Sanitation practices to reduce apple scab inoculum in orchards * Piet Creemers, Alida Vanmechelen, Kjell Hauke RSF-Royal Research Station of Gorsem, De Brede Akker 13, B-3800 Sint-Truiden (Belgium) * Research subsidized by the Ministry of Small Enterprises, Traders and Agriculture.

Administration for Research and Development Abstract: Economical and pomological factors have increased the sensitivity of modern orchards for fungal diseases. Different strategies must be integrated in the control measures to achieve a sustainable production system. The disposal of modern fungicides with a high performance is only one pillar in disease management. Sanitation, optimal timing of spray treatments, adequate application techniques and resistance risk modifiers are the main tools to obtain an integrated durable fruit production structure. Besides this agricultural approach, the consumer attaches more importance to the side effects of pesticides on the environment and eventually the residues left on the fruits.

Scab (Venturia inaequalis on apple and Venturia pirina on pear) is the key parasite on pipfruits. On the most commercial varieties, more than 60 % of treatments are related with scab control. The evolution to monocultures of one variety, and the ban of plant growth regulators to shorten the growth period and in this manner the infection period, has increased the infection pressure to a level that one mistake in the spray program can lead to an economic disaster. In the scab risk assessment, biological factors as inoculum, varietal susceptibility, ascospore release and leaf growth are incorporated in Mills infection periods to improve scab-warning services. The determination of biotic parameters is enormously labour consuming and here software image analysing systems and simulation models can contribute to better estimate the infection risk.

Several practices can be used to reduce the amount of inoculum in the orchard and to enhance the efficacy of the fungicide program. Spray coverage and distribution are improved by pruning the tree in an open canopy. Moreover this improves penetration of air and light, which shorten wetness periods on leaves and fruits. Reducing or eliminating apple scab inoculum on overwintering leaves reduces disease pressure in spring. In our research program we have carried out different trials to limit the overwintering population. Key words: apple scab, sanitation, fungicides, anti-resistance Introduction Each year, a new infection cycle of scab begins with the release of ascospores after the overwintering of the fungus, mainly in leaf litter (Creemers & Vanmechelen, 2000). A number of sanitation practices can diminish this release by means of reducing the apple scab inoculum in orchards (MacHardy, 2000). The purpose of all these practices is the same, namely to decrease the development of pseudothecia. This development occurs during the sexual phase (in the winter period on fallen leaves) and begins when a fusion of the two different mating types of Venturia inaequalis takes place (MacHardy et al., 2001).

One can avoid these fusions by stimulating the leaf decomposition. In experiments, a pre-incubation of the leaves under conditions of 20°C and a relative humidity of 100% showed us a major improvement of decomposition compared with a pre-incubation at a lower relative humidity. Of course, practically this is of little meaning but it gives us an insight of the role of the climate in the decomposition process. Colonisation of leaves with Athelia bombacina, an antagonist of Venturia inaequalis gives good results to prevent the

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colonisation of scab itself (Carisse et al, 2000, Fiaccadori & Cesari, 2000). This is coherent because it is the pre-incubation that provides the necessary conditions for the colonisation of these micro-organisms.

Another experiment concerned the effect of “Digester”, a nonviable blend of biologically produced fermentation extracts and selected minerals. This seemed to stimulate the conversion of organic material into humus. The action is based on the degradation of cellulose and the stimulation of fungi and bacteria which accelerates the decomposition of organic matter (Bereford et al., 2000). Also, a comparison was made between the decomposition on an herbicide strip and a grass strip.

Leaf fall is also a very critical period for infection with Nectria which causes canker. The products with the highest effect against these infections are the benzimidazoles and coppersynthesis. However, these are of negative influence on the population of earthworms, which are very important organisms concerning leaf decomposition and the ventilation of the soil (Aalbers, 2001). Apart from this ecological reason they are under a lot of pressure because of possible toxicological side effects. A possible alternative for these products is imazalil, which isn’t harmful for earthworms. Imazalil is a member of the group of DMIs (Demethylation Inhibitors). Imazalil was also combined with a new generation of surfactants, the trisiloxanes, based on silicones. In order to work in a more biological way, trials were carried out using biodegradable surfactants instead of trisiloxanes.

Next to stimulating the decomposition of leaf litter by means of pre-incubation at high relative humidity or treatment with “Digester” cutting the leaves into bits also accelerates the decomposition process (Sutton et al., 2000). The effect of leaf shredding on scab is twofold: on the one hand there is a faster leaf decomposition but at the other hand the chance that two compatible mating types meet together in such small leaf pieces is much lower.

In all the previous methods the aim was to have as little leaf litter as possible at bud burst and in that way few pseudothecia. We can also diminish the number of pseudothecia using DMIs (Heijne et al., 2000). This is the most extensive group of fungicides used in curative control against scab infections. The members of this group all share a common mode of action in the inhibition of fungal sterol biosynthesis. Treatment of scabbed leaves with DMIs seems to have a positive effect on diminishing the number of pseudothecia. However, to avoid the development of resistance, the use of DMIs in the sexual phase is not recommended. Material and methods Effect of pre-incubation at high relative humidity on the decomposition The rate of digestion was monitored using digital photography and an image analysing program (Kontron 300). A pre-incubation period of three weeks in an infection chamber with a temperature of 20°C and relative humidity of 100% was applied. After incubation these leaves were placed outside on the ground and the decomposition was observed. Treatment was on 12/11/97 and the incubation period lasted until 03/12/97. The assessment was done on 26/02/98. By way of comparison incubation was carried out at lower relative humidity with the intention to approach more natural conditions. During the day the conditions were 70% RH (Relative Humidity) and 20°C and by night they were 90% RH and 15°C. The use of the antagonist Athelia bombacina The strain we used came from the University of Bologna (Prof. Ricardo Fiaccadori) and is characterised by a very fast growth under perfect conditions. Scabbed leaves were plunged in a watery solution of mycelium of the antagonist Athelia bombacina. The mycelium of 5 petridishes of a 14 days old culture on PDA (Potato Dextrose Agar) was mixed in 600 ml of

17

distilled water. The same solution was used as well to spray scab leaves. Then, a portion of the leaves was first pre-incubated at 20°C and at RH of 100% before being placed outside while the rest was placed outside immediately after treatment without being pre-incubated at first. After pre-incubation one could clearly see the colonisation of the fungus as a white fluff. During spring we evaluated the pseudothecia on the leaves. This was done by checking 10 pseudothecia per leaf for a total of 5 leaves. The fruiting bodies were not always from apple scab, but sometimes from Pleospora herbarum. Stimulation of leaf decomposition by “Digester” Along with the experiments with pre-incubation at different RH and temperature a comparison was made between the decomposition of leaf litter on a herbicide strip and a green (grass) strip. Here, we also applied “Digester”. Alternatives for the treatment of canker on fruit-trees Because of the variable infection pressure in orchards and the requirement of very large plots to demonstrate a difference between the treatments tests were executed with artificial infections. Treatment with imazalil was carried out before (preventive) or after (curative) inoculation. In these trials, imazalil was used separately or in combination with a surfactant based on silicones, namely LI 103 (chemical family: Sticker). 24 injuries were made on twigs of 2-year old Jonagold trees, planted in containers under plastic. Afterwards they were infected with a suspension of conidiospores (150.000 spores/ml) of Nectria galligena. The plastic cap was kept frost-free during winter and at the beginning of the growing season the trees were placed outside. In spring the number of wounds was counted and classified into groups according to the size of the canker.

This experiment was repeated the next year. Only now we made an additional combination of imazalil with biodegradable surfactants based on cellulose. Leaf shredding A local constructor (Jammaers R.) built a pilot machine. Two sweepers at both sides bring the leaves from the herbicide strip to the middle of the alley and then a type of flail-mower shreds the leaves. An area of about 1 ha was divided into 2 times 4 quadrants. The leaf litter was shredded in 4 of these 8 quadrants. The quadrants were separated by wire netting to prevent the leaves from spreading by the wind. After the winter period 5 two-year old trees were planted in the middle of each quadrant and were covered up during spraying. Of these, the sepals, rosette leaves and the shoot leaves were checked on the presence of scab lesions in the beginning of May.

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In the results we worked with TH-values (Townsend-Heuberger). These values are used when the result of an observation is divided into different classes. Mostly 4 classes of infection are applied.

SHREDDED NOT SHREDDED

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∑ (n x v) Townsend-Heuberger formula: THv max = ___________ x 100 vmax x N THv max = degree of affection v = infection classes (0,1,2,3,4) vmax = highest infection class n = number of leaves in each class N = total number of leaves (n0x0)+(n1x1)+(n2x2)+(n3x3)+(n4x4) example: TH4 = ________________________________________________ x 100 4xN Suppression of pseudothecial development by the use of DMIs Different methods of treatment with DMIs were used on leaves infected with scab. On the one hand we dipped with difenoconazole and bitertanol and on the other hand we sprayed with 300 l/ha or 1000 l/ha of the same products. The leaves were collected per treatment and placed in a net. The overwintering of the leaves is the same as on the ground and in spring the development of pseudothecia was evaluated. Per object 10 pseudothecia of again 5 leaves were checked on the presence of ascospores. At the same time the release of ascospores, at the moment of highest ascospore maturity, was followed. This was done by wetting a total of 5 leaves with a sprayer. This causes the release of the ascospores from the ascus. They were collected on microscope slides and counted. Tests were carried out with Jonagold and Golden.

Figure 1. Leaf decomposition, incubation after leaf fall.

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Results Effect of pre-incubation at high relative humidity on the decomposition The results (Figure 1) show that the decomposition of leaves was much better after a pre-incubation period at high relative humidity.

On the contrary pre-incubation at lower relative humidity has rather a negative effect on the decomposition even in comparison with no pre-incubation at all. The use of the antagonist Athelia bombacina When placed in pre-incubation the leaves that where inoculated with Athelia bombacina gave a total inhibition of development of pseudothecia. The success of using this technique appears to be very much depending on this pre-incubation period. Not all the 50 checked fruity bodies were pseudothecia from apple scab, but sometimes high amounts were from Pleospora herbarum origin.

Table 1. Evaluation of the development of pseudothecia in scabbed leaves after treatment with

the antagonist Athelia bombacina.

Athelia Total number of pseudothecia of 5 leaves bombacina and 10 pseudothecia per leaf Conservation Check Dipping Spraying Directly outside 48 42 50 Climate chamber 26 0 0 14 days of incubation

Stimulation of leaf decomposition by “Digester” As indicated in Table 2 “Digester” gives a remarkable acceleration on the decomposition process.

Table 2. Evolution of leaf decomposition when treated with “Digester”

Treatment % leaf litter on % leaf litter on 08/02/00 21/03/00 11/04/00 08/02/00 21/03/00 11/04/00 G r a s s s t r i p H e r b i c i d e s t r i p

Check 96.7 a 81.8 c 69.3 b 89.4 a 71.3 b 49.0 b

Digester 89.6 a 55.3 b 27.3 a 90.0 a 34.1 a 10.7 a

Digester + urea 92.4 a 27.7 a 14.9 a 81.4 a 45.8 a 24.3 a

In a period of about 2 months we have a reduction on the grass strip of 62.3% with

Digester versus 27.4% with the untreated leaves. On the herbicide strip this is 79.3% versus 40.4%.

Treatment: 18/11/1996 Assessment: April-May 1997

20

In general, the decomposition is faster on the herbicide strip than on the grass strip as shown in the untreated object.

Table 3. Control of canker (Nectria galligena) on Jonagold with artificial infection of bark

wounds with preventive and curative treatments.

Fungicide Active ingredient Formulation Dose g a.i./ha Efficacy (%)

Preventive

Bavistin carbendazim 50.0 WG 450.0 96.8 a

Fungaflor imazalil 200.0 EC 200.0 91.9 a

Fungaflor + LI 103 imazalil 200.0 EC 200.0

1400.0 100.0 a

Curative

Bavistin carbendazim 50.0 WG 450.0 96.8 a

Fungaflor imazalil 200.0 EC 400.0 -20.4 b


1400.0 77.4 a

Making of bark wounds: preventive 14/12 and curative 16/12/98 Treatment: preventive 14/12 and curative 18/12/98 Artificial infections: 16 and 17/12/98 Assessment: 12/04 and 19/05/99

Table 4. Control of canker (Nectria galligena) on Jonagold with artificial infection of bark

wounds with preventive and curative treatment.

Fungicide Active ingredient Formulation Dose g a.i./ha

Efficacy (%)

Preventive

Topsin M thiophanate-methyl 70.0 WG 892.5 75.9 a

Fungaflor imazalil 100.0 EC 400.0 94.4 a


1400.0 94.4 a

Fungaflor imazalil + cellulose 100.0 EC 400.0 2000.0 100.0 a

Curative

Topsin M thiophanate-methyl 70.0 WG 892.5 61.1 a

Fungaflor imazalil 100.0 EC 400.0 11.1 b


1400.0 50.0 b

Fungaflor imazalil + cellulose 100.0 EC 400.0 2000.0 77.8 a

Making of bark wounds: preventive 20/12 and curative 15/12/99 Treatment: preventive 20/12 and curative 17/12/99 Artificial infection: preventive 22 and 23/12/99; curative 15 and 16/12/99 Assessment: 21/03/00

21

Alternatives for the treatment of canker on fruit trees Under good conditions for canker infections 56.3% of the untreated wounds were affected. Carbendazim gave a very good control preventively as well as curatively. As indicated in Table 3, imazalil gave an efficacy of 91.9% when used preventively while curatively no control was observed, even not with the use of a double dose.

The adding of the surfactant LI 103 to imazalil clearly improved its action. No cankers developed with preventive treatment and the curative control was a bit weaker in comparison with the reference carbendazim.

In Table 4 we can observe that the use of biodegradable surfactants gives us an even better result than with the ones based on silicones. Moreover they have the advantage of doing no harm to the plant at all.

Also the DMI tebuconazole has given promising results against Nectria galligena. Leaf shredding In a trial with 4 treated and untreated blocks, the scab infestation was reduced in the next year with 88, 82 and 75 % respectively on sepals, rosette and shoot leaves. The differences disappear at the end of the season as a result of secondary infections on shoot leaves. Table 5. Reduction of scab attack in spring after leaf shredding.

Treatment Block Sepals Rosette leaves Shoot leaves

%A Mean %A TH4 Mean %A TH4 Mean

1 0.00 8.77 22.92 6.77 7.81 27.87 8.74 8.77

2 1.32 29.36 9.86 29.55 9.23

3 23.17 35.00 9.00 42.29 12.00 Shredding

4 10.61 21.51 5.65 17.51 5.08

1 65.38 72.19 75.48 43.23 43.91 64.23 37.76 35.47

2 70.48 78.26 41.03 64.02 37.30

3 69.88 86.52 48.60 58.48 35.09 Check

4 83.00 80.72 42.77 59.34 34.75

Suppression of pseudothecial development by the use of DMIs Dipping of the leaves in a solution of fungicides gives us as a result that no pseudothecia at all developed. When the same products were applied outside with a spraying a weaker activity was observed. However, the same test was repeated the next year. In this case, the treatment outside as well as the dipping gave a total inhibition of development of pseudothecia.

The fact that in the sexual development of scab no difference between the efficacy of difenoconazole and bitertanol was noticed is striking, as bitertanol is no longer used in spring because of its scab resistance. This means that there is no correlation in resistance status between ascospores and the pseudothecial development

22

Table 6. Evaluation of the effect of DMIs on the development of pseudothecia.

fungicide dose Ascospore release Scab pseudothecia mg a.i./l Total of 5 leaves 10 pseudothecia/5 leaves Jonagold Golden Jonagold Golden difenoconazole: – dipping: 50.0 0 0 0 0 25.0 0 0 0 0 – spraying: 1000 l/ha 37.5 701 16 11 33

300 l/ha 250.0 0 234 30 19 300 l/ha 125.0 44 1714 28 44

bitertanol: – dipping: 375.0 0 0 0 0 187.5 0 0 1 0 – spraying: 300 l/ha 1875.0 90 0 24 1 937.5 1719 5 42 40 Untreated 2586 2428 48.9 45.9

Treatment: 03/11/1995 Assessment: April-May 1996 Discussion As shown in these results sanitation practices can be of great importance to reduce the scab inoculum in an orchard.

Pre-incubation of the leaves at high humidity stimulates the decomposition of leaf litter and at the same time provides a better environment for the colonisation of the antagonist Athelia bombacina. The use of this antagonist resulted in a total inhibition of development of pseudothecia when pre-incubation was carried out. On the other hand treatment with an antagonist of scab such as Athelia bombacina could be a possible method to reduce the apple scab inoculum in an orchard.

Also, the use of imazalil for the treatment of Nectria as an alternative for benzimidazoles and copper compounds has a positive effect on the decomposition because it isn’t harmful for earthworms, which are known to contribute in a large extent to the decomposition of leaf litter. This could be of great importance especially when taken into account that probably more and more products will be prohibited in the near future. Also the possibility of combination with the biodegradable surfactants is an important advantage for this alternative. Also tebuconazol has given a promising activity against European Nectria canker.

A more physical way is to shred the leaf litter into very small pieces. The affection of scab was significantly different in the shredded quadrants in comparison with the unshredded quadrants. A reduction in the number of treatments during the primary infection period is probably not possible in orchards where leaf shredding is applied. Savings using this method will be rather made during the secondary infection period because the primary infections were controlled better. This offers the advantage that it isn’t necessary to spray in a very short period before harvest and in this way ruling out the danger of presence of residue on the fruit. This sanitary treatment must be carried out on greater surfaces, because ascospores and also infected leaves can be transported by wind from one orchard to another. Nevertheless, this cultural method fits very well in resistance management. Indeed, in orchards with scab problems an intensive spray program, also with uni-site fungicides, is often used to prevent

23

the spread of scab on fruits. This can lead to a selection within the scab population of less sensitive strains. Leaf shredding can help to avoid the persistence of that sub-population.

Treatment with DMIs prevents the development of pseudothecia. In a first trial the results after dipping were much better than with spraying outside in the field. When repeated the next year the dipping as well as the treatment outside gave a total inhibition of development of pseudothecia. Therefore we can conclude that the weather conditions and in particular the temperature and the relative humidity during and after the treatment are a determinant factor for a proper action of DMIs against pseudothecial development in the orchard.

References Aalbers, P. 2001: Earthworms, partners in scab control. Fruitteelt Den Haag. 31: 12-14. Bereford, R.M., Horner, I.J., Wood, P.N. & Zydenbos, S.M. 2000: Autumn applied urea and

other compounds to suppress Venturia inaequalis ascospore production. New Zealand Plant Protection 53: 387-392.

Carisse, O., Philion, V., Rolland, D. & Bernier, J. 2000: Effect of fall application of fungal antagonists on spring ascospore production of the apple scab pathogen, Venturia inaequalis. Phytopathology 90: 31-37.

Creemers, P. & Vanmechelen, A. 2000: Scab infection risks in relation to biological factors and the optimum spray-timing of modern fungicide families. Proceedings of the international conference on integrated fruit production, Leuven, Belgium, eds. Müller, W., Polesny, F., Verheyden, C., Webster, AD., Acta Horticulturae 525: / IOBC-wprs Bulletin 23 (7): 133-140.

Fiaccadori, R. & Cesari, A. 2000: Sanitation from Venturia inaequalis using an antagonist Athelia bombacina to reduce ascospore inoculum. Proceedings of the international conference on integrated fruit production, Leuven, Belgium, eds. Müller, W., Polesny, F., Verheyden, C., Webster, AD., Acta Horticulturae 525 / IOBC-wprs Bulletin 23 (7): 245-250.

Heijne, B., Balkhoven Baart, J.M.T., Veens, T.S.G.M. & Anbergen, R.H.N. 2000: Field evaluation of pre-leaf applications to reduce the amount of apple scab inoculum. Proceedings of the international conference on integrated fruit production, Leuven, Belgium, eds. Müller, W., Polesny, F., Verheyden, C., Webster, AD., Acta Horticulturae, 525 / IOBC-wprs Bulletin 23 (7): 251-256.

MacHardy, W.E. 2000: Action thresholds for managing apple scab with fungicides and sanitation. Proceedings of the International Conference of Integrated Fruit Production. Leuven Belgium, eds. Müller, W., Polesny, F., Verheyden, C., Webster, AD., Acta Horticulturae 525 / IOBC-wprs Bulletin 23 (7): 123-131.

MacHardy, W.E., Gadoury, D.M. & Gessler, C. 2001: Parasitic and biological fitness of Venturia inaequalis: Relationship to disease management strategies. Plant Disease 85: 10: 1036-1049.

Sutton, D.K., MacHardy, W.E. & Lord, W.G. 2000: Effects of shredding or treating apple leaf litter with urea on ascospore dose of Venturia inaequalis and disease build-up. Plant Disease 84: 1319-1326.


pp. 25 - 29

25

Effect of apple cultivar mixtures on the epidemic of Venturia inaequalis in a treated orchard F. Didelot1, L. Brun2, S. Clément1, L. Parisi1 1 I.N.R.A., U.M.R. PaVé, BP 57, 49071 Beaucole CEDEX, France 2 Present address: I.N.R.A., U.E.R.I., Domaine de Gotheron, 26320 St Marcel Les Valence,

France Abstract: In an experimental orchard comprising 2 different cultivar mixtures, a fungicide applications protocol with a treatment threshold was applied. However, difficult climatic condition caused a decrease of the efficiency of 3 treatments. In this situation of fungicide treatments failure, the within-row mixture efficiently reduced the apple scab epidemic on fruits and leaves of the Smoothee cultivar compared to monoculture, but was not efficient to reduce significantly scab on fruits at harvest. The scab epidemic in the between-row mixture was not so high than in the monoculture, but the differences observed were not significant. This work showed that within-row mixtures can reduce the scab epidemics when the fungicides action does not control completely the disease. Introduction Apple scab is the most important disease in commercial orchards, and 10 to 20 fungicides applications can be necessary to the control of the disease. However, the negative impact on the environment and the consumer pressure render necessary to find alternatives to control apple scab. One of them, studied for several years in annual crops, is the use of mixture of cultivars different in their susceptibility to a pathogen (Wolfe, 1985). This planting strategy may strongly reduce the disease level, as Zhu et al. (2000) shown for blast disease in rice. In spite of this, few articles report the employ of cultivar mixtures with perennial crops to control fungal diseases. For apple, few studies relate the influence of cultivar mixtures on the epidemic of V. inaequalis in non treated orchards, evaluated in experimental trials (Didelot et al., 2000, Bousset et al., 1997) or by computer simulations (Blaise & Gessler, 1994). In this work, our objective was to know if the plantation of cultivar mixtures could facilitate a reduction of fungicides applications in the orchard, and what could be the fungicide application threshold in this case. Materials and methods The experimental orchard included Smoothee, considered as moderately susceptible to scab in France (Parisi & Trillot, 1993) and Baujade, a Vf resistant INRA selection (Lespinasse et al., 1992). Each cultivar was planted in monoculture plots, and in two different mixtures : between-row mixture and within-row mixture. The design was a block with 3 replications; each block comprised 4 plots with 6 rows of 25 trees. Each plot was surrounded by a hedge planted with V. inaequalis non host species. The total area of the orchard was 1,2 ha. The treatment threshold was the level of the risk of contamination following Mills & Laplante (1951) and Olivier (1986), as proposed by Lefeuvre (1995): light or severe according to the level of ascospore ejections (Table 1).

26

Table 1. Treatment threshold taking into account the cultivar susceptibility, the autumn inoculum, the rate of ascospores discharge and the level of infection period: Lefeuvre’s recommendations (adapted from Olivier, 1986).

Cultivar susceptibility Very susceptible Susceptible to

Moderately Susceptible Weakly susceptible

Lesions density on leaves in autumn

low moderate dense low moderate dense low moderate dense

Low m m l s m/s l/m (-) (-)/s m/s Rate of ascospores discharge

High l a a l l l g m l

Level of risk : a : angers; l :light; m : moderate; s : severe

Four fungicides families were used in this experiment : DMI fungicides (Hexaconazole and Fluquinconazole), an Anilino-pyrimidine (Pyrimethanil, associated with Fluquin-conazole), and two contact fungides (Captan and Mancozeb). For Smoothee, 8 assessments of scab symptoms on leaves and 4 on fruits were done between April and July 2001, according to the risk and incubation periods detected by the software Pulsowin (Figure 1).

0

1

2

3

4

1/3 16/3 31/3 15/4 30/4 15/5 30/5 14/6 29/6 14/7 29/7

Leve

l of t

he M

ills

and

Oliv

ier i

nfec

tion

perio

ds

020406080100

cum

ulat

ive

% o

f eje

cted

acso

pore

s infection periods cumulative percentage of acsospores

1 : Angers2 :Light3 : Moderate4 : Severe

Figure 1. Infection periods and ascospore ejections from March to August 2001.

The area under the disease progress curve (AUDPC) of incidence (% of scabbed leaves or fruits) was evaluated in each planting system. In September, the incidence on the fruits at harvest was evaluated on 6 trees per plot. Results Between the 24 of March and the 17 of July, 13 periods of risk were registered, 12 of them reached the threshold ; 9 fungicides applications were necessary to protect the orchard (Table 2).

However, difficult climatic conditions caused a decrease of the efficiency of 3 treatments. In this situation of fungicide treatments failure, the within-row mixture efficiently reduced the apple scab epidemic on fruits and leaves of Smoothee compared to monoculture (Figures 2 and 3, table 3), but was not efficient to reduce significantly scab on fruits at harvest (Table 4). The scab epidemic in the between-row mixture was not so high than in the monoculture, but the differences observed were not significant. For Baujade, no symptom of scab was detected on leaves or fruits.

27

Table 2. Dates of infection periods and dates of fungicide applications from the 24th of March to the 17th of July 2001.

Date of

contamination Level of infection

risk Fungicide (active

ingredient) Date of

fungicide application

March, 24 Moderate April, 4 Severe Hexaconazole April, 8 April, 15 Moderate Hexaconazole April, 17 April, 26 Angers April, 27 Severe April, 29 Severe May, 2 Severe

Pyrimethanil + Fluquinconazole

April, 30

May, 9 Light Mancozeb May, 10 June, 7 Moderate Captan June, 8

June, 15 Severe Hexaconazole + Captan

June, 18

July, 4 Severe Captan July, 2 July, 13 Severe Captan July, 11 July, 17 Severe Captan July, 17

0%

10%

20%

30%

40%

50%

60%

17/4 24/4 1/5 8/5 15/5 22/5 29/5 5/6 12/6 19/6 26/6 3/7 10/7 17/7 24/7 31/7 7/8

Perc

enta

ge o

f sca

bbed

leav

es

Monoculure Between-row mixture Within-row mixture

Figure 2. Evolution of the incidence on leaves of the cultivar Smoothee in monoculture and cultivar mixtures from April to July 2001.

Discussion This work showed that the within-row cultivar mixtures strongly reduced the epidemic of the disease in the particular situation of fungicide failure, while the between-row cultivar mixture did not induce significant reduction of the disease. The inoculum level measured in this orchard at the autumn 2000 was high, and this explain the high level of disease observed in this experiment. However, situation of fungicides failure often occur in commercial orchards and can induce important economic losses. This work showed the interest of cultivar mixture to reduce the epidemic of the disease in this case. The differences observed on the fruits at

28

harvest were not significant, so the interest of the cultivar mixtures to reduce economic losses cannot be demonstrated. However, we observed 13 % less scabbed fruits in the within-row mixtures than in monoculture; if this reduction is due to the mixture effect, it is not a negligible difference from the economic point of view. Due to the difficult management of cultivar mixtures, and especially that of within-row mixtures, their interest was not demonstrated by this work in a conventional agricultural system. However, this work demonstrated a synergy between the planting system and the fungicides treatments for reducing the epidemic of the disease; combining different factors to reduce the impact of a disease is the objective of the integrate production. Moreover, planting cultivar mixtures could be interesting in organic farming, because apple scab remains difficult to control in this kind of production. This kind of planting system could facilitate the culture of susceptible cultivars with a high commercial value, but difficult to protect against scab with the fungicides allowed in organic farming, in mixture with resistant or less susceptible cultivars. These results are in accordance with those of Zhu et al. (2000) obtained for the rice culture in a large experimental scale, with a very reduced amount of fungicides treatments.

0%

10%

20%

30%

40%

24/5 31/5 7/6 14/6 21/6 28/6 5/7 12/7 19/7 26/7Per

cent

age

of s

cabb

ed fr

uits

Monocultre Between-row mixture Within-row mixture

Figure 3. Evolution of the incidence on fruits of cultivar Smoothee in monoculture and cultivar mixtures from May to July 2001.

Table 3. AUDPC(area under disease progression curve) of the incidence in leaves and fruits

of Smoothee in different planting systems.

Planting systemMonoculture 27,9 a 12,9 aBetween-row mixture 23,7 a 10,4 abWithin-row mixture 14,7 b 6,2 b(a) : values with differents letters were significantly different at p = 0,05 (LSD test)

AUDPC (leaves) AUDPC (fruit)

Table 4. Incidence on Smoothee apples at harvest in different planting systems.

Planting system Incidence Monoculture 27%Between-row mixture 17%Within-row mixture 14%

29

References Blaise, P.H. & Gessler, C. 1994: Cultivar mixtures in apple orchard as mean to control apple

scab? Norw. J. Agric. Sci. 17 :105-112. Bousset, L., Blaise, P., Kellerhals, M. & Gessler C. 1997: Mixtures of apple cultivars in

orchards: effect on the scab epidemics. IOBC/WPRS Bulletin 20 (9): 42-48. Didelot, F., Delhaye, K., Brun, L. & Parisi L. 2000: Analysis of 1998 scab epidemic in an

experimental apple orchard planted with cultivar mixtures. IOBC/WPRS Bulletin 23 (12): 207-210.

Lefeuvre, M. 1995: Lutte raisonnée contre la tavelure du pommier: validation d’un modèle d’aide à la décision. Mémoire d'ingénieur diplômé par l'état. Ecole Nationale d'Ingénieurs des Techniques de l'Horticulture et du Paysage, Angers, France.

Mills, W.D. & Laplante, A.A. 1951: Diseases and insects in the orchard. Cornell Ext. Bull. 711: 100 pp.

Lespinasse, Y., Lespinasse, J.M., & Le Lezec, M. 1992: Baujade. Une nouvelle variété de pommier résistante à la tavelure. L'Arboriculture Fruitière 454: 15-16.

Olivier, J.M. 1986: La tavelure du pommier, conduite d'une protection raisonnée. Adalia 1: 3-19.

Parisi, L. & Trillot, M. 1993: Variétés de pommier: Résistance et sensibilité à la tavelure, à l'oïdium et aux maladies de conservation. INFOS-CTIFL 89: 29-30.

Wolfe, M.S. 1985: The current status and prospects of multiline cultivars and variety mixtures for disease resistance. Ann. Rev. Phytopathol. 23: 251-273.

Zhu, Y.Y., Chen, H.R., Fan, J.H., Wang, Y.Y., Li, Y., Chen, J.B., Fan, J. X., Yang, S.S., Hu, L.P., Leung, H., Mew, T.W., Teng, P.S., Wang, Z. & Mundt, C.C. 2000: Genetic diversity and disease control in rice. Nature 406: 718-722.


pp. 31 - 36

31

Phytotoxic effect of lime sulphur on apple and pear Bart Heijne1, Peter Frans de Jong1, Imre Janos Holb2

1Applied Plant Research (PPO), P.O. Box 200, 6670 AE Zetten, The Netherlands; 2Department of Plant Protection, Centre of Agricultural Sciences, University of Debrecen, P.O. Box 36, 4015 Debrecen, Hungary Abstract: The total area of cluster leaves and the general leaf condition were reduced by the preventive lime sulphur schedules on apple, but not for the curative lime sulphur schedule. Russeting of fruits increased by the preventive lime sulphur schedule on apple in 2000 and to a minor extent in 2001. The curative lime sulphur schedule promoted russeting in apple in 2000, but not significantly in 2001. Consequently, the percentages of first class apples were reduced by the lime sulphur schedules. There was no effect on total production.

On pear, the total area of cluster leaves tended to be smaller in the preventive lime sulphur schedule in 2000. This effect was significant in 2001. There was no effect of the lime sulphur schedule applied at high spray volume (1000 l/ha) on pear leaf condition in 2000, but the leaf condition was affected in 2001. However, lime sulphur schedules did not affect the leaf condition when applied at a spray volume of 200 l/ha or at half the standard dosage. Lime sulphur schedules produced more russeting of pear fruits. The production decreased in the lime sulphur schedule in 2000, but not significantly in 2001.

It was concluded that lime sulphur could be phytotoxic on both apple and pear under the Dutch climate conditions. Introduction Governments promote organic production of fruits all over Europe. For scab control on apple and pear sulphur is the cornerstone in organic production. Under cold conditions sulphur is not enough effective (Ellis et al., 1998). Copper is quite effective under these cold conditions. However, due to environmental reasons copper is banned in the Netherlands and Denmark. Therefore, other products are sought for to improve scab control in organic orchards. One of the options is the use of lime sulphur. Lime sulphur is one of the oldest fungicidal products. It has been described in 1802 in England. It is prepared by boiling dehydrated lime (CaO) and sulphur (S) in water and it is composed mainly of calciumpolysulphides.

The efficacy of lime sulphur against scab was investigated in the beginning of the twentieth century. For example, it was proved to be as effective as copper fungicides (Hamilton, 1931). Moreover, Hamilton (1931) and Mills (1947) demonstrated that lime sulphur gave sufficient control when applied within 30 to 72 hours after inoculations. More recently, Ellis et al. (1994) claimed that reduced rates of lime sulphur gave better scab control during summer than sulphur. Also in Europe, authors like Trapman and Drechsler-Elias (2000) and Holb & Heijne (2001) found that lime sulphur has a good efficacy against scab and might replace copper.

However, good efficacy was in some cases accompanied by increased russeting of apples. Therefore, we started new experiments to investigate the possible phytotoxicity of lime sulphur on apple and pear with the aim to find possible strategies to circumvent adverse effects.

32

Materials and methods On apple and on pear, experiments were conducted in 2000 and 2001 at Randwijk, the Netherlands in orchards maintained according guidelines of organic production. The planting system of the apple orchard was two rows of cultivar (cv.) Jonagold alternated with two rows of cv. Schone van Boskoop both on M.9 rootstock at a planting distance of 3 x 1.25 m. Trees were planted in 1996. The planting system of the pear orchard was two rows of cv. Conference alternated with one row of cv. Gieser Wildeman both on quince C rootstock at a planting distance of 3.5 x 1.5 m. Trees were planted in 1999. Data presented in this paper are only for cv. Jonagold and cv. Conference.

All applications were made with a hand-held spray gun with ceramic hollow cone nozzle. Sprayings were applied at 1.1 – 1.2 MPa with a spray volume of 1000 l/ha, except for one treatment in pear, which was applied at a spray volume of 200 l/ha.

The experimental layout was a randomised block design with five replicates. The treatments are summarised in Table 1. The conventional fungicide was Captosan (80 % a.i. captan) applied before and after 1 May at 0.11 % and 0.15 % respectively. Sulphur was applied as Thiovit S (80 % a.i.) at 0.4 % when temperatures were higher than 15 °C and at 0.8 % when lower than 15 °C. Lime sulphur was applied as Polisolfurio di Calcio (27 % a.i. polysulphides) at 2 % and 1.5 % before and after bloom respectively. In one treatment on apple and pear, lime sulphur was applied at half the dose when the relative humidity was over 85 % (rcLS, rpLS). Another treatment on pear consisted of a low volume (200 l/ha) application of lime sulphur (lpLS). The products were applied in a preventive spay schedule with the policy to apply the product shortly before an expected scab infection period. Other treatments were applied within 48 hours after a calculated infection period; these are called curative schedules. Treatment schedules started end March and all treatment schedules changed to a schedule of 0.5 % sulphur at the end of the ascospore season from half June till harvest. Table 1. Treatments

apple pear code product dose schedule 2000 2001 2000 2001U untreated - X X X X pC conventional fungicide 0.11-0.15 % preventive X X X X pS sulphur 0.4-0.8 % preventive X X X X pLS lime sulphur 1.5-2 % preventive X X X X cLS lime sulphur 1.5-2 % curative X X rcLS lime sulphur 1.5-2 % curative X rpLS lime sulphur 1.5-2 % preventive X lpLS lime sulphur 1.5-2 % preventive X

Leaf area was assessed by collecting 12 clusters per tree at the end of May. Individual leaf areas were measured and the average total leaf area per cluster was calculated as the sum of the total leaf area per plot divided by the number of clusters sampled per plot. Fruit russeting was recorded based on a scale of 1 to 4: class 1 = no russet; class 2 = 1-11 %; class 3 = 12-33% and class 4 > 33% of surface russeted. A russeting index was calculated according to the formula:

33

5.665.2260 4321 •+•+•+•=t

class

t

class

t

class

t

class

NN

NN

NN

NNRI

in which RI = average percentage of fruit surface russeted; Nclass1, Nclass2, Nclass3 and Nclass4 represent the number of fruits in each class of russeting; Nt = total number of fruits. The number 0, 6, 22.5 and 66.5 in the equation represent the percentage of the russeted fruits expressed as a mean of the lower and upper boundary of class1, class 2, class 3 and class 4, respectively. All harvested fruits were classified and the percentage of first class fruits was determined. First class fruits were defined as fruits belonging to russeting class 1 and 2, irrespective of all other parameters.

All data were subjected to analysis of variance using the Genstat 5 Release 4.1 statistical package. No transformations were necessary. Significant F-tests were followed by Least Significance Difference (LSD)-test for pair wise comparisons. Results The leaf condition of apples was measured on a scale from 0 (no adverse effects) to 5 (severe damage). The leaf condition was significantly affected by the use of lime sulphur in both years, whereas the sulphur treatment schedules were not significant different from the untreated and the conventional fungicide schedule. This accounted for both the preventive and the curative lime sulphur schedule.

The total leaf area of cluster leaves was reduced by lime sulphur schedules compared to untreated, conventional and sulphur schedules (Figure 1). This effect was most prominent in the preventive lime sulphur schedules. This effect was found for apple and pear and in both years.

Figure 1. Total cluster leaf area (in cm2) and fruit russeting index for apple and pear treated

with different scab control schedules U, pC, PS, pLS, cLS, rcLS and lpLS (see also table 1).

bb b

a a

cc c

a

bab

4

6

8

U pC pS pLS cLS rcLS

leaf

are

a

20002001

apple

aaaa

aababcc

bc

4

6

8

10

12

14

U pC pS pLS rpLS lpLS

leaf

are

a

20002001

pear

cb

aa

a bbbababa

0

1

2

3

4

5


russ

etin

g in

dex

20002001

apple

cc

a

b

ccc

ba

b

2

4

6


russ

etin

g in

dex

20002001

pear

34

The russeting index increased by the use of a lime sulphur spray schedule compared to the conventional spray schedules (Figure 1). The curative lime sulphur schedule caused more russeting in apple than the preventive lime sulphur schedule in year 2000. This was a year with much russeting, also in practice. Russeting in pear is of less importance economically, but the sulphur as well as the lime sulphur schedule increased russeting significantly in both years (Figure 1).

The percentage first class fruits of apple was reduced dramatically by the lime sulphur schedules, both preventively and curatively applied as a result of russeting (Figure 2). This has major economic implications.

Figure 2. The percentage first class apples as a result of different scab control schedules: U,

pC, pS, pLS, cLS and rcLS (see also table 1).

The production of apples increased with the conventional fungicide schedule compared to all other schedules (Figure 3) in both years. The production of pears did not change significantly in 2001. However, there was a pronounced effect of the different spray schedules in 2000. The production was strongly reduced by applying the sulphur schedule. The effect of application of the preventive lime sulphur schedule was even more dramatic. The production was reduced by 61 percent compared to the conventional schedule (Figure 3).

Figure 3. Production of fruits of apple and pear treated with different scab control schedules

U, pC, pS, pLS, cLS, rcLS, rpLS and lpLS (see also table 1). Discussion Lime sulphur schedules showed an increase in phytotoxic effects both on apple and pear in the presented study. This was the case not only compared to a conventional spray schedule,

a

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but also compared to a sulphur schedule. Subhash (1988) and Tate et al. (2000) also claimed that lime sulphur is toxic to plants. Although the mechanism of lime sulphur phytotoxicity is insufficiently understood, it is believed that soluble sulphide components could be responsible for the plant injury.

Some authors noted that an increased phytotoxic effect occurred when the relative humidity was high or the leaf wetness period prolonged after spraying (Subhash, 1988; Tate et al., 2000). We incorporated these ideas in the experiments of 2001. For this purpose, we included treatment schedules where the dosage of lime sulphur was half the rate when the relative humidity was high. In another treatment schedule fungicides were applied at low spray volume, which almost immediately dries upon contact with the foliage. Therefore it was anticipated that these schedules rpLS, rcLS and lpLS would cause less phytotoxic effects. Although the percentage first class apples was higher, neither the leaf area reduction nor the russeting was alleviated by these treatment schedules in both apple and pear.

It has been found that the curative activity of lime sulphur might be dependent on wet conditions after spraying (Trapman & Drechsler-Elias, 2000). The idea was that sulphide components from the lime sulphur formulation are the active ingredient for the curative activity. The water-soluble sulphides might penetrate the fungal mycelia in the water phase. Since the soluble sulphide is thought to be also the phytotoxic component for plants (Subhash, 1988; Tate et al., 2000), this creates a insoluble dilemma.

In earlier studies on the fruit production, it was assumed that lime sulphur was injurious to the tree and thereby reduced yield (Hamilton, 1931). Recently Holb & Heijne (2001) demonstrated that tank mix combinations of lime sulphur and sulphur did not affect yield, when sprayed before full bloom. Lime sulphur was sprayed also during flowering at reduced rates in the present study. Since the present study demonstrated a reduced yield in pear, it is suggested that lime sulphur might damage the pistils of flowers.

It is concluded that there is too little knowledge about the physiological background of both the phytotoxic and fungitoxic effect of lime sulphur. This makes it difficult to combine the good efficacy of lime sulphur with circumventing phytotoxic effects at the same time. For the fruit grower this poses a serious dilemma. References Ellis, M.A., Madden, L.V., Wilson, L.L. & Ferree, D.C. 1994: Evaluations of organic and

conventional fungicide programs for control of apple scab in Ohio. – Ohio Agricultural Research Development Centre Research 298: 63-68.

Ellis M.A., Ferree, D.C. & Madden, L.V. 1998: Effects of an apple scab-resistant cultivar on use patterns of inorganic and organic fungicides and economics of disease control. – Plant Disease 82: 428-433.

Hamilton, J.M. 1931: Studies of fungicidal action of certain dust and sprays in the control of apple scab. – Phytopathology 21: 445-523.

Holb, I.J. & Heijne, B. 2001: Evaluating primary scab control in organic apple production. – Gartenbauwissenschaft 66: 254-261.

Mills, W.D. 1947: Effects of sprays of lime sulphur and of elemental sulphur on apple in relation to yield. – Ithaca, N.Y. Cornell University Agricultural Experimental Station 273: 38.

Subhash, C.V. 1988: Nontarget effects of agricultural fungicides. – London, UK: CRC Press, 443 pp.

36

Tate, K.G., Manktelow, D.W., Walker, J.T. & Stiefel, H. 2000: Disease management in Hawke’s Bay apple orchards converting to organic production. – New Zealand Plant Protection 53: 1-6.

Trapman, M. & Drechsler-Elias, E. 2000: Die kurative Wirkung von Schwefelkalk gegen Apfelschorf. – Obstbau 25: 559-561.


pp. 37 - 41

37

Pome fruit storage diseases Joana Henriques CEFA - IICT, Storage Pests Research Center - Tropical Scientific Research Institute, Trav. do Conde da Ribeira, 9, 1300-142 Lisboa, Portugal Abstract: Pome fruits are one of the main Portuguese cultures with great economical importance. They can be stored for very long periods, sometimes reaching nine or ten months. Several fungi often cause diseases in fruits during this period, although they can be originated in the orchard or in the store. The aim of this work is to quantify and describe the mycological pathologies in apples and pears stored in refrigerated chambers and under several conditions related with different atmosphere contents and post-harvest fungicide treatments. Samples of different kinds of fruits ("Rocha" pear, Golden, Gala Must, Royal Gala, "Reineta" and Starking apple) were taken and, after being accounted, the pathogenic fungi were isolated from them. Alternaria, Aspergillus and Penicillium were the most common identified fungi but several other fungi were isolated, mostly saprophytic ones. To store fruits for a long time, the use of controlled atmospheres and the treatment with fungicides are profitable. Under such conditions, rotting decreases and fruit quality remains better. Controlling post-harvest diseases is an integrated system which involves the hygiene of the orchard and the store chamber, the handling and the storage programme, all carefully planned in accordance with the storage period. Key words: pome fruits, fungi, controlled atmosphere, fungicide Introduction Fruits are one of the most important crops of Portugal. Apples (Malus domestica Borkh) and pears (Pyrus communis L.) are among the main cultures in economical means, especially in the west zone of Portugal (GPPAA, 2001).

Lots of researches have been done about the physiology and pathology of pome fruits, so the influencies of the environmental and cultural factors of the orchard on fruits development and their storage potencial are well known (Snowdon, 1990).

After the decade of forty, with the overuse of pesticides and pome fruit production valorization, a concern with environmental and toxicological problems originated the use of Integrated Pest Management (IPM) and subsequently Integrated Production systems on pome fruit cultures (Amaro, 1993; Oberhofer, 1992a in Clemente, 1994). Amaro et al (1993) define Integrated Production of fruits as "an economical production of high quality fruit, using mainly methods with ecological concerns and minimizing the application of pesticides and its secondary effects, in order to improve the environment protection and human health".

Apples and pears are commercially harvested before they become ripe for eating. Fear of loss through abscission, senescence and pathogen attack encourages growers to harvest immature fruits (Knee, 1993). In all major apple and pear growing areas the bulk of the crop is stored for several months to maintain availability for as long as economically desirable (Knee, 1993). The conservation of fruits for long periods causes several sanitary problems that one needs to know in order to avoid losses during its storage time or commercialization (Bondoux, 1992).

Fruit losses during the storage period are due to either physiological or mycological problems (Bondoux, 1992). In the research only mycological diseases will be considered.

38

During the thirties more than ninety species of fungi were identified as apple post-harvest pathogens (Heald & Ruehle, 1931 in Conway & Rosenberg, 1986) and about sixty species were identified as pear pathogens (English, 1940 in Conway & Rosenberg, 1986).Several other fungi have been isolated in pome fruits since those first reports (Conway & Rosenberg, 1986). However, with the current storage methods, only a few fungi cause relevant losses such as Penicillium, Botrytis and Rhizopus (Julio & Carvalho, 1993; Lund & Snowdon, 2000).

Fruit contamination can occur either in the orchard or during its storage time. Fungi can infect fruit during the bloom, through the carpels and the stamens and, later, through fruit natural openings such as lenticels and pedunculum. An important way of post-harvest contaminations is the injuries that fruits suffer during harvest, transport and conservation handling (Bondoux, 1992; Julio & Carvalho, 1993; Malheiro, 1972).

The development of pathogenic agents that enter into natural openings of flowers and fruits only happens when fruits are mature enough, so the warehouse conditions not only avoid fruits´ fast ripening (which enable fungi to develop in them) but also avoid fungi germination and growth. The technology of fruit storage has become complex and expensive, evolving refrigerated systems, different atmospheres and chemical treatments with pesticides and growth regulator products (Knee, 1993).

Preservation of pome fruits with minimum losses of fruit quality has been possible since chemical and physiological reactions that occur during fruit ripening get reduced (Simas, 1998). Conservation in refrigerated chambers with normal atmosphere conditions is based on the beneficial effects of low temperatures and high relative humidity, reducing the metabolic activity of fruits and losses of weight, respectively (Franco, 1995). Conservation in controlled atmosphere conditions consists on putting fruits in isolated refrigerated chambers which have low oxygen levels and relatively high carbon dioxide concentration, comparing with normal air composition (Will et al., 1989 in Franco, 1995).

The greatest advantages of controlled atmosphere conservation are the extension of the storage time of fruits, keeping fruits quality and reducing diseases caused by fungi. Its bigger defect is the high cost comparing with normal atmosphere storage (Franco, 1995).

Lots of products are applied to fruits in order to control post-harvest diseases (Lund & Snowdon, 2000). It is essential guarantee the protection of fruits that are stored for long periods, by treating them preventively with fungicides (Calixto & Carvalhão, 1993). Although their effectiveness against latent pathogens, post-harvest treatments are mainly used to protect fruits from the injury by pathogens (Julio & Carvalho, 1993). Lots of organic compounds for post-harvest treatment of fruits have been analysed during the last thirty years (Eckert, 1983 in Snowdon, 1990), however one must consider which they are according to IPM rules.

Considering the real economical importance of pome fruits in Portugal and their post-harvest problems, the aim of this work research is to quantify and identify the main pathogenic fungi to the fruits. Material and methods Fruit sampling Apples and pears were sampled in two different warehouses (A and B) in the west region of Portugal from 27/10/99 to 24/03/00. Those fruits had been harvested during August to September of 1999 and stored under different conditions of atmosphere composition and post-harvest fungicides treatments. The fungicides applied were imazalil and folpet which belong to imidazole and carboximide classes respectively. Folpet interferes with fungi’s energy production, while imazalil´s action involves sterols biosynthesis (Ragsdale, 1994). In table 1 are described the conditions that the fruits were submitted during their storage period.

39

Table 1. Fruit conditions in each of the warehouses (A and B).

A B without treatment without treatment

Fungicides Folpet (50% [200g/100L]) Imazalil (7.5% [0.5L/100L])

Folpet (50% [200g/100L]) Imazalil (7.5% [0.5L/100L])

Temperature 0ºC -0.5ºC Relative Humidity 90% 95%

Normal Atmosphere Normal Atmosphere Atmosphere composition Controlled Atmosphere

(0.7-1% CO2 / 3-4% O2) Controlled Atmosphere (0.7% CO2 / 2.5% O2)

The fruits sampled belonged to the following cultivars of apples: Golden Reineta, Gala Must, Starking and Royal Gala, and Rocha pear.

Fruits were sampled randomly when they were passing through the gauge and then separated according to the presence of diseases symptoms or not and quantified by weight and number. The infected fruits were incubated at 20ºC to promote fungi’s development. Fungi isolation and identification The pathogenic fungi were isolated from infected fruits and cultivated in Potato Dextrose Agar (PDA) growing medium. The inocula were previously disinfected in sodium hypoclorite 25% and sterilized distilled water. Fungi cultures were incubated at 28ºC and high relative humidity.

Fungi identification was based on the observation of reproductive asexual structures using Riddel chambers that allow a better visualization of those structures.

The fungi that were most frequently isolated were reinoculated in pome fruits in order to verify the symptoms that they cause. Fresh fruits were disinfected with sodium hypoclorite 25% and sterilized distilled water and, after being inoculated were incubated at 20ºC to stimulate the development of the illness symptoms but to prevent fruit’s senescence. Results and discussion The fruits sampled during this research were accounted according to the presence of symptoms of disease and related with the different storage conditions and period of conservation that they were submitted. In figure 1 is represented the evolution of fruit contamination during the sampling period.

After the harvest, fruit susceptibility to fungi infections increases gradually as the ripening process of fruits proceeds (Julio & Carvalho, 1993). By looking at figure 1, one can verify that for some cases (Rocha pear from warehouse A and Golden apple), during the first seventy days of storage, there is an increasing of contaminated fruits. After that first period, the fruits sampled were stored under different conditions (indicated in the figure) that had protected fruits from fungi’s contamination. The fruits that were intended for a longer storage period were conserved under controlled atmosphere and were previously treated with post-harvest fungicides. Those steps accomplished positive results, once by the end of the sampling period (which corresponded with the last fruits stored) the levels of contaminated fruits remained relatively low. Those results are in agreement with Julio & Carvalho (1993) and Knee (1993) who suggested that fruit destined to extend conservation should be treated with fungicides and stored under controlled atmosphere in order to prevent fruits´ senescence

40

and contamination. Both fungi’s variability and quantity were influenced by fruit post-harvest treatment and atmosphere composition of the warehouse.

Figure 1. Evolution of fruit contamination according to the cultivars sampled and the different storage conditions of the warehouses.

The line that represents Rocha pear from warehouse B evolution (in figure 1) shows some oscillations that correspond to a lot of fruits that had some problems during their transport from the orchard to the warehouse. Those fruits had high contamination levels which emphasize the importance of a careful handling of fruits avoiding injures that would facilitate the fungi's entry.

Apples from Reineta, Gala Must, Starking and Royal Gala cultivars were sampled only once. All those apples didn’t suffer any post-harvest fungicide treatment. They all show very high contaminations levels. Apples are more sensitive to fungi’s infections due to their enhanced apical cavity and abundant lenticels (Clemente, 1994). Golden apples had lower contaminations levels because they were treated with fungicides.

Most identified fungi belonged to Genera Alternaria, Aspergillus and Penicillium. Acremonium, Botrytis, Fusarium, Monilia, Stemphylium, Trichoderma and Ulocladium were also identified. Other different moulds were isolated from infected fruits but didn’t produce reprodutive structures enabling their identification. The most frequent pathogen of Rocha pear was Alternaria while for apples was Penicillium.

The symptoms that Alternaria causes in fruits are initially a black drought spot that, as fruits ripe, develops very fast in a round, brown wet spot which can form a dense brown tuft of conidial chains. Aspergillus causes light brown spots that develop a white mycelium with yellow little spots that are the conidial structures. Fruits´ infection by Penicillium is very fast, it begins as a round wet light brown spot that forms large blue-grey masses of spores on fruit surface.

Controlling post-harvest diseases is an integrated system that involves the hygene of the orchard and warehouse, the handling and storage programme, all planned in accordance with the storage period.

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References Amaro, P., Oliveira, P., Silva, A.V., Soeiro, A., Fernandes, A.M.S., Mexia, A., Oliveira, A.

Matias, C., Costa, C.A., Caldeira, J.M., Silva, J.M., Silva, J.V., Jardim, J., Clemente, J., Sousa, M.M.D., Morais, M.M. & Filipe, N. 1993: Projecto de normas portuguesas de produção integrada de pomóideas. – Colóquio de produção integrada em pomóideas, 13-14 Maio 1993: 8-22.

Bondoux, P. 1992: Maladies de conservation des fruits à pépins pommes et poires. – INRA. Calixto, J. & Carvalhão, F. 1993: Tratamento pós-colheita em pomóideas. – Gazeta das

Aldeias, 3003: 25-26. Clemente, J.M.R. 1994: Proposta para um programa de produção integrada em maceira

(Malus domestica Borkh.) na região do oeste. – Tese de mestrado em proteccão integrada, ISA/UTL.

Conway, W.S. & Rosenberger, D.A. 1986: Evaluating fungicides for control of postharvest decay of pome fruits. – In: Methods for Evaluating Pesticides for Control of Plant Pathogens, ed. Hickey, K.D., APS Press: 88-91.

Franco, M.J.B. 1995: A conservação e a qualidade da pêra Rocha - Influência da rega, data de colheita e tipos de conservação. – Tese de mestrado em Produção Vegetal, ISA/UTL.

GPPAA 2001: Anuário Hortofrutícola 2000. – Ministério da agricultura, do Desenvolvimento rural e pescas.

Julio, E. & Carvalho, A. 1993: Doenças de conservação em maçãs e pêras. – Revista de ciências agrárias, 16(1-2-3): 151-162.

Knee, M. 1993: Pome fruits. – In: Biochemistry of Fruit Ripening, eds. Seymor, G.B., Taylor, J.E. & Tucker, G.A., Chapman & Hall: 325-346.

Lund, B.M. & Snowdon, A.L. 2000: Fresh and processed fruits. – In: The Microbiological Safety and Quality of Food, Vol. 1, eds.Lund, B. M., Baird-Parker, T. C. and Gould, G. W., Aspen Publishers, Inc: 738-757.

Malheiro, M.R.A.L. 1972: Doenças mais notórias de maçãs e pêras conservadas em câmara frigorífica, Rel. final do curso de engenheiro agrónomo. – ISA/UTL.

Ragsdale, N.N. 1994: Fungicides. – In: Encyclopedia of Agricultural Science, Vol. 2, eds. Arntzen, C.J. & Ritter, E.M., Academic Press: 445-453.

Simas, A.F.P. 1998: Conservação de pêra Rocha em câmaras de atmosfera normal e controlada. Relatório de fim de curso de Engenharia Agro-Industrial. – ISA/UTL.

Snowdon, A.L. 1990: A Colour Atlas of Post-Harvest Diseases & Disorders of Fruits & Vegetables, Vol. 1. – Wolfe Scientific Ltd.


pp. 43 - 48

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Durable disease resistance and high fruit quality, a challenge for apple breeding Markus Kellerhals1, Cornelia Sauer1, Ernst Höhn1, Barbara Guggenbühl1, Jürg Frey1, Robert Liebhard2, Cesare Gessler2 1 Swiss Federal Research Station, P.O. Box 185, CH-8820 Wädenswil, Switzerland; 2Federal Institute of Technology, Institute of Plant Science/Phytopathology, ETH Zentrum, CH-8092 Zürich, Switzerland Abstract: High fruit quality and durable disease resistance are crucial aspects in apple breeding directed towards sustainable production systems. Results of consumer tests with new disease resistant apple cultivars confirm that progress has been achieved in improving fruit quality and, as a consequence, in consumer acceptance. Fruit firmness, juiciness and aroma are among the most important criteria. The challenge of breeding durable disease resistant varieties is approached by developing efficient molecular techniques that allow to detect the combined presence of the target resistance genes in a seedling. First examples for molecular selection towards genotypes with pyramidised genetic resistance against scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotricha) are promising. Key words: Apple, breeding, apple scab, powdery mildew, molecular markers Introduction Apple breeding at Wädenswil is aimed at combining high fruit quality with good orchard performance and durable disease resistance for sustainable production systems. Ariwa is a commercially available scab (Venturia inaequalis) and mildew (Podosphaera leucotricha) resistant cultivar (Vf and Pl1) developed at Wädenswil. Further advanced selections are evaluated in trials including organic growing conditions.

Apple breeders mainly used single resistance genes to achieve good field resistance towards scab and mildew. They originate from wild species such as Malus floribunda 821, M. zumi and M. robusta. However, in recent years this type of resistance has shown to be rather vulnerable since it can be overcome by new pathogen races. Today the main emphasis is on the combination of functionally different major resistance genes and the use of quantitative resistances to achieve durability. Molecular markers were developed which permit the detection of major resistance genes. The combined presence of the target resistance genes in a seedling can be determined by PCR-based molecular analysis used in marker-assisted selection (MAS). Currently similar markers for loci on the genome which determine qantitative resistance (QTL) are being developed.

Molecular tools for early selection of disease resistance and fruit quality are rapidly developing. In Europe common efforts have been made within the D.A.R.E project (Durable Apple Resistance in Europe, 1998-April 2002), partly supported by the European Union and national funds (e.g. in Switzerland). It was devoted to the development of plant material, pathogen reference strains, genetic maps and marker-assisted selection, the basis for the creation of new apple varieties carrying durable resistance against scab and powdery mildew (Lespinasse et al., 2000).

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Advances in Marker-assisted selection Molecular markers were developed which allow to detect the Vf scab resistance (Tartarini et al., 1999). Several large progenies were analysed for phenotypic scab classification in the glasshouse and for presence or absence of the ALO7 allele in coupling with the Vf scab resistance. Class 4 symptoms according to Chevalier et al., 1991 corresponded extremely well with the absence of the expected band, while classes 0 to 3b generally amplified the band.

A certain number of plants classified as 3a and 3b (5-10% of all progeny plants) although phenotypically resistant to scab do not amplify the expected band. An additional analysis of these specific plants with a second molecular marker for Vf (M18, Gianfranceschi et al., 1996) usually confirm the absence of the Vf gene. As AL07 and M18 are flanking markers of the Vf gene at 0.3 cM and 1.2 cM repectively (Patocchi et al., 1999b) the probability of double recombinants is very small (7.2 x 10-5). These plants must therefore be considered not double recombinant but incorrectly classified for the resistance. However, it can be concluded that the inheritance of the markers linked to the Vf resistance and the phenotypic analysis correspond well (Kellerhals et al., 2000a).

Molecular markers linked to Vbj originating from Malus baccata jackii (Gygax, personal communication) and close to three apple scab resistance genes detected in Russian seedling (Vr, Vx and Vr2) were also developed (Hemmat & Brown, 2002; Patocchi et al., 2003). Interestingly, Vr, Vr2 and Vbj are all located on the linkage group 2 (map of Liebhard et al., 2003), Vr2 maps on the opposite side of of the likage group than Vr and Vbj. The two closest SSR’s linked to Vr and Vbj, CH02b10 and CH05e03 respectively, on the map of Liebhard et al., 2003 map 4.2 cM apart. Hemmat & Brown. (2002) mapped Vr 7.8 cM from CH02b10 while Vbj was mapped 0.9cM from CH05e03 (Gygax, personal communication). Since genetic distances are frequently variable depending on progenies used, it is also possible to suggest the hypothesis that Vr and Vbj could be allelic or could belong to a cluster of apple scab resistance genes.

Table 1 shows results of marker analysis in two populations crossed in the year 2000 and evaluated in 2001 and 2002 at the phenotypic and molecular level. Table 1. Results of marker-assisted selection in two apple progenies including the scab and

mildew resistance genes Vf, Vbj and Pl2, respectively. FAW 8027 (Vf, Pl2) x FAW

8476 (Vbj) FAW 8244 (Vf, Pl2) x FAW 8476 (Vbj)

Number of plants 290 430 ALO7 (Vf) present 145 (50%) 219 (50.9%) N18 (Pl2) present 115 (39.6%) 289 (67.5%) T6 (Vbj) present (only when AL07 present)

67 (46.8 %)

Z13 (Vbj) present (only when AL07 present)

95 (66.4 %)

These progenies include the resistance genes Vf and Vbj against scab and Pl2 against powdery mildew. For Vf a clear 1:1 segregation was confirmed when using the marker ALO7. The SCAR marker N18 linked to the Pl2 mildew resistance and developed by Seglias & Gessler (1997) was present in 39.6 and 67.5 % of the progeny plants respectively. A squed distribution with a rate of more than 50% presence of the marker in one of the progenies can

45

currently not be explained. Work carried out within the EU-project DARE using the enriched map around Pl2 and the field data of Seglias & Gessler (1997), the Pl2 locus explains about 50-55% of the variability of a mildew scoring data set (calculated with MapQTL). The microsatellite marker CH04H02 was located close to the N18 marker. Considering these results, it is questionable whether Pl2 has to be considered as a major gene or a large-effect QTL. However, the correspondence between phenotypic scoring of powdery mildew in the two year old apple seedlings and the molecular results was very good (Figure 1 and 2).

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FAW 8027 (Vf, Pl2) x FAW 8476 (Vbj).

020406080

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Figure 2. Mildew susceptibility distribution for plants carrying the N18 marker in the progeny

FAW 8244 (Vf, Pl2) x FAW 8476 (Vbj).

The markers for Vbj were only tested on the plants carrying ALO7. T6 and Z13 did not completely correpond. We conclude that these markers need further improvement or replacement by more reliable markers, respectively. This work is one of the first examples for molecular selection towards genotypes with pyramidised genetic disease resistance.

In the framework of the EU-project DARE a large range of new molecular markers for genes coding for scab and mildew resistance and potentially useful for MAS were developed.

46

However, several markers are not yet ready for use in high-throuput MAS. Moreover, QTL markers to detect partial resistance against scab and mildew are being developed (Kellerhals et al., 2000b). To detect QTL loci, a genetic linkage map was constructed (Liebhard et al. 2003, in press). A population with about 400 seedlings of Fiesta x Discovery was planted at three locations in Switzerland: Wädenswil, Conthey (VS) and Cadenazzo (TI). Both parents are moderately susceptible to scab. Discovery has low susceptibility to mildew but Fiesta is very susceptible to mildew. A linkage map of the two parents has been made and about 350 polymorphic markers were grouped in 17 linkage groups. On these populations we also did some preliminary studies on fruiting and fruit quality parameters. Fruit quality: a very important criterion Fruit quality is a crucial aspect for the success of a new variety in the market. The aims of the apple breeding programme at Wädenswil are to develop cutivars with

- high fruit quality - good and regular yields - disease resistance (scab, mildew, fireblight)

Apples are a healthy food as they contain numerous minerals, trace elements, vitamins and secondary plant components with antioxidative effect (Mayr & Treutter, 1996; Müller & Treutter, 2000).

In the last decades many apple breeding programmes worldwide have considered disease resistance as being a high priority breeding aim (Alston, 1989; Kellerhals et al., 1998). Disease resistant varieties allow significant reduction of pesticide inputs in orchards and are therefore supposed to further improve the image of apples being a healthy food. However none of those disease resistant varieties has yet achieved a breakthrough in the market comparable to varieties such as Golden Delicious, Gala or Braeburn. Sensory studies including consumer tests were conducted to examine the market potential of several new disease resistant cutivars and possible reasons for their rejection by consumers. Consumer tests with new disease resistant apple varieties Consumer tests were performed in march and april 2000 (n = 280) and in march 2001 (n = 184) at different supermarkets in Switzerland (Wädenswil & Sion in 2000; Thalwil & Bulle in 2001). In all tests each consumer evaluated one apple of three different cultivars, two disease resistant and a well-known commercial variety as standard. In order to increase the variation in fruit quality and to examine the response of the varieites to stress, different storage and shelf-life conditions were applied. Samples were stored under controlled-atmosphere (CA) conditions, others in ususla cold store. Additionally, in 2001 some samples were put at room temperature for one or two weeks, respectively, before the test. The apples were presented to the consumers according to a William Latin Square design and evaluated for their appearance as well as their overall liking on a nine-point hedonic scale (dislike extremely = 1, like extremely = 9). In order to further characterise the sensory attributes of the apples, a ‘just-about-right‘ scale with five categories anchored with „too weak“, „too strong“ and „just-about-right“ was presented for the evaluation of firmness, juiciness, aroma-intensity, sweetness and acidity. Each individual fruit tested by the consumers was subsequently analysed instrumentally for firmness, acidity, sugar and juice content.

The consumer tests showed that Swiss consumers have a determined opinion about which quality traits are important in apples (Figure 3). Firmness was among the most important attributes. Overall, the scab resistant apple varieties Ariwa and Topaz tested in 2000 were similarly well accepted as the popular control variety Royal Gala (data not shown). In 2001, the two scab resistant cultivars Goldrush and Otava got an even higher rating for most attributes than Golden Delicious (example for overall acceptance given in Figure 4).

47

Figure 3. Quality criteria relevant to consumers (n= 280, year 2000).

1 2 3 4 5 6 7 8 9

overall acceptance

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Golden DeliciousGoldrushOtava

Figure 4. Overall acceptance of the new scab resistant apple cultivars Goldrush and Otava

compared to Golden Delicious (1= very low, 9=very high, n=184, year 2001).

While relating sensory properties and instrumental data, we can conclude that only a small part of the variation in sensory perception of apples can be explained by instrumental data. It might therefore be necessary to think also about other factors such as aroma components or psychological aspects related to sensory perception .

Progress is being made in the molecular analysis of fruit quality components (King et al., 2001). Genetic linkage maps of the apple are established. However, most quality criteria in apple are determined by several genes. In this situation the QTL analysis (quantitative trait loci) and QTL mapping are suitable.

Conclusion The prospects are good to successfully combine durable disease resistance and fruit quality requirements of the market and the consumers by using modern breeding techniques.

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48

Acknowledgments The authors acknowledge technical support by Beatrice Frey and financial support by Swiss Federal Office of Education and Science (DARE project). References

Alston, F. 1989: Breeding pome fruits with stable resistance to diseases: selection techniques

and breeding strategies. – In: Integrated control of pome fruit diseases, eds. Gessler, Butt and Koller. IOBC/wprs Bulletin 12(6): 90-99.

Chevalier, M., Lespinasse, Y. & Renaudin, S. 1991: A microscopic study of the different classes of symptoms coded by the Vf gene in apple for resistance to scab (Venturia inaequalis). – Plant Pathol. 40: 249-256.

Gianfranceschi, L., Koller, B., Seglias, N., Kellerhals, M. & Gessler, C. 1996: Molecular markers in apple for resistance to scab caused by Venturia inaequalis. – Theor. Appl. Genet. 93: 199-204.

Hemmat, M. & Brown, S.K. 2002: Tagging and mapping scab resistance genes from R12740-7A apple. – J. Amer. Hort. Sci. 127: 365-370.

Kellerhals, M., Viviani, A., Goerre, M. & Gessler, C. 1998: New challenges for apple breeding. – Acta Hort. 484: 131-134.

Kellerhals, M., Dolega, E., Koller, B. & Gessler C. 2000a: Advances in marker-assisted apple breeding. – Acta Hort. 538: 535-540.

Kellerhals, M., Gianfranceschi, L., Seglias, N. & Gessler, C. 2000b: Marker-assisted selection in apple breeding. – Acta Hort. 251: 255-266.

King, G.J., Maliepaard, C., Lynn, J.R., Alston, F.H., Durel, C.E., Evans, K.M., Griffon, B., Laurens, F., Manganaris, A.G., Schrevens, T., Tartarini, S. & Verhaegh, J. 2001: Quantitative genetic analysis and comparison of physical and sensory descriptors relating to fruit flesh firmness in apple (Malus pumila Mill.). – Theor. Appl. Genet. 100 (7): 1074-1084.

Liebhard, R., Koller, B., Kellerhals, M., Pfammatter, W., Jermini, M., Roth, H.R. & Gessler, C. 2003: Mapping quantitative field resistance in apple against scab. – In press.

Lespinasse, Y., Durel, C.E, Parisi, L., Laurens, F, Chevalier, M. & Pinet, C. 2000: An European project: D.A.R.E - Durable Apple Resistance in Europe (FAIR 5 CT97-3898). Durable resistance of apple to scab and powdery mildew: one step more towards an environmental friendly orchard. – IOBC/WPRS Bulletin 23(12): 257-260.

Mayr, U. & Treutter, D. 1996: Vorkommen und Gehalte von Flavanolen in Apfelfrüchten und -säften. – In: Deutsche Gesellschaft für Qualitätsforschung, XXXI. Vortragstagung, Kiel, 113-118.

Müller, C. & Treutter, D. 2000: Phenolische Verbindungen in Apfelsaft, Apfelwein und Apfelessig. – In: Deutsche Gesellschaft für Qualitätsforschung, XXXV. Vortragstagung, Karlsruhe, 121-125.

Patocchi, A., Biegler, B., Koller, B., Liebhard, R., Kellerhals, M. & Gessler, C. 2003: Genome scanning approach (GSA): a fast method to find molecular markers associated to any trait; the example of apple scab resistance gene Vr2 of Russian seedling(R12740-7A). – In press.

Seglias, N.P. & Gessler, C. 1997: Genetics of apple powdery mildew resistance derived from Malus zumi (Pl2). – IOBC/WPRS Bulletin 20(9): 195-208.

Tartarini, S., Gianfranceschi, L., Sansavini, S. & Gessler, C. 1999: Development of reliable PCR markers for the selection of the Vf gene conferring scab resistance in apple. – Plant Breeding 118: 183-186.


pp. 49 - 52

49

Geographical distribution of Venturia inaequalis strains virulent to the Vf gene in Europe L. Parisi1, F. Laurens2, F. Didelot1, K. Evans3, C. Fischer4, V. Fouillet1, F. Gennari5, H. Kemp6, M. Lateur7, A. Patocchi8, H. Schouten9, C. Tsipouridis10

1 INRA, UMR PaVé, BP 57, 49071 Beaucouze CEDEX, France; 2INRA, Unité d’Amélioration des Espèces Fruitières et Ornementales (same address); 3HRI, East Malling, Breeding and Genetics, ME19 6BJ, West Malling, United Kingdom; 4BAZ-IOZ, Pillnitzer Platz, 01326 DRESDEN, Germany; 5DCA-BO, Università di Bologna, Via Filippo Re n°6, 40126 BOLOGNA, Italy; 6PPO, sector Fruit, PO Box 200, 6670 AE Zetten, The Netherlands; 7CRA, State Plant Pathology Station, Chemin de Liroux, 5030-Gembloux, Belgium; 8ETHZ, Institute of Plant Science, Universitaetstrasse 2, 8092 ZURICH, Switzerland; 9PRI, PO Box 16, 6700 AA Wageningen, The Netherlands; 10 NAGREF, Pomology Institute, PO Box 122, 592 00 Naoussa, Greece. Abstract: The results obtained in the frame of the DARE project, added to the previous published data, allow the distribution of the V. inaequalis strains virulent to the Vf gene to be mapped in Europe. Nine years after the first report of the presence of race 6 in Germany, and 8 years after the first report of the presence of race 7 in England, the results showed that races 6 and 7 were present in 7 European countries, situated mainly in the Northern part of Europe. Strains combining the virulences of races 6 and 7 were detected in the Netherlands. In this country, as in France and Denmark, the presence of virulent strains was reported in commercial orchards. These data question the stability of the Vf gene resistance in Europe and the management of the Vf resistant cultivars. Introduction The Vf gene is the main source of scab resistance used in apple breeding programmes. Since the first detection of strains of V. inaequalis virulent to the Vf gene in Germany and England (Parisi et al., 1993; Roberts & Crute, 1994), few reports on the presence of these strains in Europe were published. Race 7 was detected in the Netherlands (Schouten & Schenk, 1997) in France (Parisi et al., 2000) and races 6 and 7 in Denmark (Bengtsson et al., 2000). One of the objectives of the DARE (Durable Apple Resistance in Europe) project was to evaluate the importance of the overcoming of the Vf gene. The results obtained during the DARE project, added to the reports of previous publications, allowed the distribution of these strains to be mapped in Europe. Material and methods The pathogenicity of races 6 and 7 was defined on a set of 5 cultivars, 4 of them carrying various scab resistance genes or a combination of them: Golden Delicious (Vg), Priscilla (Vf), Prima (Vf and Vg), Malus floribunda 821 (Vf and Vfh) (Parisi & Lespinasse, 1996; Bénaouf & Parisi, 1997; Bénaouf et al., 1997; Durel et al., 2000) and the cultivar Gala which carries no known major gene of resistance. Table 1 shows the pathogenicity of these 2 races and that of a strain of race 1 avirulent to the Vf gene.

50

Table 1. Pathogenicity of races 1, 6 and 7 of V. inaequalis on 5 cultivars or species of Malus.

Cultivars or species of Malus Races of V. inaequalis

Gala Golden D. (Vg gene)

Priscilla (Vf gene)

Prima (Vf and Vg

genes)

M. floribunda 821

(Vf and Vfh genes)

Race 1 S S R R R Race 6 S S nt S R Race 7 S R S R S

nt: not tested, S: susceptible, R: resistant

GB

*NL

D

DK

CH

F

I

GR

Race 6

Data published before the DARE projectDARE data

Race 7

Data published before the DARE projectDARE data

Strains cumulating virulences of races 6 and 7Strains virulent to the Vf gene(race unknown, R. Stievenard, personnal communication)

*

Figure1. Geographical distribution of V. inaequalis strains virulent to the Vf gene in Europe.

In the DARE project, we collected information on the presence of these virulent races by 2 different approaches :

51

1) In a network of core-orchards planted in 8 European countries (Belgium, France, Germany, Greece, Italy, Switzerland, the Netherlands and United Kingdom) 20 cultivars including the set of 5 differential hosts were planted. Susceptibility/resistance assessments on these orchards were performed in 2000 and 2001.

2) The pathogenicity of 39 strains of V. inaequalis, originating from different orchards of 7 from the 8 countries (Belgium, France, Germany, Greece, Italy, Switzerland, the Netherlands) was tested on a set of cultivars which comprised of 4 of the 5 differential hosts : Gala, Golden Delicious, Malus floribunda 821 and Prima. This test was performed in controlled conditions (growth-chamber).

Results and discussion This work showed that races 6 and 7 are mainly present in the Northern part of Europe: Denmark, Northern Germany, the Netherlands, Belgium, Northern France and England (Figure 1).

However, the presence of race 7 in the Eastern part of Germany and Switzerland could indicate a spread in the Eastern and central parts of Europe. Strains virulent to the Vf gene were not detected in Italy and Greece. These results have to be interpreted carefully. Only one core-orchard was planted in each country, so we have no exhaustive data on each region of each country. Furthermore, the number of strains tested in the growth chamber is not high, because the pathogenicity test needs time and space. This work is therefore not a complete investigation on the presence of races 6 and 7 in each region of the 8 European countries, but a synthesis of the current situation with the data available today.

The observed distribution of race 7 could support the hypothesis of a natural spread from England to the continent by the dominant winds. However, the long-distance dissemination ability of this fungus has not been demonstrated and remains controversial (MacHardy, 1996). The possibility of human contribution to the spread of these virulent strains cannot be excluded.

Race 6, firstly only present in Germany, is now present or suspected in 4 countries. Strains combining the virulence to the Vf, Vg and Vfh genes were found in the Netherlands, showing that the strategy of pyramiding major resistance genes in one cultivar does not increase significantly the durability of its resistance. Only 10 years were necessary for the fungus to combine the ability to overcome these 3 resistance genes, under good climatic conditions for the disease but in absence of a strong selection pressure. In fact, as Golden Delicious is an important cultivar in Europe, the Vf resistant cultivars are not intensively cultivated and the ornamental crab-apple Malus floribunda 821 is present only in gardens and experimental orchards.

The presence of strains virulent to the Vf gene in commercial orchards in France, the Netherlands and Denmark strongly points to the need for strategies for the management of the Vf resistant cultivars. How do we have to manage the Vf resistant cultivars in the countries where virulent strains are present ? What kind of treatments or cultural practices will increase the durability of this resistance ? To answer these questions, we need more information on the origin and the dissemination of the virulent strains, and more knowledge on the management of resistant cultivars. References Bénaouf, G. & Parisi, L. 1997: Pathogenicity of Venturia inaequalis strains from Malus flori-

bunda 821: comparison with race 6 on apple clones. – IOBC/wprs Bulletin 20(9): 8-11.

52

Bénaouf, G., Parisi, L. & Laurens, F. 1997: Inheritance of Malus floribunda clone 821 resistance to Venturia inaequalis. – IOBC/wprs Bulletin 20(9): 1-7.

Bengtsson, M., Linhard, H. & Grauslund, J. 2000: Occurrence of races of Venturia inaequalis in an apple scab race screening orchard in Denmark. –IOBC/wprs Bulletin 23(12): 225-229.

Durel, C.E., Weg, W.E., Van De, Venisse, J.S. & Parisi, L. 2000: Localisation of a major gene for apple scab resistance on the European genetic map of the Prima x Fiesta cross. – IOBC/wprs Bulletin 23(12): 245-248.

MacHardy, W.E. 1996: Apple Scab Biology, Epidemiology, and Management. – APS Press, St Paul, Minnesota, USA.

Parisi, L. & Lespinasse, Y. 1996: Pathogenicity of Venturia inaequalis strains of race 6 on apple clones (Malus sp.). – Plant Dis. 80: 1179-1183.

Parisi, L., Durel, C.E. & Laurens, F. 2000: First report on the presence of Venturia inaequalis race 7 in french apple orchards. – IOBC/wprs Bulletin 23(12): 99-104.

Parisi, L., Lespinasse, Y., Guillaumès, J. & Kruger, J. 1993: A new race of Venturia inaequa-lis virulent to apples with resistance due to the Vf gene. – Phytopathol. 93: 533-537.

Roberts, A.L. & Crute, I.R. 1994: Apple scab resistance from Malus floribunda 821 (Vf) is rendered ineffective by isolates of Venturia inaequalis from Malus floribunda. – Norw. J. Agric. Sci. 17: 403-406.

Schouten, H.J. & Schenk, A. 1997: Schurftresistentie op sommige plaatsen doorbroken. – Fruitteelt 87 (31): 14-15.


pp. 53 - 58

53

Factors influencing deposition of Venturia inaequalis ascospores on apple trees Vittorio Rossi1, Simona Giosuè1, Riccardo Bugiani2 1UCSC, Istituto di Entomologia e Patologia vegetale, Via Parmense 84, I-29100 Piacenza; 2Servizio Fitosanitario Regionale, Via di Saliceto 81, I-40128 Bologna Abstract: A model simulating the deposition of Venturia inaequalis ascsospores on apple leaves was used to determine the effect of rainfall, wind, distance from the inoculum source, and tree canopy density (as LAI, leaf area index) on the number of ascospores deposited per leaf area unit. LAI and rainfall had a significant effect on ascospore deposition. The effect of wind is closely related to the distance from inoculum source, and can be considered of little importance at an orchard scale. Using data from model simulations, the proportion of airborne ascospores deposited on apple leaves in relation to LAI and rainfall was estimated by a combination of two regression equations. The first equation accounts for the proportion of spores released into the orchard air from pseudothecia that remain in the air layer between the ground and the lowest leaves; since these spores do not reach the apple canopy, they are removed from the airborne ascospore dose. The second equation determines the proportion of ascospores deposited onto the apple leaf surface. Deposition of all the ascospores which are airborne within the canopy is possible only when LAI is equal to or greater than 1, and there is rain. Since the primary inoculum season usually advances when there is little foliage on the trees, and ascospore ejection, though initiated by rain, continues with no rain, the findings from this work have a practical importance and should be used to improve estimation of the risk level for scab infection. Key words: apple, Venturia inaequalis, simulation model, weather, Leaf Area Index, ascospore deposition Introduction Warning systems for controlling apple scab during the primary inoculum season are based on the dynamic of overwintering pseudothecia, on ascospore maturation and ejection, and on meteorological conditions favouring ascosporic infection (MacHardy, 1996). An important step in the infection cycle of the pathogen receives little attention, i.e. the aerial concentration of ascospores and their deposition on the host tissue. Since there is a highly significant correlation between the scab severity and the exposure to airborne ascospores (Aylor & Kiyomoto, 1993), information about the rate at which ascospores are deposited on the host tissue could contribute to better estimating the potential number of scab lesions developing from each ascospore discharge event.

There are two different approaches for studying spore deposition: i) direct measurement of deposition by spore- or plant-traps; ii) use of mathematical models based on the physical laws that regulate spore transport and deposition processes (MacCartney & Fitt, 1985). Models account for the different variables that affect spore deposition, particularly meteorological conditions and the horticultural characteristics of the orchard; thus, they are applicable in many different situations. Unfortunately, models are generally complex, so their direct use in epidemiological studies or, more, in disease management, is limited.

In the present work, we used a model simulating deposition of V. inaequalis ascospores on apple leaves to: i) determine the most important variables influencing the phenomenon, ii) produce a simplified tool to be used in warning systems for apple scab control.

54

Materials and methods Simulation model The model simulating the aerial concentration of V. inaequalis ascospores and the deposition processes on apple tissue was described in a previous work (Rossi et al., 2003a). It includes two main parts (Fig. 1).

The first part of the model simulates the aerial concentration of ascospores within the

apple canopy (SIA, ascoSpores Into the Air, in spores/m3 air·hour). The model is initiated when ascospores are ejected into the air from scabbed leaves on the ground (SRA, ascoSpores Released into the Air, in spores/m2 soil·hour). After ascospores are released into the air, they are simultaneously transported in the downwind direction by the horizontal motion of the wind (W, in m/s) and diffused in the vertical direction by the turbulent fluctuations of the wind. As a result of these diluting actions of the wind (SDR, ascoSpore Dilution Rate, which is composed by VD and HD, Vertical and Horizontal Dilution, respectively), the aerial concentration of ascospores decreases with downwind distance (d, in m) and the vertical distance (h, in m) from the point of release. The proportion of SIA within the canopy depends on the vertical extension of the canopy, from H1 (height above the ground of the lowest leaves) to H2 (height of the top leaves).

The second part of the model simulates deposition of ascospores from the air to the leaf surface (SDL, ascoSpores Deposited on Leaves, in spores/cm2 leaf·hour). Ascospores are removed from the air and deposited on plants and on the ground by a combination of wet and dry deposition processes (WDR and DDR, Wet and Dry Deposition Rates, respectively). Since both wet and dry depositions are relatively small, it is assumed that the two processes act independently.

Wet deposition results from ascospores that are washed down by rain (R, in mm/hour) from the air column above the trees; the proportion of spores removed by rain that reaches the leaf surface depends on the LAD (Leaf Area Density, in m2/m3) projected onto the horizontal plane (fx), where LAD depends on LAI (Leaf Area Index, in m2/m2). The model does not include the washoff of the deposited spores, due to the raindrops impacting on the leaf blade and splashing off from the leaf. This will likely overestimate the number of ascospores remaining on the leaves.

sv

h

SDR

SRA

VDSIA

DD

RW

DR

SED IMP

W

E

LAD

sv

W

LADR

SDL

d

HD

H2

H1

fxfz

fz

LAI

sv

h

SDR

SRA

VDSIA

DD

RW

DR

SED IMP

W

E

LAD

sv

W

LADLADRR

SDL

d

HD

H2

H1

fxfxfzfz

fzfz

LAI

Figure 1. Relational diagram of the model simulating aerial concen-tration and deposition of V. inaequalis ascospores. Simpli-fied from Rossi et al. (2003a); see text for explanation of acronyms

55

Dry deposition involves ascospores that are contained in the air but not in raindrops; these spores are deposited by gravitational settling, or sedimentation (SED), and by inertial impaction (IMP). SED depends on the settling velocity of ascospores (sv, in m/s) and predominates on horizontal leaf elements (LAD, fz). IMP predominates on vertical leaf elements (LAD, fx) and depends mainly on W and the efficiency of impaction (E). E is defined as the ratio between the rate at which spores impact upon a surface and the rate at which they pass through the same area as the surface projected horizontally; it depends mainly on W and sv of spores. Model running and data analysis The model was operated by changing some variables acting into the model, i.e. LAI, R, W, and d, while other variables were kept constant, i.e. H1 and H2 (0.5 m and 3 m, respectively), and SRA (1000 spores/m2 soil).

To simulate the effect of these variables on the number of V. inaequalis ascospores deposited per unit of apple leaf surface (SDL), the model was run with combined changes in LAI (0.1 to 2.5 m2 leaf / m2 soil), R (0 to 15 mm/hour) and W (0.02 to 15 m/s); in this case, d was equal to zero (i.e. inoculum has a uniform distribution on the ground under the apple canopy). Data generated by the model were analysed by a factorial analysis of variance to test the significance of LAI, R, W, and their interactions, and to determine their relative contribution to the total variation of SDL. Other simulations were generated by changing distance from the inoculum source.

To determine the potential impact of ascospore deposition on the development of scab lesions, values of SDL were calculated for a representative shoot of a McIntosh apple tree (Aylor & Kiyomoto, 1993) (Fig. 2), for trees with different LAI, under different rain conditions; W was kept at 1 m/s and d at zero. Then the numbers of scab lesions per leaf and per shoot were calculated by the lesion-causing efficiency of ascospores (as average number of scab lesions per ascospore) found by Aylor & Kiyomoto (1993).

Figure 2. Representative shoot of a McIntosh apple tree (from Aylor & Kiyomoto, 1993).

No scab lesions develop on +4 or older leaves.

Finally, the proportion of ejected ascospores landing on apple leaves was calculated as

the ratio SDL/SRA, by running the model with changing LAI and R; W was kept at 1 m/s and d at zero. This ratio was used as dependent variable in a non-linear regression analysis.

Age class of leaves

Leaf area (cm2)

Lesion-causing efficiency of ascospores

+3 27.7 0.015 +2 28.6 0.039 +1 24.1 0.210 0 17.3 0.211 -1 9.8 0.227 -2 4.3 0.099

5 cm

+2

0

-1 -2 +1

+3

+4

5 cm5 cm

+2

0

-1 -2 +1

+3

+4

56

Results and discussion

Influence of LAI, rainfall, wind, and distance from inoculum source on ascospore deposition The AOV showed that LAI, R and their interaction had a significant (P<0.01) effect on SDL; on the contrary, neither W nor its interactions affected the response variable significantly. The most influential variable was R, which accounted for about 90% of total variance of the experiment; SDL increased as the amount of rain increased (Fig. 3C). LAI accounted for about 2% of total variance; deposition increased when LAI increased from 0.01 to 1, then SDL progressively decreased when LAI was greater than 1 (Fig. 3A). In these simulations, SDL did not change with wind speed (Fig. 3B).

The above-mentioned results were obtained by setting d=0. In the simulations made by changing d, wind affected deposition only at greater distances from the inoculum source. When there is no wind, SDL decreased steeply when distance from inoculum increased; otherwise, the reduction of SDL occurred at d greater than 10 m. As a consequence, the role of wind on ascospore deposition can be considered of little importance for the scab that occurs in an orchard.

Figure 3. Changes in the number of V. inaequalis ascospores deposited per apple leaf surface

unit (SDL), simulated by changing LAI, wind speed and rainfall. Values represent the average effect of each variable, when the others change within the showed range.

Influence of LAI and rainfall on scab lesion development The SDL calculated for a representative apple shoot changed according to LAI and R. When LAI increased from 0.5 to 1, SDL increased by 12%, while when it increased from 1 to 1.5 it decreased by 26%. When R was kept at 0.2, 1, 3 and 9, SDL increased from 5 ascospores per shoot to 7, 11 and 21, respectively. The above-mentioned differences in ascospore deposition determined differences in the potential number of scab lesions per leaf and per shoot (Table 1).

Differences due to both LAI and R were greater for the leaves aged between +1 and –1 than for younger or older leaves, because on the former group of leaves the asospores had a greater lesion-causing efficiency (Fig. 2). The effect of R on the total number of scab lesions per shoot was greater than that of LAI; scab lesions increased by 4.3 times when rain fell during ascospore release increased from 0.2 to 9 mm/hour.

00.020.040.060.080.100.120.14

0 0.5 1 1.5 2 2.5LAI (m2/m2)

SDL

(n/c

m2

leaf

)

0 3 6 9 12 15Rain (mm/hour)

0

0.05

0.10

0.15

0.20

0.25

0 3 6 9 12 15Wind (m/s)

0

0.04

0.08

0.12

0.02

0.06

0.10

A B C0

0.020.040.060.080.100.120.14

0 0.5 1 1.5 2 2.5LAI (m2/m2)

SDL

(n/c

m2

leaf

)

0 3 6 9 12 15Rain (mm/hour)

0

0.05

0.10

0.15

0.20

0.25

0 3 6 9 12 15Wind (m/s)

0

0.04

0.08

0.12

0.02

0.06

0.10

00.020.040.060.080.100.120.14

00.020.040.060.080.100.120.14

0 0.5 1 1.5 2 2.50 0.5 1 1.5 2 2.5LAI (m2/m2)

SDL

(n/c

m2

leaf

)

0 3 6 9 12 150 3 6 9 12 15Rain (mm/hour)

0

0.05

0.10

0.15

0.20

0.25

0

0.05

0.10

0.15

0.20

0.25

0 3 6 9 12 150 3 6 9 12 15Wind (m/s)

0

0.04

0.08

0.12

0.02

0.06

0.10

0

0.04

0.08

0.12

0.02

0.06

0.10

A B C

57

Table 1. Numbers of scab lesions on the apple shoot of Fig. 2, calculated on the basis of the lesion-causing efficiency of the ascospores deposited on leaves, by changing LAI and rainfall.

Age class of leaves (see Fig. 2) +3 +2 +1 0 -1 -2 Shoot

LAI (m2/m2) 0.5 0.04 0.12 0.52 0.38 0.23 0.04 1.34 1 0.05 0.13 0.59 0.43 0.26 0.05 1.50

1.5 0.04 0.10 0.44 0.31 0.19 0.04 1.11 Rain (mm/hour)

0.2 0.02 0.05 0.22 0.16 0.09 0.02 0.55 1 0.03 0.07 0.31 0.22 0.14 0.03 0.79 3 0.04 0.11 0.49 0.36 0.22 0.04 1.26 9 0.08 0.20 0.93 0.67 0.41 0.08 2.36

Relationship between LAI, rainfall and fraction of the ejected ascospores which is deposited on leaves The relationship between SDL/SRA (dependent variable), LAI and R (independent variables) was fitted by a combination of two regression equations, as follows:

SDL/SRA = (1.017 · 0.374H1) · λ [1] λ = 1 / {1 + exp[2.575 – 0.987 · LAI · (5.022 · R0.063)]} [2]

These equations accounted for about 99% of experimental variability (R2 = 0.985) and residues between observed and estimated values of the dependent variable did not show systematic deviations (not shown). Equation [1] accounts for the proportion of SRA that

remains in the air layer between the ground and the lowest leaves (Aylor, 1995): these spores do not reach the apple canopy and must be removed from the airborne ascospore dose. Equation [2] determines the proportion of such ascospore dose deposited onto the apple leaf surface, as a function of LAI and rainfall. Deposition of all the ascospores which are airborne within the canopy is possible only when LAI is equal to or greater than 1, and there is rain (Fig. 5).

This finding has practical importance, because: i) the primary inoculum season usually continues for less than two months from green tip, when there is little foliage on the trees; ii) once ascospore release is initiated by rain, ascospores continue to be ejected for some time

1.5

0

0.3

0.6

0.9

1.2

LAI(

m2 /m

2 )

10 0.5 1.5 2 2.5 3Rain (mm/hour)

0.1

0.30.50.7

0.9

1.5

0

0.3

0.6

0.9

1.2

LAI(

m2 /m

2 )

10 0.5 1.5 2 2.5 3Rain (mm/hour)

0.1

0.30.50.7

0.9

10 0.5 1.5 2 2.5 3Rain (mm/hour)

0.1

0.30.50.7

0.9

Figure 5. Influence of rain and LAI on the proportion of airborne ascospores within the apple canopy deposited on leaves, according to equation [2].

58

though rain has stopped, so that most ascospores are released during hours with no rain (Aylor & Sutton, 1992; Rossi et al., 2001). The equations produced using the model simulating deposition of V. inaequalis ascospores on apple leaves should be used to improve estimation of the risk level for scab infection. MacHardy & Jeger (1982) defined the main components determining the scab lesion density expected from an ascospore infection period: i) the potential ascospore dose (PAD), i.e. the total season’s ascospores available for discharge, ii) the ascospore dose that actually become airborne, iii) the number of airborne ascospores deposited on susceptible apple tissue, and finally, iv) the number of deposited ascospores that cause lesions to develop. Methods have been developed to estimate PAD (Gadoury & MacHardy, 1986) and the rate of maturation (Gadoury & MacHardy, 1982) and discharge of ascospores (Rossi et al., 2000). These methods provide an estimate of the number of ascospores that will potentially discharge per m2 of orchard floor during a rain event; the proportion of such ascospore dose discharged per hour can also be estimated as a function of temperature (Rossi et al., 2003b).Thus, at the moment, it is possible to estimate the dose of ascospores actually becoming airborne, which is similar to SRA, the starting-point for the present model. Now, SDL produces an estimate for the 3rd component indicated by MacHardy & Jeger (1982), i.e. the number of airborne ascospores deposited on apple tissue. Acknowledgements This work was funded by the Emilia-Romagna Region and co-ordinated by CRPV. References Aylor, D.E. 1995: Vertical variation of aerial concenration of Venturia inaequalis ascospores

in an apple orchard. Phytopathology 85:175-181. Aylor, D.E. & Sutton, T.B 1992: Release of Venturia inaequalis ascospores during unsteady

rain: relationship to spore transport and deposition. Phytopathology 82: 532-540. Aylor, D.E. & Kiyomoto, R.K. 1993: Relationship between aerial concentration of Venturia

inaequalis ascospores and development of apple scab. Agric. For. Meteorol. 63: 133-147. Gadoury, D.M. & MacHardy, W.E. 1982: A model to estimate maturity of ascospores of

Venturia inaequalis. Phytopathology 72: 901-904. Gadoury, D.M. & MacHardy, W.E. 1986: Forecasting ascospore dose of Venturia inaequalis

in commercial apple orchards. Phytopathology 76: 112-118. MacCartney, H.A. & Fitt, B.D.L. 1985: Construction of dispersal models. Adv. Plant Pathol.

3: 107-143. MacHardy, W.E. 1996: Apple scab. APS Press, St. Paul, Minnesota. MacHardy, W.E. & Jeger, M. 1982: Integrating control measures for management of primary

apple scab, Venturia inaequalis (Cke.) Wint. Prot. Ecol. 5: 103-125. Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 2000: A new model estimating

the seasonal pattern of airborne ascospores of Venturia inaequalis (Cook) Wint. in relation to weather conditions. J. Plant Pathol. 82: 111-118.

Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 2001: Environmental factors influencing the dispersal of Venturia inaequalis ascospores in the orchard air. J. Phytopath. 149: 11-19.

Rossi, V., Giosuè, S. & Bugiani, R. 2003a: A model simulating deposition of Venturia inaequalis ascospores on apple trees. EPPO Bull. 33: 407-414.

Rossi, V., Giosuè, S. & Bugiani, R. 2003b: Influence of air temperature on the release of ascospores of Venturia inaequalis. J. Phytopath. 151: 50-58.


pp. 59 - 65

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A Chorus tolerant population of Venturia inaequalis found in a South African apple orchard Wolf Schwabe The Fruit Doctor, 24 Drommedaris Road, Somerset West 7130, South Africa Abstract: At the farm, Ouplaas, in the Agter Witzenberg area, South Africa, Chorus 50 WG was used for the control of apple scab from the 1996/97 to the 2000/01 growing seasons. During the 1996/97 season it was applied six times at pre-blossom stage as well as at the early post-blossom stage, while during the other seasons application was only at the pre-blossom stage and never more than three times per season. In the 2000/01 growing season severe fruit scab was experienced in an Early Red orchard at Ouplaas. The first symptoms were observed a fortnight to three weeks after the last Chorus application. In order to determine whether Chorus tolerance of the scab fungus was the reason for the severe scab incidence, the following investigation was conducted. Scab infected leaves and fruit were collected in the problem orchard and the scab fungus was multiplied on fungicide free potted MM109 rootstock trees. Three groups of potted MM109 trees each were sprayed with Chorus 50 WG at 30g/hl (1X), at ½X and at ¼X respectively. Unsprayed control trees were kept as well. One day later half of the trees of each group were inoculated with a conidial suspension of the Ouplaas scab fungus line, while the second half was inoculated with a conidial suspension of a reference fungus line. The latter was known to be sensitive to all fungicides used for the control of scab in South Africa. All rates of Chorus gave more than 90% scab control on trees inoculated with the reference fungus line, while on trees inoculated with the Ouplaas fungus line, scab control varied between 71 and 37% on trees sprayed with the 1X, ½X and ¼X of Chorus. After withholding Chorus applications for two seasons from orchards at Ouplaas, scab infected leaves and fruit were collected again in the problem orchard in January 2002, the second part of the 2001/02 growing season. Then scab control by Chorus was compared on three scab fungus lines, i.e. the reference fungus line, the 2000/01 collection and the 2001/02 collection. Control of the sensitive reference fungus line was more than 97% on trees sprayed with any of the three rates of Chorus, while control varied between 26% and 43% on trees inoculated with the two fungus lines from the Ouplaas orchard. There was virtually no difference between control of the 2000/01 and the 2001/02 collections from the Ouplaas orchard except that at the highest rate of Chorus control of the 2001/02 collection was significantly better. Excessive use of Chorus has to be avoided for the control of scab. It should not be applied more than twice per season. Once Chorus tolerance has been demonstrated in an orchard, Chorus or any other anilino-pyrimidine fungicide should not be used for the time being. Key words: Venturia inaequalis, ineffective control, tolerance to Chorus. Introduction In the late 1970s Schwabe (1977, 1979) first reported on tolerance of the apple scab fungus, Venturia inaequalis (Cke) Wint., to dodine and benzimidazole fungicides in South African apple orchards. The excessive use of these fungicides led to the increase of tolerant scab fungus lines. Tolerance levels to the benzimidazole fungicides were exceptionally high, whereas in the case of dodine there was more a shift towards reduced sensitivity. This resulted in the use of benzimidazole fungicides being discontinued, while dodine had to be applied at higher dosages. Similar reports were received from other parts of the world (Wicks, 1974; Jones & Walker, 1976; Shabi & Ben-Yephet, 1976).

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When ergosterol biosynthesis inhibiting (EBI) fungicides appeared on the market for scab and mildew control in the late 1970s and the early 1980s, the experience with benzimidazole tolerance was still very fresh. Consequently, strict precautions were taken to prevent a repetition of the fiasco of the 1970s. It was recommended that EBIs should never be applied alone, but always in combination with an effective contact fungicide. Excessive use of EBIs was also discouraged. However, during the 1980s and early 1990s the EBIs were the only fungicides with an excellent curative action, and therefore, no real restriction was put on the number of applications permitted per season per orchard in South Africa. In the mid-1980s trials were carried out to determine the risk of development of tolerance to the EBIs. Schwabe, Shabi & Hurter (1988) and Schwabe & Shabi (1994) found that excessive exposure of V. inaequalis to EBIs, especially at sub-lethal dosages, led to the selection of scab lines with reduced sensitivity to EBI fungicides. These findings supported the continuation of the anti-tolerance strategy.

During the 1996/97 growing season apple scab control was unsatisfactory on a few farms were EBIs had been used excessively. Investigations confirmed the existence of EBI-tolerant fungus lines on those farms (Schwabe & van der Rijst, 1997). Since then fungus lines, tolerant to EBIs, were found on an additional number of farms in South Africa (Schwabe, unpublished data).

Chorus, an anilino-pyrimidine, was released for scab control in the mid-1990s. This fungicide had also curative activity. When during the 2000/01 growing season after application of Chorus severe fruit scab was experienced in an Early Red orchard at Ouplaas, Agter Witzenberg, an investigation was conducted to determine the existence of Chorus tolerant scab fungus lines in the scab population. Materials and methods In order to demonstrate the existence of a scab fungus population tolerant to Chorus, glasshouse trials were conducted on actively growing potted MM109 rootstock apple trees with one to two actively growing shoots per tree. The fungicide, Chorus 50WG (cyprodinil 500g/kg), was used. Sample A was supplied by Syngenta SA (Pty) Ltd, Halfway House and sample B by Terason (Pty) Ltd, Paarl. 2000/01 Trial Preparation of inoculum On 15 January 2001 scab infected leaves and fruit were collected from an Early Red orchard at Ouplaas, Agterwitzenberg where Chorus tolerance was suspected. Collected leaves and fruit were stored at 0.5°C. In order to obtain fungicide free fresh inoculum, a conidial suspension was prepared from collected leaves and fruit a month later and potted MM109 rootstock apple trees were inoculated. Simultaneously the fungicide sensitive Stellenbosch reference fungus was multiplied on another group of potted MM109 rootstock trees. After incubation for 2-3 weeks fungicide free conidial inoculum was available on leaves of inoculated trees. Spraying and inoculation of trees Hundred and forty potted MM109 trees were divided into seven groups of 20 trees each and treated as follows:

1. Unsprayed control 2. Chorus 50WG A 30g/hl 5. Chorus 50WG B 30g/hl 3. Chorus 50WG A 15g/hl 6. Chorus 50WG B 15g/hl 4. Chorus 50WG A 7.5g/hl 7. Chorus 50WG B 7.5g/hl

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Fungicides application was to run-off by a Honda Backpack Sprayer WJR2225. Ten trees of each group were inoculated with a conidial suspension of the Stellenbosch reference fungus (68,000 viable conidia per ml suspension) 24h after fungicide application, while at the same time the remaining 10 trees of each group were inoculated with a conidial suspension of the Ouplaas fungus (65,000 viable conidia per ml suspension). Inoculated trees were exposed to a wetting period of 48h at 17.5°C. Permanent wetting was by an overhead irrigation system being activated for 40s every 30min. During the incubation period of 12-16 days trees were kept in a glasshouse were temperatures fluctuated between 16 and 27°C. Scab assessments and calculation of scab control Assessments of scab development were made by the method described by Schwabe (1977). Percentage infection was calculated using the method of Kremer & Unterstenhöfer (1967). The percentage of scab control for each treated (sprayed) tree inoculated with a conidial suspension from a specific source was calculated by expressing the percentage infection on that tree as a percentage of unsprayed control trees inoculated with a conidial suspension of the same source. Percentage scab control for each fungus line was subjected to one-way analysis of variance. Student’s least significant difference (LSD) was calculated at the 5% level of probability to compare treatment means. 2001/02 Trial No Chorus was applied in the problem orchard after the 1999/2000 growing season. In order to determine whether withholding Chorus from the orchard had an effect on its V. inaequalis population a further trial was conducted in 2002. Scab infected leaves and fruit were collected in the problem orchard on 10 January 2002. After storage for a month at 0.5°C the fungus from collected leaves and fruit as well as the conidia from the two fungus lines from the previous season were multiplied on MM109 rootstock apple trees as described above. When fungicide free fresh conidial inoculum from the three fungus lines was available 2-3 weeks later a similar comparison trial as during the previous season was carried out. Chorus from sample A was used for the 2002 trial. Further the same procedure was followed as in the2001 trial. Chorus spray history at the Ouplaas Early Red /Royal Gala orchard During the 96/97, 97/98, 98/99, 99/00 and 00/01 growing seasons Chorus was applied 6x, 2x, 3x, 3x and 3x respectively in the problem orchard, i.e. a total of 17 applications. Results and discussion The effect of different dosages of Chorus on the control of scab from the two sources as demonstrated in 2000/2001 season, is shown in Figures 1, 2 and 3. In both tests carried out, control of the Ouplaas fungus line was significantly less effective than control of the reference fungus line. This was shown when Chorus was applied at full, ½ and ¼ rate of the commercially recommend dosage. There was a tendency that on trees sprayed with the lowest Chorus dosages, the poorest control was achieved with the Ouplaas fungus line. Furthermore it was shown that both samples of Chorus were equally effective or ineffective against both scab fungus lines. This was as expected, since both Chorus samples were manufactured by Syngenta and the date of manufacturing was virtually similar.

In Figure 4 data are shown on scab control on trees sprayed with three dosages of Chorus (1X, ½X and ¼X) of the commercially recommended rates and inoculated with three fungus lines, i.e. the scab fungus from the 2001 collection, the 2002 collection and the Stellenbosh reference scab fungus line. Both orchard collections were controlled significantly less

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Chorus tolerance 2000/01 (Syngenta)

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Figure 2. Scab control on leaves of potted MM109 rootstock apple trees sprayed with Chorus

50WG (cyprodinil) at 30g, 15g and 7.5g/hl and inoculated with a conidial suspension of Venturia inaequalis from two different sources one day later. Chorus was supplied by Terason (Pty) Ltd, Paarl.

Figure 1. Scab control on leaves of potted MM109 rootstock apple trees sprayed with Chorus 50WG (cyprodinil) at 30g, 15g and 7.5g/hl and inoculated with a conidial suspension of Venturia inaequalis from two different sources one day later. Chorus was supplied by Syngenta SA (Pty) Ltd, Halfway House.

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Chorus tolerance 2000/01 (Pooled)

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50WG (cyprodinil) at 30g, 15g and 7.5g/hl and inoculated with a conidial suspension of Venturia inaequalis from two different sources one day later. Data from trees sprayed with Chorus from the two suppliers were pooled.

Chorus tolerance 2001/02

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50WG (cyprodinil) at 30g, 15g and 7.5g/hl and inoculated with a conidial suspension of Venturia inaequalis from three different sources one day later. Chorus was supplied by Syngenta SA (Pty) Ltd, Halfway House.

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effective than the fungicide sensitive Stellenbosch reference fungus line. At the highest dosage (30g/hl) the fungus from the 2002 collection was significantly more effectively controlled than the fungus from the 2001 collection. This indicates a shift of the tolerance level after withholding Chorus for two seasons. In future further tolerance trials have to be conducted to confirm this phenomenon.

Braun (1994) reported from Canada that after using only broad-spectrum fungicides in an orchard with an EBI resistance factor of 10.2 in 1989, the resistance factor declined to 3.5 in 1992. Pommer & Lorenz (1982) and Fourie (1996) found in European and South African vineyards respectively that dicarboximide-tolerant lines of Botrytis cinerea were not as fit as sensitive lines. By ceasing the use of dicarboximide fungicides in vineyards, the portion of the tolerant B. cinerea population decreased rapidly.

Conclusion Seventeen applications of Chorus, applied over a period of six growing seasons, led to the selection of a scab fungus population that was tolerant to Chorus. This resulted in ineffective commercial scab control. Consequently, Chorus and any other anilino-pyrimidines had to be withheld from this orchard. For the time being Chorus or chemically related fungicides should not be used in this orchard. There are indications that by withholding Chorus from this orchard, the Chorus tolerant population is shifting towards the normal population. However, the last statement has to be reconfirmed and regular tolerance tests have to be conducted to determine when the scab population has normalized and it will be save using anilino-pyrimidines for effective scab control. Acknowledgements I thank Syngenta SA (Pty) Ltd, Halfway House for funding this investigation. References Braun, P.G. 1994: Development and decline of a population of Venturia inaequalis resistant

to sterol-inhibiting fungicides. – Norw. J. Agric. Sci. Supplem. No. 17: 173-184. Fourie, P.N. 1996: Resistance in Botrytis cinerea to dicarboximide fungicides. – M.Sc.Thesis,

University of Stellenbosch, Stellenbosch. Jones, A.L. & Walker, R.J. 1976: Tolerance of Venturia inaequalis to dodine and

benzimidazole fungicides in Michigan. – Pl. Dis. Reptr. 60: 40-44. Kremer, W. & Unterstenhöfer, G. 1967: Computation of results of crop protection

experiments by the method of Townsend and Heuberger. Höfchenbr. – Bayer PflSchutz-Nachr. 20: 625-628.

Pommer, E.-H. & Lorenz, G. 1982: Resistance of Botrytis cinerea (Pers.) to dicarboximide fungicides – a literature review. – Crop Protection 1: 225-230.

Schwabe, W.F.S. 1977: Tolerance of Venturia inaequalis to benzimidazole fungicides and dodine in South Africa. – Phytophylactica 9: 47-57.

Schwabe, W.F.S. 1979: Resisance of the apple scab fungus (Venturia inaequalis) to benzimidazole fungicides. Decid. – Fruit Grow. 29: 418-422.

Schwabe, W.F.S. Shabi, E. & Hurter, C. 1988: Tolerance of Venturia inaequalis to ergosterol biosynthesis inhibiting fungicides in South Africa. – Phytophylactica 20: 107.

Schwabe, W.F.S. & Shabi, E. 1994: Tolerance of Venturia inaequalis to ergosterol biosynthesis inhibiting fungicides in South Africa. – Norw. J. Agric. Sci. Supplem.17: 171-172.

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Schwabe, W.F.S. & van der Rijst, M. 1997: Tolerance of Venturia inaequalis to ergosterol biosynthesis inhibiting fungicides in South African commercial apple orchards. – Decid. Fruit Grow. 47: 303-307.

Shabi, E. & Ben-Yephet, Y. 1976: Tolerance of Venturia pirina to benzimidazole fungicides. – Pl. Dis. Reptr 60: 451-454.

Wicks, T. 1974: Tolerance of the apple scab fungus to benzimidazole fungicides. – Pl. Dis. Reptr 58: 886-889.


pp. 67 - 74

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Evaluation of in-vitro grown apple shoot sensitivity to Venturia inaequalis using a detached leaf assay E. Silfverberg-Dilworth, Andrea Patocchi, Cesare Gessler Plant Pathology, Institute of Plant Science, Swiss Federal Institute of Technology, ETH Zurich, Switzerland Abstract: Assessment of scab susceptibility or resistance of apple plantlets, grown in-vitro, is important for early assessment of individuals transformed with putative scab resistance genes. Useful clones can be selected and only these plants need be grown further and acclimatised to the greenhouse. Here we describe a detached leaf assay carried out using in-vitro grown shoots of the cultivars Florina, Enterprise and Gala. We define resistance reaction classes observed on a microscopical level that enable us to distinguish resistant (Vf) cultivars from susceptible ones. Under the conditions described statistically significant differences are seen between Florina (Vf) and Gala. Also tested are Florina shoots that have been re-generated from calli, as transformed plantlets would be. No significant variation was observed between the results from these plants and those of Florina shoot tip cultures. Key words: Venturia inaequalis, detached leaf assay, in-vitro propagated apple. Introduction Resistance of many modern apple varieties to the fungal pathogen Venturia inaequalis, the causal agent of apple scab, originates from the wild variety Malus floribunda 821 (Vf- Venturia floribunda). Recently a cluster of genes, encoding transmembrane leucine rich repeat proteins, were cloned from the apple variety Florina (Vinatzer et al., 2001). These genes map in the Vf region and are similar to the Cf resistance genes of tomato hence are believed to be involved in apple resistance to Venturia inaequalis. They were named HcrVf, homologs to C. fulvum resistance genes of the Vf region. To investigate the function of HcrVf gene products, analysis of the pathogenic interaction between the scab fungus and plants (originally possessing no Vf resistance) transformed with an HcrVf open reading frame is required. As the process of growing the plants until they can be maintained under greenhouse condition is long, an earlier test on in-vitro grown plants is desirable. Shoot tip cultures from Gala, Enterprise (possessing the Vf resistance gene) and Gala transformed with HcrVf2 were used in a detached leaf assay to assess sensitivity to scab (Barbieri et al., 2002). After 4-5 days the samples were evaluated microscopically, each sample was classified into resistance classes A, B, C, D or E (described below). Problems occurred whilst trying to define a threshold for resistance and susceptibility amongst the classes.

Microscopical studies of the interaction between apple and the scab fungus show that on both susceptible and resistance apple cultivars V.inaequalis is able to germinate, form appressoria, penetrate the host cuticle and that differences in virulence are first observed at a subcuticular level (Nussbaum, 1938; Gessler & Stumm, 1984). In susceptible varieties penetration of the epidermal cells is followed by the formation of subcuticular primary stroma, extended secondary hyphae, which advance to form a criss-cross of running hyphae and finally conidiophores forming conidia which complete the asexual cycle. In resistant varieties primary stroma forms, this sometimes gives rise to branched hyphae but if so the expansion of the hyphae is limited (Chevalier et al., 1991). Chevalier et al. (1991) also

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reported that in weakly resistant samples (Class 3a- macroscopic symptoms being described as the formation of necrotic lesions, some chlorotic lesion and very little sporulation) secondary running hyphae expand and the formation of conidiophores is possible but that they soon abort.

Pathogenicity tests on shoot-tip cultures showed that although macroscopic symptoms are comparable to those on greenhouse grown plants, symptoms develop more slowly (Yepes & Aldwinkle, 1993) on in-vitro grown shoot tip and detached leaves. This was confirmed by a further study where both macroscopic and microscopic symptoms were observed (Ivanicka et al., 1996). They showed that shoot tip cultures and detached leaves from shoot tip cultures of susceptible and resistant cultivars could be distinguished by the presence and absence of macroscopic scab symptoms and that although primary stroma was observed in all samples no secondary was observed in the resistant cultivar even after 28 days of incubation.

Considering these points a detached leaf assay using leaves from in-vitro grown shoot tip cultures may require an incubation time longer than five days and previous studies have shown that longer incubation times are possible without leaf senescence occurring. Care must be taken that the controls are representative and do not lead to bias, i.e. they should have, as far as possible, under gone the same treatment as any of the tested samples. Avoiding misleading results due to ontogenic resistance must also be considered by selecting only the youngest leaves. Ontogenic resistance is already present, to some degree, in the third leaf of greenhouse grown plants (Gessler& Stumm, 1984). Selecting the first or second leaf from in-vitro grown shoot tip cultures is not so straight forward as the shoots do not always grow in an apically dominant fashion. Therefore only shoots with clearly identifiable young leaves should be used for inoculations.

Here we describe a detached leaf assay where we attempt to find a method that will allow us to distinguish between resistant and susceptible leaves and that can be used in future to assess plantlets transformed with putative resistance genes. Material and methods In order to allow sufficient development of the fungus in susceptible varieties and to properly test the resistant varieties incubation times of up to ten days were chosen. The cultivars Florina (Vf), Enterprise (Vf) and Gala (scab susceptible) were used. In addition Florina plants that had been through the same regeneration process as Gala shoots transformed with HcrVf2 were tested to assess the effect, if any, of the regeneration process. Further scab susceptible controls were not tested as Gala was considered the best possible control, as it is the background cultivar of the HcrVf2 plants. Plant material Starting material for shoot tip cultures of Florina where kindly provided by E. Chervaux, INRA and those of Gala and Enterprise from S. Tartarini, DCA-Bologna. Shoot tip cultures were propagated on media consisting of MS media with Vitamins (duchefa) 4.4g/l, Sucrose 30g/l, Benzyladanine 0.8g/l, Ascorbic acid 5mg/l and Oxiod Agar 7g/l with pH5.7. Plants were maintained at 18-22°C with 16 hour photoperiod of 35,000lux.

Regenerated Florina plants were non transformed plantlets arising from an Agrobacteria-mediated transformation experiment. The shoots were re-generated, via callus formation from leaf segments. Shoots were separated from the callus and transferred firstly to partial light (20°C), then finally to conditions for propagation. Emerging shoots were multiplied on proliferation media (M. Barbari, personal communication).

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Inoculation of detached leaves with Venturia inaequalis The youngest leaves of shoot tip cultures were removed and placed adaxial side up on water agar. Ten leaves from Florina, Enterprise and Gala were prepared and five from regenerated Florina.

Conidial suspension was prepared by thoroughly washing scab infected leaves, collected from a Gala orchard (FAW, Switzerland), in water. Leaf debris was removed and the conidial suspension centrifuged at 3000g for 10 minutes. The supernatant was removed and conidia were resuspended in water to a concentration of 200,000/ml. Two 5µl drops of inoculum were placed upon each leaf and the leaves were incubated at 18°C with 20,000Lux continuous light for five, seven or ten days.

Figure 1. Developmental classes of Venturia inaequalis infection. "A"; secondary expanding running hyphae, often star shaped outgoing from a central plate of primary stroma, "B+"; presence of primary stroma as a plate with limited secondary hyphae starting to form, "B"; absence of the typical running hyphae but the presence of primary stroma as plate with a typical lobed structure, "C"; single hyphae extending from the appressoria, clearly larger than a germination tube, borders of the hyphae clearly lobed, "D";as stage C however hyphal walls straight, "E"; no fungal development beyond that of appressoria and penetration pegs. Note: in photographs A,B+,B and C stroma, which is clearly visible in coloured photographs, has been outlined using Microsoft Paint software.

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Figure 2. Venturia inaequalis infection on Gala, 5 and 7 days after inoculation. Running

hyphae have extended further 7 days after inoculation than 5 days afterwards. Preparation of microscope samples Whole leaves were cleared in 96% Ethanol, 4% Acetic acid at 75°C for 30 minutes, stained in 70% Ethanol, 29.9% water, 0.1% Aniline Blue also at 75°C for 7 minutes, then washed three times in 70% Ethanol and left for at least 30 minutes, at room temperature, in 50% Ethanol, 50% lactic acid. Finally leaves were mounted on a microscope slide with lactic acid and protected with a coverslip. Evaluation of severity of infection Conidia were counted and degree of fungal development from each conidium was assessed on a scale A-E (Figure 1). Each leaf was finally classified according to the most advanced class found more than once, with the exception of Class A. Even if only one conidium with Class A symptoms was found the leaf was assigned to this class. Results On all those samples which could be evaluated, conidia which had reached development stages C to E were found (Table 1). A few samples (less than 5% of total inoculated) had to be discarded as they had become infected with secondary fungi or the leaf had degraded during incubation. Class B was observed on most samples of all cultivars. Class B plus was found more frequently on Gala, Enterprise and Re-generated Florina than on Florina,

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especially after seven days (Table 1). Class A was never found on Florina and only single cases were found on re-generated Florina, where after the same incubation time 70-80 cases were found on Gala (Table 1a and 1b). On Gala samples class A was already found after five days and the running hyphae had extended further after seven days (Figure 2).

Curious were the results from Enterprise, after five days only a few cases of class A were found, however after 7 days many were found, in fact more than on Gala. It must be noted however that the number of Class A conidia found on Gala samples after 7 days may be low as the high density of running hyphae, firstly made counting difficult and secondly may have given preference to the wide expansion of hyphae from fewer conidia. The number of Enterprise samples that after 10 days incubation showed Class A symptoms was once again reduced. Indicating that for this variety the results obtained from this test are not consistent. Table 1. Stroma development after five (a), seven (b) and ten (c) days of incubation at 180C.

Shown are the total number of conidia, assigned to each infection class from all leaves of the relevant cultivar and incubation time. Florina (RG) = Florina re-generated.

a. After 5 days incubation.

Number of conidia of Class A-E Cultivar A B plus B C-E Total

Florina 0 2 174 1562 1738 Enterprise 2 12 154 2139 2307 Gala 81 57 239 1691 2068 Florina (RG) 1 72 186 1196 1455

b. After 7 days incubation

Number of conidia of Class A-E Cultivar

A B plus B C-E Total Florina 0 6 242 2406 2654 Enterprise 161 88 444 2098 2791 Gala 70 31 222 2262 2585 Florina (RG) 1 77 381 958 1417

c. After 10 days incubation

Number of conidia of Class A-E Cultivar

A B plus B C-E Total Florina 0 23 458 1884 3295 Enterprise 64 41 207 2588 2900 Gala 45 50 307 2415 2817 Florina (RG) 0 10 129 746 885

Once the leaves have been classified according to the A-E scale the results must be evaluated according to resistance or susceptibility to scab. This can be done by making a threshold for resistance and susceptibility between the classes.

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If the threshold is drawn between classes B and C (i.e. All leaves classified in classes A, B plus and B are considered susceptible and all leaves classified in classes C to E resistant) there is little correlation between the results and the phenotype of the plant material (Table 2). Using this threshold the majority of the leaves from both Vf cultivars are assessed as susceptible therefore it seems clear that Class B symptoms can not be considered symptoms of susceptibility. Table 3 indicates that this could also be true of Class B plus as when the threshold is drawn between classes B plus and B several samples from Vf cultivars are also assessed as susceptible. Table 2. The values shown represent the number of leaves from each cultivar, which were

considered susceptible or resistant, if resistance threshold 1 is used. All leaves classified as Class A, B plus and B are considered susceptible, all leaves classified in Classes C to E, as resistant.

5 days 7 days 10 days Cultivar Susceptible

A-B Resistant

C-E Susceptible

A-B Resistant

C-E Susceptible

A-B Resistant

C-E Florina 6 3 8 2 9 1 Enterprise 8 2 8 2 5 4 Gala 10 0 10 0 9 0 Florina (RG) 4 1 3 0 4 0

Table 3. The values shown represent the number of leaves from each cultivar, which were

considered susceptible or resistant, if resistance threshold 2 is used. All leaves classified as Class A, B plus are considered susceptible, all leaves classified in Classes B to E, as resistant.

5 days 7 days 10 days Cultivar Susceptible A-B plus

Resistant B-E

SusceptibleA-B plus

Resistant B-E

Susceptible A-B plus

Resistant B-E

Florina 1 8 3 7 3 7 Enterprise 3 7 6 4 4 5 Gala 6 4 7 3 9 0 Florina (RG) 3 2 3 0 2 2

Table 4. The values shown represent the number of leaves from each cultivar, which were

considered susceptible or resistant, if resistance threshold 3 is used. All leaves classified as Class A are considered susceptible, all leaves classified in Classes B plus to E, as resistant.

5 days 7 days 10 days Cultivar Susceptible

A Resistant B plus-E

SusceptibleA

Resistant B plus-E

Susceptible A

Resistant B plus-E

Florina 0 9 0 10 0 10 Enterprise 2 8 5 5 3 6 Gala 5 5 6 4 6 4 Florina (RG) 1 4 1 2 0 4

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If the threshold is drawn between class A and B, so that only leaves with class A symptoms are assessed as susceptible, there is a good correlation between that phenotype of the cultivars and the results from the detached leaf assay. All Florina samples are assessed as resistant and the majority of Gala samples as susceptible. Regardless of the incubation time statistically significant differences were observed between Gala and Florina if this threshold is chosen (Table 5). Table 5. Chi-squared values calculated from results if threshold 3 is applied. Gala is compared

to Florina and to Enterprise. The subsequent p values show that Gala is significantly different to Florina at all incubation times (At a significance level of at least 0.5%).

Chi-squared Values

Incubation time

Expected/Observed 5 days 7days 10days Gala/Florina 8.2 15 15 Gala/Enterprise 3.6 0.41 2 P values Gala/Florina 0.005<P>0.001 < 0.001 < 0.001 Gala/Enterprise 0.10<P>0.05 0.90<P>0.75 0.75<P>0.5

Increasing the incubation time from 5 days to 7 days was advantageous as a greater difference between resistant (Florina) and susceptible varieties could be observed (Tables 3, 4 and 5). This contrast did not develop further between 7 and 10 days of incubation, therefore there seems to be no particular advantage in 10 days of incubation in fact it only increases the chance of contamination from secondary infections. Discussion and conclusions In our previous study (Barbieri et al., 2002) samples were assigned to a resistance class A, B, C, D or E without counting all conidia. In the samples assessed there was a clearer distinction between Classes A and B, therefore the symptoms we now define as Class B+ were not observed very often. If they were observed they were classified as either A or B, depending on the expansion of the hyphae. Classification of individual samples functioned well and there was little disagreement between the individual assessors. Problems occurred, however, with the setting of a threshold for resistance between the classes. The results were summarised using thresholds one and three described above but from the control results of Gala and Enterprise samples it was not possible to define the correct threshold.

In the study presented here further data were collected to assist with the setting of a threshold. The results of this study show that if threshold three is used (i.e. only class A samples are considered as susceptible) then a clear difference can be observed between susceptible (Gala) and resistant (Florina) samples.

The differences between Gala and Enterprise were not so apparent and therefore we conclude that this variety is not suitable for this test, as its resistance is not sufficiently expressed under these particular experimental conditions.

A clear difference could be observed between Gala and re-generated Florina especially if the number of class A conidia found on each leaf were considered. Although only a few re-generated Florina samples could be tested, the similarity of these results with those from

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propagated Florina strongly indicate that the re-generation process has little effect on the resistance of this variety towards scab.

In conclusion we have shown that both Florina and Gala can be used as control cultivars in a detached leaf assay for in-vitro propagated and re-generated apple shoots, using the method and scale described, to assess the severity of the scab infection. Acknowledgements We would like to thank Vicente Martinez for his technical assistance. Also Elizabeth Chevreau, Stefano Tartarini and Massimo Barbieri for providing staring material for in-vitro grown apple shoot tip cultures and for giving advice on how to maintain these cultures and on apple shoot re-generation processes. This research was funded by the Swiss National Foundation for scientific research grant No. 31-58914.99. References Barbieri, M., Belfanti, E., Tartarini, S., Vinatzer, B., Sansavini, S., Silfverberg-Dilworth, E.,

Gianfranceschi, L., Hermann, D., Patocchi, A., & Gessler, C. 2002: Progress of the map based cloning of the Vf-resistance gene and functional verification: preliminary results from expression studies in transformed apple. – In press.

Chevalier, M., Lepinasse, Y. & Renaudin, S. 1991: A microscopic study of the different classes of symptoms coded by the Vf gene in apple for resistance to scab (Venturia inaequalis). – Plant Pathology 40: 249-256.

Gessler, C. & Stumm, D. 1984: Infection and stroma formation by Venturia inaequalis on apple leaves with different degrees of susceptibility to scab. – Phytopathology Z. 110: 119-126.

Ivanicka, J., Kellerhals, M. & Theiler, R. 1996: Evaluation of Scab (Venturia inaequalis (Cooke) G.Wint) on shoots and detached leaves from in vitro and greenhouse grown plants of the apple cultivars Golden Delicious and Florina. – Gartenbauwissenschaft 61(5): 242-248.

Nusbaum, C.J. 1938: A cytological study of host-parasite relations of Venturia inaequalis on apple leaves. – Journal of agricultural research 56(8): 595-617.

Vinatzer, B.A., Patocchi, A., Gianfranceschi, L., Tartarini, S., Zhang, H-B., Gessler, C. & Sansavini, S. 2001: Apple contains receptor-like genes homologous to the Cladosporium fulvum resistance gene family of tomato with a cluster of genes cosegregating with Vf apple scab resistance. – Molecular Plant Microbe Interactions 14(4): 508-515.

Yepes, L.M. & Aldwinkle, H.S. 1993: Selection of resistance to Venturia inaequalis using detached leaves from in vitro-grown apple shoots. – Plant science 93: 211-216.


pp. 75 - 81

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An adaptation of the New Hampshire degree-day model to predict ascospore release of Venturia inaequalis in Norway Arne Stensvand1, David M. Gadoury2, Terje Amundsen1, Robert C. Seem2 1Department of Plant Pathology, Norwegian Crop Research Institute, Plant Protection Centre, 1432 Ås, Norway; 2Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA Abstract: Volumetric spore traps were used to monitor ascospore release of the apple scab fungus, Venturia inaequalis, at four locations in southern Norway. Forecasts of a model of ascospore maturity, previously developed in New Hampshire (Gadoury & MacHardy, 1982), were compared to observed release. The model predicts 50, 95, and 99% spore maturation to occur at 250, 420, and 490 degree-days (DD), respectively. In the current investigation, the mean DD accumulation (base temperature 0°C from the green tip phenological stage of the apple flower bud) at the time when the seasonal spore trapping had accumulated to 50, 95, and 99% was 334, 621, and 694, respectively. In locations and years with frequent rain events throughout the season for ascospore release, the actual spore release followed the predicted maturation closely. Long periods without rain not only delayed spore release, but also spore maturation and consequently extended the season for ascospore release. The most extreme year was 1992, when DD accumulation at the time 95% of the season’s spores were trapped reached 1305 and 1092, respectively, at two sites. By halting DD accumulation during dry days, it was possible to improve the accuracy of the model. The best estimate of the spore maturation was made by halting DD accumulation after more than 4 days without rain. Key words: aerobiology, apple scab, epidemiology, spore discharge, spore maturation Introduction Apple scab is caused by the fungal pathogen Venturia inaequalis (Cooke) Winter. Ascospores formed within pseudothecia in infected, overwintered apple leaves serve as primary inoculum during periods of rain in spring and early summer. The primary season for ascospore release of V. inaequalis typically lasts six to ten weeks, however, the period of major spore release can be much shorter. The first ascospores are normally mature and ready to be released at bud break of the apple tree. Depending on the frequency of rainfalls, the peak in release is usually from the tight cluster stage to the petal fall stage of fruit bud development. The most important time of spraying against apple scab is during the major period of ascospore release, concurrent with the pre-bloom expansion of cluster leaves. When ascospore release ceases in early summer, scab sprays are usually ended or continued at extended intervals.

Gadoury & MacHardy (1982) in New Hampshire, USA, developed a model to estimate the cumulative percentage of matured ascospores, based on degree-day (DD) accumulation starting at the first appearance of mature ascospores. The model predicts 50, 95, and 99% spore maturation at 250, 420, and 490 DD’s (base temperature 0°C), respectively. Spring weather in New Hampshire is characterized by frequent periods of rain. However, periods of extreme dry weather can delay ascospore maturation in V. inaequalis (James & Sutton, 1982a, 1982b; Keitt & Jones, 1926; O'Leary & Sutton, 1986; Schwabe et al., 1989; Wilson, 1928). James & Sutton (1982a) in North Carolina, found that dry periods in spring delayed ascospore

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maturation in the field. In the laboratory, the pseudothecial development rate was retarded at a water saturation deficit greater than 85%, and there was no pseudothecial development in dry leaves. They found no pseudothecial development if rainfall was 0.25 mm or less and hours of 100% RH was 12 or less per day (James & Sutton, 1982b).

Weather in spring and early summer in the fruit growing regions of Norway varies greatly with location and year, and it is not unusal with prolonged periods of 1-3 weeks with no or very little rain in spring. Under protracted dry conditions, the accumulation of degree days alone is unlikely to accurately predict cumulative maturity of ascospores. We have studied the seasonal distribution of ascospore release in V. inaequalis in fruit growing regions of Norway in order to evaluate and possibly adapt the New Hampshire DD model (Gadoury & MacHardy, 1982) to Norwegian orchard conditions. Preliminary results from our study have been reported earlier (Stensvand et al., 1998, 2000). Materials and methods Burkard 7-day recording volumetric spore traps (Burkard Manufacturing Co Ltd., Rickmans-worth, Hertfordshire, UK) were installed at three different sites in southern Norway during the primary inoculum seasons of 1989-2000. We have 14 combined site/year observations of ascospore release. The traps were baited with a 2-3 m² area of heavily infected apple leaves placed on the orchard floor. Operation of the traps, microscopic preparation and examination was carried out as previously described (Gadoury & MacHardy, 1983).

Weather stations provided hourly records of precipitation, temperature, relative humidity, and leaf wetness. DD accumulation began at the green tip phenological stage, with a base temperature of 0°C. We defined green tip as the time when more than 50% of the flower buds on either of the cultivars ‘Gravenstein’ or ‘Summerred’ showed the first green tissue. The estimated ascospore maturity in V. inaequalis, according to the New Hampshire DD model, was compared with actual ascospore release. DD accumulation was calculated from daily mean temperatures. The model was also evaluated based upon DD accumulation computed as follows: No DD’s were assumed to have accumulated if no precipitation had occurred after more than 1, 2, 3, 4, 5, 6, or 7 days, respectively. No precipitation was defined as less than 0.2 mm, and days with less than 12 hours of free moisture occurring as dew or fog during night were considered dry days. Results and discussion The total seasonal number of airborne ascospores trapped averaged 51762, and ranged from 3511 to 200980. The mean DD accumulation at time of 50, 95, and 99% spore trapping was 334, 621, and 694, respectively. There was a variation in DD accumulation ranging from 393 to 1305 at the time 95% of the season’s spores were trapped (Table 1).

When DD accumulation was halted during dry periods, the mean number of DD’s at time of 95% spore trapping varied from 286 (DD’s halted after more than 1 dry day) to 478 (DD’s halted after more than 7 dry days) (Table 1). The best prediction of 95% ascospore trapping was provided if DD accumulation was halted after 4 dry days.The percentage of total observations within the confidence limits of the model was almost equal if DD accumulation was halted after 3, 4, 5, 6, or 7 days (Table 2). However, the number of incidences occuring below the 90% confidence limits of the New Hampshire model increased the longer the dry period was before the accumulation of DD’s was halted (Table 2).

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Table 1. Number of degree-days at the time 95% of the season’s ascospores of Venturia inaequalis were trapped; either for actual trapping or if degree-days (DD) were halted during dry periods.

DD accumulation halted during dry periods

after more than: Site/year

Actual trapping

1 day 2 days 3 days 4 days 5 days 6 days 7 daysSvelvik91 662 350 418 462 489 515 540 567 Svelvik92 1092 300 344 405 439 473 502 534 Hjelmeland93 401 176 209 247 280 307 332 359 Hjelmeland94 423 313 346 372 382 392 402 413 Ås90 672 319 385 440 474 503 523 543 Ås91 643 277 317 345 372 395 416 437 Ås92 1305 330 420 494 561 625 679 734 Ås93 467 230 296 352 378 415 428 442 Ås94 705 422 450 467 477 487 496 505 Ås95 392 272 331 360 371 379 386 392 Ås97 657 284 348 392 433 467 495 517 Ås98 448 218 257 295 325 356 387 413 Ås99 426 248 293 333 366 387 411 419 Ås00 522 268 319 344 371 385 402 419 Mean 630 286 338 379 408 435 457 478 Standard deviation 269 61 66 69 74 82 88 97

Observations within conf. limitsz

6 3 8 12 11 10 10 8

zNumber of site/year combinations within the confidence limits of the New Hampshire model (330 to 500 degree-days at 95% ascospore maturity) Table 2. Observations (%) of ascospores of Venturia inaequalis trapped in Burkard spore

trapsx that were within or outside the 90% confidence limits of the New Hampshire degree-day model; either for actual trapping or if degree-days (DD) were subtracted during dry periods.

DD accumulation halted during dry periods

after more than: Actual

trapping 1 day 2 days 3 days 4 days 5 days 6 days 7 days

Within conf. lim. 54.1 33.5 53.7 68.8 75.6 75.4 78.6 74.9

Before modely 8.7 66.0 45.8 30.6 22.7 19.1 13.4 11.8

After modelz 37.2 0.5 0.5 0.5 1.7 5.4 7.7 13.3

xMean of 14 combined site/year observations yAscospore trapping above the confidence limits of the NH model zAscospore trapping below the confidence limits of the NH model

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Thus, the fewer dry days that occurred before DD’s were halted, the lower the risk was for ascospores to remain to be released after the end of the season for ascospore release was predicted. If DD’s were halted after 1 or 2 dry days, the cessation of the ascospore release season as predicted by the maturity model came very late (on average 134 and 82 DD’s later than predicted at time of 95% spore trapping, respectively).

Details from spore trapping at all site/year combinations are not shown, and only two examples of seasonal distribution of ascospore release are presented (Fig. 1).

Figure 1. Accumulated seasonal trapping (%) of ascospores of Venturia inaequalis at two locations; Svelvik in 1992 (A) and Ås in 1997 (B). Dotted lines depict the New Hampshire degree-day model and its 90% confidence limits. Solid lines depict actual trapping and the same data if degree-days were halted after more than 4 dry days.

They show the distribution of spore trapping for actual trapping and if DD’s were halted after more than 4 dry days.At Svelvik in 1992 (Fig. 1 A), several rain and spore release events occurred in April and early May, and the actual spore trapping followed the maturity model closely. From mid May to late June there were two protracted periods where no rain occurred. According to the maturity model, rain following approximately 500 DD’s should exhaust

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most of the ascospore supply. Thus, all spores should have matured and be ready to be released when rain came during June 8 and June 12 (at ca 690 and 760 DD’s, respectively). Both days rain came during daytime (1 and 3 mm totally during the two days, respectively), and most spores would be expected to discharge. However, still 11.6% of the season’s spores were left. After the next rain June 30 to July 2, more than 99% of the season’s spores had been trapped. Very similar observations were made at Ås in 1992. The distance between the two sites is only approximately 30 km. At Ås, still 8% of the season’s spores were left to be trapped after the rain events in late June and early July, and spores were trapped during rain events until mid July (1450 degree-days). At Ås and Svelvik in 1992, DD accumulation reached 1305 and 1092, respectively, at the time 95% of the spores were trapped (Table 1).

At Ås in 1997 (Fig. 1 B), there was a protracted dry period of 13 days in late May and early June, from approximately 300 to 500 DD’s after green tip. Rain was frequent after this period. The actual spore trapping seemed to follow the maturity model at a distance similar to the prolonged dry period of 200 DD, and 95% spore trapping was reached at approximately 650 DD.

In both cases (Svelvik 1992 and Ås 1997, Fig. 1), there was a great deviation from actual trapping of spores to the spore maturation predicted by the model. However if DD’s were halted, the accumulated spore trapping coincided very closely with the maturity model. Fig. 2 shows the maturity model and actual trapping for all site/year combinations (Fig. 2 A) or if degree-days were halted after more than 7, 4, or 1 dry day (Fig. 2 B-D). By halting DD’s after more than 3 (data not shown) or 4 dry days for all 14 site/year combinations, the data for spore trapping fit closely to the maturity model.

Figure 2. Accumulated seasonal trapping (%) of ascospores of Venturia inaequalis trapped by Burkard volumetric spore traps; 14 site/year observations (black dots). Solid lines depict the New Hampshire degree-day model and its 90% confidence limits. A is actual trapping; B, C, and D are trapping data if degree-days were halted after more than 7, 4, or 1 day.

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If the first rain after a prolonged dry period had occurred during night, this could have explained some of the discrepancy between the maturity model and actual spore release, since darkness suppresses ascospore release in V. inaequalis (MacHardy, 1996). Another explanation could be that the rain lasted too short to allow all mature spores to be released. The first rain after a prolonged dry period always included rain during daytime, and within a few days it lasted long enough to release the matured spores from the pseudothecia. Neither darkness nor short rainy periods can thus explain why not nearly all the season’s spores were released during rain when more than 500 DD’s were accumulated since the green tip stage. From our investigation it is thus obvious that protracted dry periods delay spore maturation and release, and this agrees with previous authors (James and Sutton, 1982a, 1982b; Keitt & Jones, 1926; O’Leary & Sutton, 1986; Schwabe et al., 1989; Wilson, 1928).

The New Hampshire DD model and modified versions of the model, including modifications suggested by the present authors (Stensvand et al., 1998, 2000) was evaluated in a region of Italy (Rossi et al., 1999). If DD’s were halted after more than 4 dry days in the Italian study, the number of observations that where within the model were fewer than for actual spore trapping. In most years, the seasonal trapping of ascospores ceased much earlier than predicted by the maturity model if accumulation of DD’s were halted as suggested by the present authors. One discrepancy from our study was that Rossi et al. (1999) estimated the biofix according to a model developed in North Carolina that was based on climatic conditions prior to the first appearance of mature ascospores (James & Sutton, 1982b), and did not use green tip as the start of DD accumulation. Furthermore, the number of ascospores trapped in the Italian study was low. In two years the spore counts were only 360 and 555, and in only two years the total seasonal counts were above 10000 spores. In seasons when the total number of trapped spores are low, there is a great risk that no spores are trapped during rainy periods in the early and late parts of the season. Finally, no measures were taken to prevent earthworm activity and breakdown of leaf litter in the Italian study. Thus, the relative number of spores trapped in the early part of the season was probably higher than if the ground had been treated to avoid earthworms.

For a model to gain wide acceptance among growers and grower advisors, it is important to develop simple rules to aid in decisions. The degree-day model in itself is very simple, since most electronic scab warning devices calculate DD’s. However, the model was not accurate when there were protracted dry periods. We thus suggest an improvement of the model; dry days (i.e. less than 0.2 mm rain) beyond the 4th dry day should be subtracted from the DD accumulation. This will give a fairly conservative estimate of the spore maturation and much more exactly pinpoint the end of the primary inoculum season than by using the model under dry conditions as it is. Spore trap data from other countries, especially from areas which commonly have protracted dry periods during the season for ascospore release, should be compared to the New Hampshire maturity model. If subtraction of dry days make the spore trapping fit more closely to the maturity model, the New Hampshire DD model could become a more valuable tool in scab management thoughout the world. References Gadoury, D.M. & MacHardy, W.E. 1982: A model to estimate the maturity of ascospores of

Venturia inaequalis. – Phytopathology 72: 901-904. Gadoury, D.M. & MacHardy, W.E. 1983: A 7-day volumetric spore trap. – Phytopathology

73: 1526-1531.

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James, J.R. & Sutton, T.B. 1982a: Environmental factors influencing pseudothecial development and ascospore maturation of Venturia inaequalis. – Phytopathology 72: 1073-1080.

James, J.R. & Sutton, T.B. 1982b: A model for predicting ascospore maturation of Venturia inaequalis. – Phytopathology 72: 1081-1085.

Keitt, G.W. & Jones, L.K. 1926: Studies of the epidemiology and control of apple scab. – Wisconsin Agricultural Experiment Station Research Bulletin No. 73: 104 pp.

MacHardy, W.E. 1996: Apple Scab. Biology, Epidemiology, and Management. – APS Press, MN, USA: 545 pp.

O’Leary, A.L. & Sutton, T.B. 1986: The influence of temperature and moisture on the quantitative production of pseudothecia of Venturia inaequalis. – Phytopathology 76: 199-204.

Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 1999: Field evaluation of some models estimating the seasonal pattern of airborne ascospores of Venturia inaequalis. – Journal of Phytopathology 147: 567-575.

Schwabe, W.F.S., Jones, A.L., & van Blerk, E. 1989: Relation of degree-day accumulations to maturation of ascospores of Venturia inaequalis in South Africa. – Phytophylactica 21: 13-16.

Stensvand, A., Gadoury, D.M., Seem, R.C., Amundsen, T. & Falk, S.P. 1998: Some recent advances in epidemiology of apple scab. – (Abstract) 7th International Congress of Plant Pathology, Edinburgh 9-16 August.

Stensvand, A., Gadoury, D.M., Amundsen, T. & Seem, R.C. 2000: Recent research on ascospore discharge in Venturia inaequalis. – IOBC/wprs Bulletin 23 (12): 39-51.

Wilson, E.E. 1928: Studies of the development of the ascigerous stage of Venturia inaequalis. – Phytopathology 18: 375-420.


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Meteorological data for warning systems: some views concerning sensors Christer Tornéus Swedish Board of Agriculture, christer.torné[email protected] Abstract: Weather data, as a basis for estimating various biological developments, has become an important resource in our efforts to produce food with more environmental-friendly methods.

Mainly three different types of weather stations are used in Sweden; Metos, Adcon and Campbell. Some discriminating features are presented. They are used either as stand-alone instruments or in smaller networks. Whether the sensors should be placed in or near the crop, or according to the rules for “normal” stations used by public weather institutes, is another important issue.

Main items discussed in this lecture are weak points in the field part, especially leaf wetness sensors and relative air humidity sensors. Some systems calculate the leaf wetness periods and there are two types of sensors used, with two different ways of presenting the leaf wetness status. The importance of high quality system maintenance and a validation system for the database is also emphasized. Weather data, as a basis for estimating various biological developments, has become an important resource in our efforts to produce food with more environmental-friendly methods. Several soft wares deal with plant diseases and pests or are used as decision support tools for plant nutrition management. Some of those also include models for pesticide break down and plant growth models. Another important field for weather stations is irrigation management.

A management system consists of three parts; a model based on biological research, a collecting system for weather data and software for combining those into a user-friendly interface.

If the growers base their plant protection activities on a weather based prognosis system, it must be completely reliable at any time. Through many years experience I can say that no system is living up to that demand. I have learned a lot and it is my ambition to share this knowledge in order to make things better for the future. The weather stations Mainly three different types of weather stations are used in Sweden; Metos, Adcon and Campbell, but there are a lot of other brands on the market. I personally have only been working with Metos and Adcon (and I still use a couple of “Anton Paar” stations).

The weather stations can be used either as stand-alone instruments or in networks.There are many soft wares in the field and I’ll only mention some of those that I have got in touch with during the years.

“Adem”, a combined program for apple scab, apple mildew and fire blight, current status unknown.

“Schorf”, an Apple scab program, current status unknown. “Rimpro”, an Apple scab program, current status up and running, widely spread. “Plant Plus” and “Pro Plant”, for various agricultural crops, up and running, with Internet

applications.

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The practical use This is the main part of my presentation and I will tell about some problems and questions I have met with. Whether the sensors should be placed in or near the crop, or according to the rules for “normal” stations used by public institutes like SMHI (The Swedish Meteorological and Hydrological Institute), is an important issue that not ill be discussed here. My strategy for fruit orchards is that the station should be placed in the orchard and in a worst-case situation. Data transmission There are different methods to move the data from the field and the all have their problems. Cables can be cut off or damaged by corrosion. The Infra red light transfer normally works well, once it is configured which sometimes is not so easy, but it is a short-range method not suited for automatic purposes. Modem and radio (mainly relay stations) transfer can suffer from power problems. Radio transmissions are sometimes sensitive to radio interference. All systems are sensitive for lightening especially when cables are involved. The radio transfer is the best in this regard. The most important sensors

Temperature. Air temperature is an important in all biologic models. The sensor it self is not very complicated and the linearity is fairly good in the relevant intervals. The placing of the sensors could cause some questions though. Should the sensor be placed in or outside the crop? The answer is it should be placed like the sensors that the model builds on. This goes for soil temperature as well. Ground surface temperature is a special case for the Rimpro model. It is an option used to make the asco spore-maturing model better. Normally there is no special sensor available for this measuring and you have to do with a soil temperature sensor or a leaf temperature sensor.

Relative humidity. There is a very wide range of sensors for relative humidity. Many of those have a relative good linearity between 30% and 85 %. But when we come over the 85 % level this is not the case any more. The nearer to 100 % we come the worse the problem. This fact makes the humidity sensor being the worst link in the chain. In order to have an acceptable level of trust we have to go for the most expensive sensors. These sensors are also the sensors that has the shortest life span since it is very sensitive to air pollution making it even more expensive. Calibration of this sensor is a tricky thing that is often not being done. Relative humidity is an important parameter, especially when leaf wetness is calculated and therefore for it must have a high reliability.

Precipitation. Rainfall is fairly easy to measure but must have a good resolution when it comes to scab since the asco spores are released at very small amounts of rain. With a resolution of 0,2 mm or 0,1 mm we will get some problems. In order to get this high resolution we use a wide funnel and when it rains heavily the spoon flips to fast for the counter and to small values are registered. Another problem is dirt in the bottom of the funnel, including bird’s shit. With the Adcon rain gauge we have had problems with spider fixing the spoon with its web! If we are supposed to register precipitation the whole year around frost and snow can be a problem.

Leaf wetness. There are two different methods for measuring leaf wetness, paper based sensors or plated ceramics sensors. To my knowledge there is no consensus on which method is the best. Also in this case the placing of the sensors is essential for the reliability of the prognosis. When scab is concerned I am convinced that we need two sensors, one very exposed getting wet very fast and one more hidden in the tree drying up very slow. This way we can register the whole wetness period. Some models calculate the leaf wetness based on the relative humidity. This is a good method as long as the humidity sensor is very accurate

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and situated the right position. With calculated leaf wetness you miss the opportunity to measure inside the tree (unless you have a second sensor there), but for strawberries it is OK.

A minor problem is that the data output from some soft ware is decimal, while wetness is a Boolean variable, it is either wet or not! The decimal value could be useful though, regarding the fact that leaves from different plants may have different drying up time. You can then base the threshold for wet on observations. Still, the decimal value needs some further interpretation before it can be used in the model, unless the model has a special interface for this.

Wind. The sensors for wind speed and wind direction are not very complicated. Main question here is location. Are we interested in wind for spraying conditions or do we need fore water management or spreading of diseases? Wind direction has a special problem when it comes to graphic representation, namely north. Value wise north is represented of both zero and 360 and this is difficult to show on a graphic.

Light. To measure light there is the photo diode sensor that can deliver the value true or false, thus feasible for registering if it is day or night. You can also get a lux meter sensor for more accurate readings if you are dealing with insect flight for instance (forgive me for saying this in a disease conference).

Solar radiation. Not so much to say about this but I have think you have to choose the right measuring method depending on what you want to achieve For the growth models the wave length must be right. Conclusions There are several factors influencing the data quality. The placing of a station is of great importance. It does not matter how good a station you have, if you place it in the wrong place.

The right calibration methods must be used and you need to service your stations at least once a year. As a practical user I miss a good procedure for finding missing values both with Adcon and with Metos. If only a few values are missing, I would like to have the possibility to put in some copied/interpolated data from the nearby readings. If many values are missing it should be possible to put in values from a nearby station. Those ways of completing the data should be applications in the software from the producer of the station. If you are providing weather data on the Internet, I find it essential that you have a good procedure for validating the data before it is published.


pp. 87 - 94

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The simulation of ascospore release from apple scab: do we use suitable climatic data? Peter Triloff Marktgemeinschaft Bodenseeobst eG, Albert-Maier-Str. 6, 88045 Friedrichshafen, Germany Abstract: Simulation of ascospore release with RIMpro shows a good accuracy in timing and quantity of released ascospore compared to trap records, however the peak release period may be delayed compared to trap counts. Also ascospore releases under special climatic situations like cold periods, snow cover and after extended dry periods are not properly calculated by the programme under all circumstances. Using climatic data like temperature and wetness driving ascospore maturation and release from leaf litter instead from the tree canopy as usual, the overall accuracy of the simulation has been improved significantly as well as the above mentioned releases under special climatic conditions. The high value of the programme can be increased by feeding the developmental stages of the fungus taking place on the orchard floor with temperature and wetness data from the leaf litter and the infection process itself with traditional climatic data from tree canopy. Key words: apple scab, ascospore release, simulation, leaf litter temperature, leaf litter wetness Within the last decade the simulation of the primary season of apple scab has made big progress after models describing the maturation and release pattern of ascospores from apple scab have been developed in the 80ies and 90ies, as well as the infection phase on the leaf. From various simulation programs, "RIMpro", processing air temperature, leaf wetness and precipitation to compute maturation and release of ascospores, was used by the author at Lake Constance area since 1993 when the first version became available. Although the simulation resulted in a good accuracy concerning the timing of spore release and an estimate of Figure 1. Air temperature and leaf litter temperature and their relation to other climatic

factors.

Apple Scab, Lake Constance, ´99Important climatic parametres for apple scab

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ascospore numbers released, some discrepancies appeared under the climatic situation north of the alpes. In general the primary season was extended by the simulation showing significant releases beyond the end of the natural ascospore release period with lower releases in the early part of the primary season. The simulation also predicted peak spore releases later in the primary season than recorded in spore traps. It has also been found that after extended dry periods without any precipitation, simulated releases have been significantly lower than ascospore counts. Figure 2. Simulation of ascospore release with air temperature and leaf wetness compared to

spore trapping. Figure 3. Simulation of ascospore release with leaf litter temperature and leaf wetness

compared to spore trapping.

Additionally there have been some cases when spore releases have not been simulated under snow cover, however medium to high releases were recorded from spore traps. These

RIM-Trials Lake ConstanceAscospore Release ´01

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findings could not be resolved by modifications of the programme´s algorythms and thresholds so that other factors must have been responsible for the differences observed.

The fungus´s life cycle gives first hints because it takes place about 6 to 7 months on leaves, fruit and bark but also 6 to 7 months as a saprophyte in the leaf litter on the orchard floor. This phase includes the entire primary season.

So important stages like development of pseudothecia and ascospores during off season, ascospore maturation and ascospore release are driven by climatic factors other than air temperature and leaf wetness. Possible differences between temperature in the air and in the leaf litter as well as wetness on the leaves and in the leaf litter could account for the differences in ascospore releases between the simulation and the trap records, since air temperature and leaf wetness are commonly used to simulate ascospore maturation and release. Figure 4. Simulation of ascospore release with leaf litter temperature and leaf litter wetness

compared to spore trapping.

Figure 5. The influence of leaf litter temperature compared to air temperature on simulated

ascospore release under snow cover .

Apple Scab, Lake Constance, ´99Ascospore Release under Snow Cover

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Slots for rain water drainage and permeability for soil moisture

Insulation area to avoid direct contact of sensingfingers with soil surface35 µ copper sensing fingers

with 2.5 µ gold plating

Sensor base: flexible 25 µ polymid film

Connectors

Based on this assumption, in 1998 additional temperature sensors have been placed inside an artificial leaf litter bed underneath the apple trees on the bare soil of the herbicide strip. These sensors were connected to weather stations to study the influence of leaf litter temperature on ascospore maturation and release. To utilize these additional data within "RIMpro", the programme was modified using leaf litter temperature for driving maturation and release of ascospores whereas air temperature served for simulating infection and incubation as previously.

An example of the pattern of temperature measured in the air and in the leaf litter is shown in Figure 6, where temperature in the leaf litter during sunshine was up to 15°C higher than air temperature. But also during night and rain events temperature in the leaf litter is higher than in the air. These higher temperatures hasten spore maturation and release relative to air temperature.

Feeding ascospore maturation and release as well as the infection process with temperature data from the locations where they take place, improved the simulation of ascospore release remarkably in two ways: the overestimated releases in the later part of the primary season and those beyond the end of spore counts have been reduced significantly while the peak releases before bloom became stronger. So generally a better fit of the simulation with spore trappings was observed when leaf litter temperature was used for calculating spore maturation and release (Figure 2 and 3).

A special case is ascospore release under snow cover or at low air temperatures which usually is not simulated when using air temperature for calculating spore maturation and release. This artefact disappeared when feeding spore maturation and release with leaf litter temperature. Figure 5 shows an example with a simulated high ascospore release under snow cover which correlated very well with spore counts but was not at all achieved when air temperature was used for simulating ascospore release (Figure 5). Comparing heat units in this special release period, leaf litter temperature yielded 485 ADH >0,0°C while air temperature only returned 161 ADH >0,0°C.

Such a release omitted by the simulation could lead to a misinterpretation of this actual and its following release period since it may lead to an underestimation of the actual release period because the percentage of mature spores remains high pretending a high risk for a heavy release at the following rain event. This might probably result in wrong advice when infection conditions are not a limiting factor during the actual release period. Figure 6. Schematic of leaf litter wetness sensing grid.

91

Figure 7. Leaf litter wetness in relation to leaf wetness and other climatic factors under wet conditions.

Figure 8. Leaf litter wetness in relation to leaf wetness and other climatic factors under dry conditions.

But despite the use of a leaf litter temperature sensor, underestimated ascospore releases compared to trap records still have been found when a rain event followed an extended dry period during the main release period. Because very high spore releases have been recorded after such periods, the fungus must have had enough humidity in the leaf litter for continued spore maturation which was not recorded by the regular leaf wetness sensors up in the tree canopy. Currently ascospore maturation and release besides precipitation is computed with

Apple Scab, Lake Constance, 2001Leaf litter temperature and leaf litter wetness (wet period)

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data from leaf wetness sensors which are supposed to show quite a different wetness pattern compared to the humidity in the leaf litter, especially in dry regions where leaf wetness hours are low and therefore might cause an even stronger delay of simulated ascospore release compared to spore trapping.

This inaccuracy in simulating ascospore release led to the development of a leaf litter wetness sensor in order to simulate maturation and release of ascospores not only with leaf litter temperature and precipitation, but also with leaf litter wetness instead of leaf wetness.

This flexible resistive sensor with a sensing surface of 125 x 70 mm is made from a 25µ polymid film to achieve close contact to the soil surface and to keep thermal and insulating effects as small as possible. The film holds copper fingers which are gold plated to avoid electrolysis and corrosion from the soil solution. Between the fingers and an insulation area around each finger small slots are cut to drain rainwater and allow reverse water supply from the soil to the leaf litter. The sensing grid shown in Figure 1 is connected to a modified “Campbell Scientific 237 wetness sensor” and may be exchanged when broken. The sensor is placed on the bare soil which first has to be evened out and then the sensing grid is covered with a single layer of softened leaf litter. The leaves must be fixed with a plastic mesh to keep them in contact with the sensor. To avoid destruction of the sensor and the leaf litter, earth worms must be kept away from the area with benzimidazoles or other suitable compounds. In the moment the sensor cannot be run without interrupting the data logger grounding to avoid ground loops which would lead to poor readings because a galvanically separated power supply is not yet available. Since the risk for lightning strokes is very low during primary season and the sensor should be harvested after the end of ascospore release, there seems to be no risk for leaving the logger disconnected from electrical grounding. Results First data from two winter seasons and two primary seasons showed that leaf litter wetness is much more sluggish than leaf wetness. Under overcast sky after rain periods it may take even several days to drop and may occur only for one or two hours during dry nights even under heavy dew registered from the leaf wetness sensors. But leaf litter wetness also was registered at night without any dew detected on the leaves. During bright weather leaf litter wetness is terminated very rapidly with the increase of global radiation in the morning while under overcast sky the values may stay high allthough leaf wetness sensors already recorded dry conditions for a couple of hours (Figure 7 and 8).

Figure 4 shows the effect of sensors for leaf litter temperature and leaf litter wetness on the simulated ascopore release compared with spore trapings. Compared with the combinations leaf litter temperature and leaf wetness (Figure 3) and air temperature and leaf wetness (Figure 2) the improvement of the simulated release by using leaf litter temperature and leaf litter wetness becomes visible

Simulation results from 2001 and 2002 The primary season 2001 may be divided into three periods: period 1 from bud burst around March 10 to April 15, which was very wet; period 2 from April 16 through 24 where temperatures have been very low with snow cover for several days and period 3 from April 25 to end of May which was quite dry.

Using leaf litter climatic data for simulating ascospore maturation and release even under the very wet conditions in 2001 improved the results obtained from the simulation. During the cold period with snow cover the simulation with air temperature and leaf wetness did not yield in any releases while simulation with leaf litter data resulted in reasonable correlation of

93

the simulation with ascospore counts. Because of the poor releases from the simulation with climatic data from the canopy during the cold period, numbers of mature ascospores remained high and consequently led to a very much overestimated release on May 15 while simulation with leaf litter data returned a significantly lower release with again acceptable correlation with the spore trap records (Figure 9).

Figure 9. Simulation of ascospore release with leaf litter temperature and leaf litter wetness

compared to air temperature and leaf wetness in relation to spore counts in 2001 (wet primary season).

Figure 10. Simulation of ascospore release using leaf litter temperature and leaf litter wetness compared with air temperature and leaf wetness in relation to ascospore counts in 2002 (dry primary season).

RIM-Trials Lake ConstanceAscospore Release 2002


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The primary season 2002 started with an extended dry period from bud burst around

March 4 until April 13 with only one short rain event around March 19. In the second part of the primary season starting at April 14, rain events occurred more frequently and in shorter intervals.

The unexpected high release recorded in the spore traps on April 13 and 14 was simulated best by using leaf litter data while canopy data only led to a minor release. For the reminder of the primary season temperature and wetness from the tree canopy resulted in a delayed peak release and too high releases towards the end of the primary season compared with ascospore trappings. Again the use of leaf litter data for driving ascospore maturation and release resulted in a good overall correlation with the spore release records from the traps (Figure 10). Conclusions After two primary seasons of simulation of ascospore release with the simulation programme “RIMpro” comparing the use of air temperature and leaf wetness with leaf litter temperature and leaf litter wetness it may be concluded that leaf litter data significantly improved the simulation of ascospore maturation and release under various climatic conditions. Especially under cold conditions as well as after extended dry spells the use of leaf litter data proved to result in a much better correlation of the simulated ascospore release with spore trap records than with climatic data from the canopy. Provided that these results are obtained also from other regions during the next years, using leaf litter temperature and leaf litter wetness together with rain and irradiation for simulating ascospore maturation and release and air temperature and leaf wetness for the calculation of infection and incubation, the accuracy of scab warnings may improve significantly resulting in better advice for scab control on a farm level. Climatic data from leaf litter would also allow the “fine tuning” of simulation programmes which is supposed not to be successful as long as calculation of both spore release and infection are based on the same climatic data from the tree canopy.


pp. 95 - 106

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Chemical control of apple powdery mildew (Podosphaera leucotricha): mode of actions Xiangming Xu, Joyce Robinson, Angela Berrie Horticulture Research International - East Malling, West Malling, Kent ME19 6BJ, UK. Email: [email protected] Abstract: Eight fungicides (dinocap, kresoxim-methyl, myclobutanil, bupirimate, fenarimol, triadimefon, penconazole and pyrifenox) were tested for their epidemiological mode of action (e.g. protectant, anti-sporulant, curative activity) against Podosphaera leucotricha at two rates (25% and 100% of the label recommended rate) in comparison with untreated or water-treated plants. All the fungicides were effective in controlling powdery mildew when applied as a protective, anti-sporulant and curative treatment. Fungicides differed significantly in their efficacies. However, these differences were generally small compared to the differences with the controls. Protective activities (or persistence) decreased more or less linearly with the interval between fungicide application and inoculation. Fungicides had little effect on disease when leaves were inoculated 10 days after application. The curative activity varied little between applications applied 48 and 96 h after inoculation. In general, application at quarter rates significantly reduced the fungicidal activity, especially for triadimefon, penconazole and kresoxim-methyl. Pyrifenox, myclobutanil, dinocap and bupirimate were shown to be the most effective fungicides, whether applied as a protective or curative, anti-sporulant treatment. Keywords: apple mildew, curative, protectant, anti-sporulant, fungicide Introduction The main UK apple cultivars, Cox, Gala and Bramley's Seedling, are susceptible to a range of fungal diseases, including powdery mildew caused by Podosphaera leucotricha. Powdery mildew severely damages apple leaves, flowers, fruits, buds, shoots and twigs, and can cause considerable loss of fruit yield (van der Sheer, 1980; Yoder & Hickey, 1983) due to the reduction of photosynthesis intensity and decrease of leaf nutrient content (Ellis et al., 1981). Intensive applications of fungicides are usually used to control apple powdery mildew, applied from bud-burst to harvest, at 7-14 day intervals (Butt & Jeger, 1982; Berrie, 1997). Such routine programmes, though costly, have usually proved successful, reliable and simple for farm managers to operate. However, with the high level of public concern about pesticides and their possible side effects on health and the environment, and the rising costs of chemicals, such practices have become less acceptable.

For powdery mildew it is possible to optimise fungicide use, with little risk to fruit quality, by matching fungicide dose to the level of secondary infection recorded in the orchard (Butt & Barlow, 1979; Butt et al., 1983; Berrie & Cross, 1996). To help growers adopt a more rational disease control strategy, Horticulture Research International, East Malling, has built and validated an epidemiological model (Podem™) that relates the development of apple powdery mildew to biotic and abiotic factors (Xu, 1999). Podem™ has been implemented as a PC-based disease warning system, called Adem™ (Apple Diseases

96

East Malling), together with models for apple scab, Nectria canker and fruit rot, and fireblight (Xu & Butt, 1996a; Xu & Butt, 1996b).

Podem™ warns growers of potential mildew infections and spore production, thus assisting growers in timing the spray and adjusting the rate and volume more rationally. To fully utilise the warnings given by Podem™, the epidemiological mode of action (protectant, curative, and anti-sporulant) of common mildew fungicides must be fully understood. Much research has been conducted to investigate the effectiveness of chemical control of apple powdery mildew. However, most of the research was either on general efficacy of chemical control in field conditions (Lalancette & Hickey, 1985; Nowacka et al., 1985; Nowacka & Cimanowski, 1987; Nowacka, 1995) or on anti-sporulant action (Blake et al., 1982; Cimanowski, 1989; Gupta & Gupta, 1991; Cimanowski & Bielenin, 1996). The protective and curative properties of fungicides against powdery mildew have so far been neglected.

This paper reports results of investigations on the modes of action of common fungicides against apple powdery mildew. Experiments, conducted in glasshouse compartments or polytunnels, were focused on (1) protective activity and persistence, i.e. the ability to prevent mildew spores from infecting healthy leaves and how long this activity remains effective, (2) curative activity, i.e. the ability to kill the young developing, but symptomless, colonies thereby preventing them from forming visible lesions, and (3) anti-sporulant activity, i.e. the ability to reduce spore production. Materials and methods Experiments were conducted in two phases using MM106 rootstock plants. Phases 1 and 2 were conducted in 1998 and 2000, respectively. Plants were stored at 2°C, potted up in batches and grown in a ‘mildew-free’ glasshouse compartment at approximately 20°C (18-23°C) and 70% relative humidity (rh) with a 16 h light/8 h dark daily regime. There were at least three plants, each with two shoots, for each treatment. Leaf positions were identified by tagging the youngest fully unrolled leaf (i.e. the leaf ‘0’) at the time of inoculation or spraying. All the experiments were repeated once. Inoculum and inoculation Infected plants (stock plants) with fresh colonies of powdery mildew were maintained in a polytunnel and were supplemented frequently with plants used previously in experiments and carrying younger mildew colonies. Leaves with sporulating lesions were collected from the stock plants. On each plant to be inoculated, the four youngest leaves on each shoot were inoculated by shaking conidia from the mildew-infected leaves onto their surface. Fungicides Details of fungicides used in this study are given in Table 1. Fungicides were applied to the plants (shoot/leaves) until run-off using a 500 ml hand-held sprayer. Fungicides were applied at two rates: 25% and 100% of the label-recommended run-off rate (water volume 2000L/ha). In addition to these fungicides, untreated and water-treated controls were included. Protective activity and persistence

Phase 1. Persistence of the fungicides was not studied in this phase. Healthy plants were first moved to a glasshouse compartment where the spraying took place. After being sprayed, the plants were then left to dry for several hours later. Once dry, the plants were inoculated by shaking leaves with sporulating mildew colonies over the top of the shoots thus dispersing spores on the surface. Shoot tips were labelled at the time of spraying.

Phase 2. Both protective activity and persistence were studied in this phase. Plants were first sprayed and kept in a mildew-free compartment. Once the plants were dry, three plants

97

from each treatment (fungicide x rate) were moved to a polytunnel to be inoculated. The plants were then left amongst the mildew-infected plants in the polytunnel for three days and then moved to another, mildew-free, compartment. Another three plants per treatment were then taken to the tunnel for inoculation and left for three days before being moved to the mildew-free compartment. This was repeated three more times, i.e. five batches for each treatment.

Table 1. Fungicides used in the testing and some experimental details of the two phases of research.

Phase 1 Phase 2 Fungicides

Trade name

% of a.i.a

Formulationb

Dosec (a.i.)mg/l Pd C A S P C A S

Fenarimol Rubigan 12 w/w SC 264 xe x x Triadimefon Bayleton 25 w/w WP 25 x x x Bupirimate Nimrod 27.2 w/w EC 175 x x x x Penconazole Topas 10.6 w/w EC 30 x x x x Pyrifenox Dorado 20.8 w/w EC 40 x x x x Myclobutanil Systhane 6 w/w EW 26 x x x x x x x Dinocap Karathane 33.2 w/w EC 210 x x x x Kresoxim-methyl Stroby 50 w/w WG 50 x x x x a: a.i. – active ingredient; w/w - weight/weight. b: EC - emulsifiable concentrate, EW - oil in water emulsion, SC - suspension concentrate (= flowable),

WG - water dispersible granules, WP - wettable powder. c: dose at the full label-recommended rate, a.i. – active ingredient. d: P – Protective; C – Curative; A – Anti-sporulant; S – Persistence. e: x – tested in the experiments.

Curative activity Phase 1. Healthy plants were first inoculated using the same method described above and left in the diseased compartment. After two or four days the plants were moved to a mildew-free glasshouse compartment and sprayed. Two experiments were conducted. In the first, fungicides were applied at only the full rate to the plants two days after inoculation; in the second, fungicides were applied at both the full and 1/4 rate to the plants four days after inoculation. Shoot tips were labelled on the day of inoculation.

Phase 2. Similar experiments were conducted with fungicides at both the full and 1/4 rate applied to the plants two and fours days after inoculation. Anti-sporulant activity Healthy plants were first inoculated with mildew spores. Ten days later, four leaves on each plant with good sporulating colonies were selected, tagged and sprayed with the appropriate fungicides or water. Before the spray, the tagged leaves were well shaken to remove those old spores. Disease assessment Phase 1. For protective and curative studies, the plants were assessed for mildew at 10 and 14 days after inoculation. Numbers of mildew colonies were counted on each leaf of each treated shoot from the leaf ‘0’ to the tagged leaf. Leaf ‘0’ is the youngest unrolled leaf on a extension shoot. Leaves at position -1, -2, -3 etc. are older and increasingly larger than leaf 0, leaf -1 being adjacent to leaf 0. Anti-sporulant assessments were taken 10 and 14 days after spraying.

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Two of the four leaves labelled prior to spraying were assessed at 10 days after and the other two at 14 days. Pieces of "adhesive tape (sellotape)" (ca. 1 cm2) were pressed against colonies on treated leaves, then taken off and stuck onto slides for microscopic examination. Numbers of apparent ‘healthy’ spores (i.e. not deformed or shrivelled) were assessed on a scale of 0-5 (0: no spores, 1: 1-15 spores, 2: 16-30 spores, 3: 31-50 spores, 4: 51-100 spores, 5: > 100 spores in one view area under the microscope with the magnification of x100).

Phase 2. For protective activity, persistence and curative activity studies, numbers of mildew colonies were similarly assessed but only at 14 days after inoculation. Anti-sporulant assessments were also taken 14 days after spraying. Three leaves per plant were labelled prior to spraying. Pieces of "adhesive tape" (ca. 3 cm2) were pressed against the top of the treated lesions, then peeled off and stuck onto glass slides. The imprint of mildew colonies left on the sticky tape was examined under a microscope. The number of apparent ‘healthy’ mildew conidia was estimated by examining 100 spores. Data analysis Standard analysis of variance (ANOVA) with a nested design was used to analyse the data. ANOVA was first used to show whether there were significant effects of treatments compared with the two controls. Then, further ANOVA was conducted to investigate the effects of application rate. The significance of treatment effects was determined by testing against the variation between plants within treatments. Fisher’s least significant differences (LSD) were used to compare treatments. Before analysis, individual count data were logarithmically transformed. Genstat™ (Payne et al., 1993) was used for data analysis. Results Phase 1 For all the tests, statistical analysis showed that assessments of anti-sporulant activity 10 or 14 days after inoculation or spraying gave very similar results. These two assessments were therefore combined.

Protective activity. Fungicides significantly (P < 0.1%) reduced the number of lesions, compared with the two controls (Fig. 1), the reduction ranging from 77% to 98%. There were no significant differences in the protective efficacies between bupirimate, pyrifenox and myclobutanil. Triadimefon, fenarimol and penconazole did not differ significantly from each other but resulted in significantly more (P < 1%) lesions than the other three fungicides. The quarter rate resulted in more lesions than the full rate, but only for triadimefon and pyrifenox was this rate difference significant (P < 5%).

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Curative action. There were highly significant differences between the two controls and the fungicide treatments (Fig. 2). When the fungicides were applied at the full rate two days after inoculation, there were only an average 0.2 lesions per fungicide-treated leaf, compared to 1.3 and 2.1 lesions per leaf for the water treatment and the untreated, respectively. Triadimefon resulted in significantly (P < 1%) more lesions than the other five fungicides, which did not differ significantly from each other (Fig. 2a). When applied four days after inoculation, the quar-ter rate resulted in significantly (P < 0.1%) more lesions than the full rate (Fig. 2b). However, there were significant (P < 1%) interactions between the fungicides and the rate; the loss of control efficacy of the quarter rate over the full rate was proportionally much greater for triadimefon and penconazole than for pyrifenox, bupirimate and fenarimol (Fig. 2b). At the full rate, myclobutanil, penconazole and pyrifenox gave the control. Overall, myclobutanil and pyrifenox gave the best control.

Anti-sporulant activity. There were significant (P < 0.1%) differences in anti-sporulant activity between fungicides and between application rates. All six fungicides significantly reduced sporulation activities compared to the controls (Fig. 3), reductions ranging from 40% to 80%. There were no significant interactions between the fungicides and the rate. The full rate reduced sporulation activities much more than the quarter rate. The fungicides could be divided into two groups; the first includes myclobutanil, bupirimate, and pyrifenox, with the other three in the second group. The first group of fungicides was much more effective in reducing sporulation activities than the second group. Phase 2 Protective activity and persistence. There were significant differences between the treatments for the first three batches, i.e. inoculation up to 9 days after fungicide applications, mainly due to the reduction in colony numbers in fungicide treatments over the controls. Within treatment plant-to-plant variation increased with increasing intervals between spraying and inoculation. The differences between fungicides were generally small (Fig. 4) and there were only significant differences between a few pairs of comparisons. Pyrifenox appeared to be less effective when applied at the quarter rate. On average, the efficacy, measured as the reduction of colony numbers over the untreated, decreased linearly as the interval between spraying and inoculation lengthened (Fig. 4), except for pyrifenox, penconazole and bupirimate in the last batch, where this trend was reversed. Inoculating plants from 10 days onward after fungicide application generally resulted in a similar amount of disease to the controls. Overall,

Figure 1. Number of apple mildew colonies (logarithmically transformed) on leaves treated with fungicides at ¼ and full label-recommended rates, expressed as a percentage of controls (average of the untreated and water-treated was 2.06) in phase 1. The treated plants were inoculated several hours after fungicide applications

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bupirimate appeared to have the best persistence/protective activity though the other four fungicides were similar.

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Figure 2. The curative efficacy of the six fungicides, in phase 1, applied 48 or 96 h after inoculation at ¼ and full label-recommended rates, expressed as a percentage of controls. (a) Fungicides were only applied at the full rate two days after inoculation(average of the untreated and water-treated was 1.68); (b) fungicides were applied at both the full and quarter rate four days after inoculation (average of the untreated and water-treated was 1.41).

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ANOVA on the effects of application rate and fungicides, excluding the two controls,

further confirmed that generally the fungicides all had very similar persistence/protective activities. Only for batch 3 (inoculation 7-9 days after fungicide application) were there significant (P < 5%) differences between the fungicides. Applications at the quarter rate resulted in considerable loss of efficacy compared to the full rate, but this loss was only significant (P < 1%) for the second and third batches (i.e. inoculation between 4-9 days after fungicide application). This rate effect was generally consistent with all fungicides (Fig. 5). In only one case did the full rate result in significantly more colonies than the quarter rate (penconazole in batch 4).

Curative activity. There were significant (P < 1%) differences in curative activity between the two repeats. There were also significant interactions between treatments and the repeat, but accounting for far less variation than the main effects. The treatment effects were

Figure 4. Number of colonies on leaves treated with the fungicides at ¼ and full label-recommended rates in phase 2, expressed as a percentage of controls (untreated and water-treated). The treated plants were inoculated from 0-15 days after fungicide application.

Number days after spraying

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mainly due to the differences between the fungicides and the two controls. There were virtually no differences between applications at 48 or 96 hours after inoculation.

Further ANOVA excluding the two controls showed that there were significant

differences in the curative activities between the three fungicides. Kresoxim-methyl had significantly less curative ability (average 0.4 lesions per leaf) than dinocap and myclobutanil (both average 0.1 lesion per leaf). However, kresoxim-methyl still reduced the number of

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Figure 5. Number of apple mildew colonies on leaves treated with the fungicides at ¼ and full label-recommended rates in phase 2, expressed as a percentage of controls (untreated and water-treated). The treated plants were inoculated from 0-15 days after fungicide application.

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colonies by about 85% over the two controls, compared with 95% for dinocap and myclobutanil, respectively.

Application at the full rate resulted in significantly fewer lesions than the quarter rate (Fig. 6). However, there was a significant interaction between rate and fungicide, which was mainly due to the larger number of lesions in the kresoxim-methyl at the quarter rate treatment (Fig. 6). Table 2. Average percentage of healthy conidia on colonies sampled 10 days after fungicide

application over the two fungicide application rates.

Fungicides Repeat 1 Repeat 2 Averagea

Control 80.4 94.4 87.4d Water 69.3 92.7 81.0c Dinocap 66.8 61.9 64.3a Myclobutanil 69.1 59.0 63.8a Kresoxim-methyl 74.9 76.1 75.5b a: Treatments with the same letter after their means were not significantly different from each other, based on LSD tests (P = 0.05).

Anti-sporulant activity. There were significant interactions between repeat and treatment, though accounting for much less variation than the main effects. The significant interaction was mainly due to the differences in the two controls between the repeats. The three fungicides tested significantly reduced sporulation 14 days after treatment compared to the controls. Water treatment also significantly reduced sporulation compared to the untreated

F igu re 6 . T h e cu ra tiv e efficacy o f th e th ree fu n g ic id es , in p h ase 2 , ap p lied 4 8 o r 9 6 h after in o cu la tio n a t ¼ and fu ll ra tes , as a p ercen tage o f co n tro ls (av erage o f u n trea ted an d w ater-trea ted w as 1 .4 9 ).

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control (Table 2). Dinocap and myclobutanil had similar effects, reducing the sporulation by 25%, and were significantly more effective than kresoxim-methyl, which only reduced the sporulation by 10%. There were no significant effects of application rate or its interaction with fungicides. Discussion and conclusion This study has for the first time showed that all the fungicides tested have significant effects in controlling apple powdery mildew whether applied as curative as well as protectant/anti-sporulant fungicides. In all tests, applying water also consistently reduced disease development, although to a lesser extent than the fungicides. It is expected that water will reduce spore production (Sivapalan, 1993a; 1993b; 1994). However, water is also effective when applied as a protective or curative treatment, which may be due to the changes in phylloplane microflora induced by water. Despite significant differences between repeats, and large variation between plants within treatments, there were significant differences between fungicides in their efficacy. The large differences between repeats are likely to be due to the variations in plant growth rates and in inoculum quantity and concentration. To inoculate leaves with a consistent conidial dose for all treatments requires a spore suspension with the inoculum dose adjusted on the basis of counts of conidia using a haemocytomer. However, the development of powdery mildews, as a group of diseases, is generally affected adversely by the presence of free water during the initial infection stage (Butt, 1978; Sivapalan, 1993b). In addition, the tested plants were also left among heavily-mildewed plants for several days. Thus, despite the efforts in inoculating plants evenly and in placing plants randomly among those infected plants, it was inevitable that there would be some variation in quantity and quality of inoculum received by the experimental plants.

All the fungicides controlled powdery mildew very effectively when plants were inoculated within three days of fungicide application. For all the fungicides tested, their protective efficacy (i.e. persistence) decreased approximately linearly with the interval between fungicide application and inoculation; indeed fungicides had little effect in controlling disease when leaves were inoculated from 10 days onwards after fungicide application. Differences between fungicides were generally small. This study also showed considerable reductions in protection efficacy when fungicides were applied at the quarter rate, compared with the full rate; even for those plants inoculated within three days of fungicide application. This rate effect was especially large for pyrifenox and triadimefon, and smallest for bupirimate and dinocap. Thus, in the present study, it appeared that fungicides, when applied at full rate, could only maintain good protection of leaves from infection for a maximum of one week. Of course, this protection interval is dependent on the rate of extension growth of the host leaves. In the present study, the rate of leaf growth/expansion at a near constant 20°C temperature in glasshouse compartments can be expected to be greater than in field conditions, and it therefore may be expected that the protective interval would be longer than one week under 'normal' field conditions, especially in early the season when temperatures are generally low.

This study showed that the fungicides assessed possess good curative action against apple powdery mildew. The curative efficacy of the fungicides did not vary significantly with application interval, i.e. applied 48 or 96 h after inoculation, and all reduced disease, ranging from 80% to 96% over the untreated. Application at the quarter rate significantly reduced the efficacy, especially for kresoxim-methyl, penconazole and triadimefon. Overall, triadimefon

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and kresoxim-methyl had the poorest curative action. All other fungicides had similar curative activities.

All the fungicides significantly reduced sporulation of mildew colonies 10 or 14 days after application. Application of all the fungicides at the quarter rate resulted in considerable reductions in their anti-sporulant efficacy. Overall, fenarimol, penconazole, triadimefon and kresoxim-methyl appeared to be less efficacious in their anti-sporulant action than the other four fungicides. Fungicide efficacy in phase 2 testing was much less than in phase 1 testing. This is most probably due to the differences in the assessment methods. In phase 1, numbers of ‘healthy’ spores were assessed over an area of the same size for all treatments; whereas in phase 2, the proportion of ‘healthy’ spores was assessed out of the first 100 spores observed. Therefore, results in phase 1 will indicate the inhibitory effects of fungicides on both spore production and spore viability, whereas results in phase 2 will only indicate the latter aspect. Thus, using the results of myclobutanil from phases 1 and 2, the reduction of spore production was estimated to be 62%, compared to 27% reduction in spore viability. The anti-sporulant test results generally agreed with previous research (Blake et al., 1982; Cimanowski, 1989; Gupta & Gupta, 1991; Cimanowski & Bielenin, 1996). Bupirimate has consistently been shown to possess greater anti-sporulant action than the other fungicides (Blake et al., 1982; Cimanowski, 1989; Cimanowski & Bielenin, 1996). Blake et al. (1982) showed that bupirimate and triadimefon had the greatest and lowest anti-sporulant activity, respectively, whereas dinocap and fenarimol showed moderate anti-sporulant activity.

In this study, four fungicides (pyrifenox, myclobutanil, dinocap and bupirimate) were shown to be the most effective whether applied as protective or curative or anti-sporulant treatments. Of these four, pyrifenox and myclobutanil are also effective scab fungicides. Thus, when both scab and mildew control are important, pyrifenox and myclobutanil are the most likely to be used, though over-use may result in development of fungicide-insensitive fungal strains. When scab is not a priority, bupirimate and dinocap (indeed penconazole at the full rate) may be used to manage mildew. When the mildew risk is not great, and anti-sporulant action is not important, kresoxim-methyl can also be used to control both scab and mildew. It is here that disease warning systems such as Adem™ will be able to assist growers in adopting more rational strategies for using fungicides. Adem™ gives forecasts of both scab and mildew risks based on weather conditions. Furthermore, for mildew it also gives forecasts of both infection and sporulation risks, thus fungicides with good curative/protective and/or anti-sporulant action could be used depending on the relative magnitude of these two risks. Acknowledgements This work was funded by the East Malling Trust for Horticultural Research, Worshipful Company of Fruiterers, and Rohm & Haas (UK) Ltd. References Berrie, A.M. 1997: Optimising fungicide applications to control apple diseases using

Adem™. – Aspects of Applied Biology 46: 155-162. Berrie, A.M. & Cross, J.V. 1996: An evaluation of plant protection practices according to IFP

guidelines compared to current commercial practice. –IOBC/wprs Bulletin 19(4): 17-27. Blake, P.S., Hunter, L.D. & Souter R.D. 1982: Glasshouse tests of fungicides for apple

powdery mildew control. II. Eradicant and anti-sporulant activity. – Journal of Horticultural Science 57: 407-411.

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Butt, D.J. 1978: Epidemiology of powdery mildews. – In: The Powdery Mildews, ed. Spencer, D.M., Academic Press, London: 51-81.

Butt, D.J. & Barlow, G.P. 1979: The management of apple powdery mildew: a disease assessment method for growers. – In: British Crop Protection Conference - Pests and diseases, Brighton, UK, British Crop Protection Council: 77-86.

Butt, D.J. & Jeger, M.J. 1982: Decision-based management of orchard pathogens and pests in the United Kingdom. – In: The 1982 British Crop Protection Conference, Brighton, UK, British Crop Protection Council: 167-189.

Butt, D.J., Jeger, M.J. & Swait, A.A. J. 1983: Bio-meteorological forecasting scheme for apple mildew. – In: The 10th International Congress of Plant Protection, Brighton, UK, British Crop Protection Council: 184.

Cimanowski, J. 1989: Antisporulant action of the latest systemic fungicides used against the powdery mildew Podosphaera leucotricha (Ell. et Ev.) Salm. – Fruit Science Reports 16, 23-26.

Cimanowski, J. & Bielenin, A. 1996: Antisporulant activity of new apple powdery mildew fungicides. – Journal of Fruit and Ornamental Plant Research 4: 21-25.

Ellis, M.A., Ferree, D.E. & Spring D.E. 1981: Photosynthesis, transpiration, and carbohydrate content of apple leaves infected by Podosphaera leucotricha. – Phytopathology 71: 392-395.

Gupta, S.K. & Gupta, G.K. 1991: Eradicative activity of fungicides against apple powdery mildew. – Indian Journal of Mycology and Plant Pathology 21: 70-72.

Lalancette, N.K. & Hickey, D. 1985: Apple powdery mildew disease progress on sections of shoot growth: an analysis of leaf maturation and fungicide effects. – Phytopathology 75: 130-134.

Nowacka, H. 1995: Effectiveness of fluquinconazole and pyrimethanil in control of apple scab and powdery mildew. – Journal of Fruit and Ornamental Plant Research 3: 187-193.

Nowacka, H.J., Cimanowski, H. & Profic-Alwasiak, H. 1985: Effectiveness of the fungicide Topas C 50 WP in the control of apple scab and powdery mildew. – Fruit Science Reports 12: 163-171.

Nowacka, H.J. & Cimanowski, H. 1987: The effectiveness of fungicide mixtures in the apple scab and the powdery mildew control. – Fruit Science Reports 14: 35-43.

Payne, R.W., Lane, P.W., Todd, A.D., Digby, P.G.N., Thompson, R., Harding, S.A., Wilson, T.G., Leech, P.K., Welham, S.J., Morgan, G.W., White, R.P. 1993: GenstatTM 5 Release 3: Reference Manual. – Oxford University Press, Oxford.

Sivapalan, A. 1993a: Effects of impacting rain drops on the growth and development of powdery mildew fungi. – Plant Pathology 42: 256-263.

Sivapalan, A. 1993b: Effects of water on germination of powdery mildew conidia. – Mycological Research 97: 71-76.

Sivapalan, A. 1994: Development of powdery mildew fungi on leaves submerged under water. Journal of Phytopathology 140: 82-90.

van der Sheer, H.A. 1980: Threshold of economic injury for apple powdery mildew and scab. – In: Integrated control of insect pests in the Netherlands, p 49.

Xu, X.M. 1999: Modelling and forecasting epidemics of apple powdery mildew (Podosphaera leucotricha). – Plant Pathology 48: 462-471.

Xu, X.M., & Butt, D.J. 1996a: Adem™ a PC-based multiple disease warning system for use in the cultivation of apples. – Acta Horticulturae 416: 293-296.

Xu, X.M., & Butt, D.J. 1996b: A description of Adem™ - a PC-based disease warning system for apple. – IOBC/wprs Bulletin 20(9): 251-260.

Yoder, K.S., Hickey, K.D. 1983: Control of apple powdery mildew in the mid-Atlantic region. – Plant Disease 67: 245-248.


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Biological characteristics of dicarboximide-resistant isolates of Stemphylium vesicarium from Italian pear orchards Giulia Alberoni, Marina Collina, Agostino Brunelli Dipartimento di Protezione e Valorizzazione Agroalimentare, Università di Bologna, Viale G. Fanin 46, 40127 Bologna, Italy Abstract: Dicarboximide-resistant isolates of Stemphylium vesicarium have been detected in Italian pear orchards since the early 1990s. Two different resistant phenotypes were observed during a 9 year monitoring study. R1 phenotypes showed Resistance Factor (RF)> 100 towards procymidone and between 3 and 100 to the other dicarboximides (iprodione, vinclozolin and chlozolinate) whereas R2 phenotypes showed RF>100 towards all fungicides of this group. Various morphological and physiological characteristics of the different resistant isolates, such as mycelial colour, mycelial growth rates, sporulation, germination rates and pathogenicity on detached leaves, were compared to those of sensitive isolates. No significant differences could be noted among the phenotypes. Competitive abilities of sensitive and R1 resistant isolates were then evaluated in vitro on unamended medium through mixed inoculations of R/S conidia in 75:25, 50:50 and 25:75 ratios. R1 resistant isolates did not seem to be less competitive in vitro than sensitive ones in the absence of dicarboximide fungicides. Key words: Stemphylium vesicarium, dicarboximides, resistance, fitness, pear Introduction Brown spot, caused by Stemphylium vesicarium, is the most important fungal disease on pear in North-eastern Italy. All the green parts of the plant: leaves, young stems and fruits can be infected throughout the entire season. Symptoms are particularly serious on fruit, where necrotic areas can rot before, during or after harvest, causing yield losses up to 100%. Besides some cultural practices, its control relies on preventative fungicide applications from petal fall to fruit ripening. Dicarboximides (mainly procymidone) were the fungicides most utilised against this pathogen but the occurrence of resistant isolates in the early 1990s further complicated disease management (Brunelli et al., 1997; Collina et al., 2002). A survey carried out on 516 samples collected from 1995 to 2003 in Po valley showed a widespread occurrence of S. vesicarium isolates resistant to dicarboximides in all the regions considered. In addition, two different resistant phenotypes (R1 and R2) were observed. R1 phenotype showed Resistance Factor (RF)>100 towards procymidone and between 3 and 100 to the other dicarboximides (iprodione, vinclozolin and chlozolinate) whereas R2 phenotype showed RF>100 towards all fungicides of this group. The incidence of the two phenotypes was very different; R2 resistant isolates were, in fact, very rare (20/516) (Alberoni et al., 2005).

Fitness of pathogen isolates resistant to fungicides is an important consideration in disease management. If fitness costs are associated with fungicide resistance, the frequency of resistant isolates will decline in the absence of the fungicide. Furthermore mixtures or alternations with fungicides of different modes of action could be instituted to delay the selection for fungicide resistance (Raposo et al., 2000). If the relevant fungicides are withdrawn, or reduced as number of application, the frequency of the resistant strains remains high in the population only if they have similar virulence and fitness to sensitive strains.

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Dominance in field populations depends on several other factors, including the ability to compete pathogenically and saprophytically with sensitive strains. Virulence and fitness characteristics, such as growth rate and sporulating capacity, of resistant isolates may be useful for prediction of competitiveness with sensitive isolates (Dekker, 1982; Elmer & Gaunt, 1994).

In the case of dicarboximides several experimental studies show a contradictory picture for different pathogens. Dicarboximide-resistant isolates of Botrytis cinerea (Davis & Dennis, 1981; Raposo et al., 1996), Alternaria brassicicola (Iacomi-Vasilescu et al., 2004), Monilinia fructicola (Sztejnberg & Jones, 1979; Penrose et al., 1985), Alternaria alternata (Biggs, 1994) were found to be as competitive as sensitive ones, whereas other authors observed a lower fitness in dicarboximide-resistant strains of Botrytis cinerea (Romano et al., 1983; Hisada et al, 1984; Leroux & Clerjeau, 1985; Lorenz & Pommer 1985; Fourie & Holz, 2003), Monilinia fructicola (Ritchie, 1983; Elmer & Gaunt, 1994), Alternaria brassicicola (Huang & Levy, 1995) and Penicillium expansum (Rosenberger & Meyer, 1981; Gullino & De Waard, 1984).

In this paper we report preliminary studies on various morphological and physiological characteristics to evaluate differences among Stemphylium vesicarium phenotypes in order to clarify how the development of resistance is related to the fungus vitality. In some cases resistant strains have been found in field after several years in which dicarboximides were no longer applied, suggesting they did not have a low fitness. Materials and methods Isolates and culture conditions The isolates considered in this study were selected from a collection of 652 isolates recovered between 1995 and 2004 from symptomatic pear fruits of different cultivars (mainly Abbé Fétel, Conference but also Doyenne, Kaiser and Passe Crassane) in Po Valley.

Isolates were cultured on V8 Agar made of 20% V8 (vegetable juice, Campbell’s Grocery Ltd.), 1.5% technical agar (Agar Grade A, Becton Dickinson), 0.4% Calcium Carbonate (Fluka) in dH2O amended with 50 mg l-1 of streptomycin sulphate. They were incubated at 23°C under fluorescent light (12 hours of photoperiod). Morphological study Two hundred and seventy seven isolates (141 resistant, 136 sensitive to dicarboximides) were considered for their morphology. They were collected in two different years: 84 isolates, during the first year of observation, and 193 isolates during the second year. They were cultured on V8 agar and incubated for 7 days at 23°C under fluorescent light (12 hours photoperiod). Mycelial colour and colony shape were then evaluated. Fitness tests Fitness parameters, such as mycelial growth, sporulation, conidial germination and pathogenicity, were studied on 6 isolates, chosen for their different sensitivity to dicarboximides: 2 sensitive, 3 R1 resistant and 1 R2 resistant.

To evaluate mycelial growth, three V8 agar plates were inoculated with each isolate (5 mm diameter inverted plug from a 7 day old colony) and were incubated at 23°C under fluorescent light (12 hours photoperiod). Evaluation of mycelial growth was carried out after 3, 4, 5, 6 and 7 days after inoculation measuring two perpendicular diameters. The assays were repeated three times.

To evaluate sporulation and germination rates a conidial suspension was prepared in 10 ml of distilled water with each isolate from 7 day old plates after 2 days of 12 h alternating periods of NUV (near-ultraviolet radiation light (TL 40 W/05 Philips)) and dark (Leach,

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1967). The amount of conidia was determined twice with a haemocytometer. The spore suspensions were then diluted and 20 µl drops were placed onto a 1.5 % water agar plate. Percentages of spore germination (100 spores for each drop) were estimated under a microscope, after 1, 2 and 20 hours of incubation at 23°C, under fluorescent light. The germ tube number and the length of the longest germ tube per conidium (15 germinated spores for each drop) were evaluated after 20 hours of incubation.

To evaluate isolate pathogenicity, three pear (cv. Abbé Fétel) detached leaves per isolate were disinfected in 1% sodium hypochlorite for 30 s and rinsed in distilled water. They were then placed with the lower surface in the upper side in Petri dishes onto the surface of 1% water agar amended with 40 mg l-1 benomyl (Benlate 50WP, Du Pont) and 30 mg l-1 streptomycin sulphate. The leaves were inoculated with spore suspensions (105 conidia ml-1) obtained from 7 day old colonies after two days of 12 h alternating periods of NUV and dark. After 2 days the percentages of necrotic area were estimated. The assays were repeated three times.

All results were processed with analysis of variance (ANOVA) and significant differences were identified using the Duncan test (P≤0.05). Competitive ability tests The competitive ability between sensitive and R1 resistant isolates was determined by inoculating untreated 9 cm diameter V8 Petri dishes with 0.5 ml of defined mixture of conidia (4.5 x 102 conidia ml-1) from resistant and sensitive strains to produce ratios of 25, 50 and 75% of resistant isolates (three replicates per ratio). The same mixed suspensions were also inoculated on V8 plates amended with procymidone (10 mg a.i. ml-1). At this concentration only resistant colonies were able to develop. Inoculated plates were incubated for 5 days at 23°C under fluorescent light (12 hours photoperiod) and then for 2 days under NUV lights (12 hours of photoperiod). After the first 2 days of incubation, the R/S ratio was evaluated in each plate, comparing colonies developed on treated and untreated plates. Pure conidial suspensions of each phenotype were used as control. Plates were then sub-cultured until one of the two phenotypes became dominant. Results and discussion Morphological study The 277 isolates, considered in two years for their colour and shape, showed four different types of morphologies: Dark, Zonate with White Star-like arrangements (DZWS); Cream, Zonate with Circle arrangements (CZC); Dark, Zonate with Circle arrangements (DZC); Cream, Zonate with Dark Star-like arrangements (CZDS) (Table 1). DZWS and CZC were the most diffuse morphological types. They represent 70% among all types in the first year of observation and 86% in the second year. Differences in colony morphology of resistant isolates and wild type were not striking. DZWS appeared more frequently in resistant isolates in both years but not with the same intensity. In fact, there were 30 resistant isolates out of 36 isolates with this morphology in the first year but only 63 out of 116 in the second year. CZC, on the other hand, was the morphology with the highest percentage of sensitive isolates in both years (96% and 76% respectively). Morphology and sensitivity to dicarboximides are therefore only slightly related and further studies are needed to better clarify this relationship. Fitness tests The following fitness studies carried out with 6 isolates (2 sensitive, 3 R1 resistant and 1 R2 resistant) made it possible to evaluate mycelial growth, sporulation, conidial germination and pathogenicity of strains of different classes of sensitivity to dicarboximides.

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Table 1. Number of isolates belonging to the two sensitivity classes (R=resistant isolates, S=sensitive isolates) and to the four different types of morphologies (DZWS=Dark, Zonate with White Star-like arrangements; CZC=Cream, Zonate with Circle arrangements; DZC=Dark, Zonate with Circle arrangements; CZDS=Cream, Zonate with Dark Star-like arrangements).

First year Second year Morphology R S R+S R S R+S

DZWS 30 6 36 63 53 116 CZC 1 22 23 12 38 50 DZC 14 5 19 4 4 8

CZDS 3 3 6 14 5 19 All morphologies 48 36 84 93 100 193

Figure 1. Mycelial growth of the different phenotypes on unamended V8 medium (Evaluation at 3, 4, 5, 6 and 7 days after inoculation).

In all three phenotypes no significant differences in mycelial growth could be observed in any evaluation made 3, 4, 5, 6 and 7 days after inoculation (Figure 1). Sporulation, evaluated as the amount of conidia, and germination rates, evaluated as number of germ tubes produced by conidium and as the length of the longest germ tube of 15 conidia, also did not change according to phenotypes (Figure 2).

The ability to infect host tissue was also comparable. Sensitive, R1 and R2 resistant isolates had the same pathogenicity on untreated detached leaves, in fact leaf necrosis caused by fungal inoculation had the same areas in all three cases for all trials (Figure 2). This behaviour was comparable to what was observed in other fungi such as Alternaria alternata (Biggs, 1994) but has to be confirmed by plant and field tests that are currently in progress.

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Figure 2. Comparison of sporulation (amount of conidia), germination rates (number of germ

tube per conidium and length of the longest germ tube at 20 h) and pathogenicity on detached leaves of the different phenotypes.

Competitive ability tests R1 resistant isolates were able to overcome sensitive ones in few passages when their percentage was 50% or 75%. But if the R/S ratio was 25:75 the results were contradictory (Table 2). R1 resistant isolates thus seemed to have a higher competitive ability than sensitive ones if they represented the majority, but they did not always succeed in predominating when they were less frequent in the population. This result was consistent with what was observed in the previous fitness tests so it can be concluded that resistant isolates seem in vitro to have a fitness not lower than sensitive ones. Table 2. Number of passages in vitro to obtain a dominant sensitive or resistant phenotype

from different mixtures of sensitive (S) and resistant (R1) isolates

Percentage of resistant conidia

Number of passages on unamended medium Final dominant phenotype

75% 1 Resistant 50% 3-4 Resistant 25% 3-4 Sensitive or Resistant

The various aspects considered in this study (colony morphology, fitness and competitive

ability) contributed to a first characterization of S. vesicarium dicarboximide-resistant phenotype behaviour compared to sensitive behaviour. No striking differences were observed among phenotypes; in particular fitness and competitive ability in vitro of resistant isolates were not significantly lower. These findings were also confirmed by field observations where resistant strains have been found in some orchards after several years of suspension of dicarboximide applications.

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leaf necrotic area (% )

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References Alberoni, G., Collina, M., Pancaldi, D. & Brunelli, A. 2005: Resistance to dicarboximide

fungicides in Stemphylium vesicarium of Italian pear orchards. – European Journal of Plant Pathology. In press.

Biggs, A.R. 1994: Mycelial growth, sporulation and virulence to apple fruit of Alternaria alternata isolates resistant to irpodione. – Plant Disease 78: 732-735.

Brunelli, A., Gherardi, I. & Adani, N. 1997: Reduced sensitivity of Stemphylium vesicarium, causal agent of pear brown spot, to dicarboximide fungicides. – Informatore Fitopato-logico 47: 44-48.

Collina, M., Gherardi, I. & Brunelli, A. 2002: Acquired resistance of Stemphylium vesicarium to procymidone on pear in Italy. – Acta Horticulturae 596: 547-549.

Davis, R.P. & Dennis, C. 1981: Studies on the survival and infective ability of dicarboximide-resistant strains of Botrytis cinerea. – Annals of Applied Biology 98: 395-402.

Dekker, J. 1982: Can we estimate the fungicide-resistance hazard in the field from laboratory and greenhouse tests? – In: Fungicide Resistance in Crop Protection, eds. Dekker and Georgopoulos: 128-138.

Elmer, P.A.G. & Gaunt, R.E. 1994: The biological characteristics of dicarboximide-resistant isolates of Monilinia fructicola from New Zealand stone-fruit orchards. – Plant Pathology 43: 130-137.

Fourie, P.H. & Holz, G. 2003: Fitness on grape berries of Botrytis cinerea isolates belonging to different dicarboximide sensitivity classes. – South African Journal for Enology and Viticulture 24: 1-10.

Gullino, M.L. & De Waard, M.A. 1984: Laboratory resistance to dicarboximides and ergosterol biosynthesis inhibitors in Penicillium expansum. – Netherlands Journal of Plant Pathology 90: 177-179.

Hisada, Y., Kato, T. & Noda, C. 1984: Biological properties of procymidone-resistant field isolates of Botrytis cinerea. – Annals of the Phytopathological Society of Japan 50: 590-599.

Huang, R. & Levy Y. 1995: Characterization of iprodione-resistant isolates of Alternaria brassicicola. – Plant Disease 79: 828-833.

Iacomi-Vasilescu, B., Avenot, H., Bataillé-Simoneau, N., Laurent, E., Guénard, M. & Simoneau, P. 2004: In vitro fungicide sensitivity of Alternaria species pathogenic to crucifers and identification of Alternaria brassicicola field isolates highly resistant to both dicarboximides and phenylpyrroles. – Crop Protection 23: 481-488.

Leach, C.M. 1967: Interaction of near-ultraviolet light and temperature on sporulation of the fungi Alternaria, Cercosporella, Fusarium, Helminthosporium and Stemphylium. – Canadian Journal of Botany 45: 1999-2016.

Leroux, P. & Clerjeau, M. 1985: Resistance of Botrytis cinerea Pers. and Plasmopara viticola (Berk. & Curt.) Berl. and de Toni to fungicides in French vineyards. – Crop Protection 4: 137-160.

Lorenz, G. & Pommer, E.H. 1985: Morphological and physiological characteristics of dicarboximide-sensitive and resistant isolates of Botrytis cinerea. – Bulletin OEPP/EPPO Bulletin 15: 353-360.

Penrose, L.J., Koffmann, W. & Nicholls, M.R. 1985: Field occurrence of vinclozolin resistance in Monilinia fructicola. – Plant Pathology 34: 228-234.

Raposo, R., Delcan, J., Gomez V. & Melgarejo, P. 1996: Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses. – Plant Pathology 45: 497-505.

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Raposo, R., Gomez V., Urrutia, T. & Melgarejo, P. 2000: Fitness of Botrytis cinerea associated with dicarboximide resistance. – Phytopathology 90: 1246-1249.

Ritchie, D.F. 1983: Mycelial growth, peach fruit-rotting capability and sporulation of strains of Monilinia fructicola resistant to dichloran, iprodione, procymidone and vinclozolin. – Phytopathology 73: 44-47.

Romano, M.L., Gullino, M.L. & Garibaldi, A. 1983: Tests of competition in Petri dishes and on grapes in the laboratory between Botrytis cinerea Pers. strains sensitive and resistant to dicarboximides. – La difesa delle piante 2: 75-80.

Rosenberger, D.A. & Meyer, F.W. 1981: Postharvest fungicides for apples: development of resistance to benomyl, vinclozolin and iprodione. – Plant Disease 65: 1010-1013.

Sztejnberg, A. & Jones, A.L. 1979: Resistance of the brown rot fungus Monilinia fructicola to iprodione, vinclozolin and procymidone. – Phytoparasitica 7: 46 (Abstract).


pp. 117 - 121

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Control of brown spot of pear in organic pear orchard Loredana Antoniacci, Riccardo Bugiani, Rossana Rossi Servizio Fitosanitario (Plant Protection Service), Regione Emilia-Romagna. Via Corticella 133, I-40129 Bologna, Italy. Abstract: Due to the Reg. CEE n. 473/2002 it is necessary nowadays for farmers to reduce the amount of copper applied in the orchard. Following the Regulation, copper applied in the orchard must not exceed 38 kg/ha over the years 2002-2006 and progressively be reduced to 30 kg/ha from 2006 to 2010 and on.

Therefore the efficacy of some copper-based formulations applied at different rates and calcium poli-sulphyte were tested against pear brown spot in an organic pear orchard.

Trial was carried out over the years 2001-2004 in Emilia-Romagna region on cv. Conference particularly susceptible to Stemphylium vesicarium.

Bordeaux mixture, copper hydroxide and copper sulphate were compared. In 2001 and 2002, bordeaux mixture applied at 50 and 25 g (cu ions)/lt of and ca polysulphyte were compared. In 2003, bordeaux mixture applied at the same rates of the previous years were compared with copper hydroxide at 52,2 and 26,25 g (cu ions)/lt and tri-basic copper sulphate at the rate of 29,25 g (cu ions)/lt. In 2004 the same products were tested but only at lower rates.

Treatment started in may when a volumetric spore-trap recorded the first peak of S. vesicarium conidia and continue with a weekly interval until pre-harvest. On the whole 12, 12, 8 and 11 chemical applications were carried out in 2004, 2003, 2002 and 2001 respectively. At harvest disease incidence on fruits were evaluated.

In 2001 and 2002 disease incidence on fruits was relatively low (5,4 and 5,6 respectively on untreated plots), while in 2003 and 2004 the disease caused severe damages (21,6 and 29,5 respectively). Results show that calcium polysulphyte was not statistically different from the check. All the copper-based formulations proved to be effective (except the bordeaux mixture at 25 g/hl in 2001) but were not statistically different from each other. The highest efficacy (65%) was obtained applying bordeaux mixture at 50 g/lt but lower rates of copper (25-26 g/lt) provided at most 50% efficacy.

An average of 10-12 copper applications per season against brown spot, at the higher dose as used in the trial would deliver in the orchard from 6-7.2 hg copper. If we consider further copper applications needed to control fire blight and scab, the amount of copper distributed in the orchard will easily overcome the threshold imposed by the CEE Regulation. Key words: Stemphylium vesicarium, brown spot, organic pear, disease, integrated control Introduction Brown spot caused by Stemphylium vesicarium (Wallr. Simm.) is the one the most dangerous disease for pear cultivars most susceptible to the disease (Abbée Fétél, Conference). In organic pear production, several copper sprays are applied to contain damages on fruits. Sprays are usually carried out from may to pre-harvest at 7-10 interval depending on weather conditions. Therefore for cultivar such as Abbéè Fétél, which is harvested in the second week of september, the number of sprays and the amount of copper applied is very high.

In recent years the use of copper is going to be limited due to public concerns about its toxic accumulation in the soil. E.U. Reg.n. 473/2002 defines limitations of copper

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formulations and amount that have to be used for surface unit. In particular for annual crops, no more than 8 kg/ha/year can be applied within 31dicember 2005, and 6 kg/ha/year from 1 january 2006. On perennial crop on the other hand no more than 38 kg/ha can be applied in a five year period (23 march 2002 – 31 december 2006). Such amount must be furtherly reduced to 30 kg/ha during the following five years (2006-2010).

This situation has led, on one hand, technicians and farmers to rationalize copper dosage and applications and, on the other hand, agrochemical firms to optimize copper formulations. This work aimed to evaluate the efficacy of reduced copper doses and new copper formulations in controlling brown spot on pear.

Material and methods Trials were carried out over the years 2001-2004 in the Emilia-Romagna region on a susceptible pear cultivar (Conference). In 2001, 2002, 2003 trials were located in an organic pear orchard in Ravenna, while in 2004 it was located in Ferrara. Plot characteristic and ex-perimental design are described in table 1. Copper formulations tested are reported in table 2. Table 1. Characteristics of trial field and experimental design.

Year 2001-02-03 2004 Location Conselice (RA) Migliarino (FE) Site plain plain Farm Massari Sorgeva Cv conference conference Root-stock Self-rooted cydo Age 15 7 Training system spindle spindle Planting space (m) 4,5x2 3,8x1,3 Soil condition grass-covered grass-covered Irrigation Micro-irrigation under herd Micro-irrigation under herd Experimental design R.B.D. R.B.D. Replicates (n.) 4 4 Plants/plot (n.) 5 7 Distribution equipment Back-sac Atomizer KWH Back-sac Atomizer KWH Water distributed (l/ha) 14 12

Chemical applications started in may as soon as volumetric spore trap (Lanzoni VPPS 2000) recorded the first peak of 30 spore per day. Sprays were carried out until pre-harvest with 5-15 days interval depending on weather conditions and disease pressure. On the whole, 11, 8, 12 and 12 sprays were carried out in 2001, 2002, 2003 and 2004 (Tab.3).

At harvest, the disease incidence on fruits was estimated (percentage of affected fruits). Data were arcsine transformed and analysed using ANOVA and S.N.K. mean separation test. Table 2. Characteristics of products used in several years.

Year Formulation Active ingredient (% o g/l ) Rate Rate a.i.

119

a.i. formulation (g-ml/hl)

(Cu++ ion)(g/hl)

2001-02-03 Poltiglia Bordolese Disperss

Bordeaux mixture 20 250 50

2001-02-03-04

Poltiglia Bordolese Disperss

Bordeaux mixture 20 125 25

2003 Kocide 2000 Copper hydroxide 35 150 52,5 2003-04 Kocide 2000 Copper hydroxide 35 75 26,25 2003-04 Cuproxat Tribasic copper

sulphate 195 150 29,25

2001-02 Polisolfuro di Ca Polisenio

Ca polisulphite 380 1300 --

Table 3. Timetable of treatments and harvest date.

Year Treatments date Harvest date 2001 4-9-19-25/05; 4-17-24/06, 3-10-17-26/07 21/08 2002 22-27/05; 5-12-26/06; 2-18-24/07 24/08 2003 6-12-19-22-30/05; 5-12-20-30/06; 7-15-22/07 18/08 2004 24/05; 1-3-10-18-25/06; 2-9-16- 23-30/07; 6/08 26/08

Results and discussion In 2001 and 2002 disease pressure was not heavy. The percentage of affected fruits in the unsprayed plot was 5.4 and 5.6 in 2001 and 2002 respectively. On the contrary, in 2003 and 2004, 21.6 and 29.5 percent of fruits were affected in the unsprayed plot respectively.

The analysis of the data showed that calcium polysulphite tested in two years, was not effective in the disease control. All copper formulations at different dosages proved to be affective compared with the unsprayed check but not among them (except bordeaux mixture at the dose of 25 g/hl of ion copper in 2001).

However, a tendency of efficacy reduction with dosage reduction was always recorded. Besides, the best efficacy (65%) was obtained with bordeaux mixture at 50 g copper ion

/hl. Copper applications at dosages of 25-26 g/hl never gave more than 50% of efficacy. On the other hand, considering 10-12 average sprays with a volume of 12/ha, the use of dosages of 50 g ion copper /hl cannot be feasible in that 6-7.2 kg of copper would be applied.

To these, further copper sprays should be carried out during the season to control scab and fire blight. It is therefore clear that the seasonal copper limitation stated by the European regulation would be easily overcome.

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Figure 1. Efficacy of different rates of copper formulation and the calcium polisulphite

against pear brown spot.

Figure 2. Efficacy of different copper formulations and copper polysulphite to control pear brown spot.

At present, copper application is the best active substance against pear brown spot in organic pear orchards. In Emilia-Romagna, with weather conditions conducive for the disease the use of copper to contain brown spot is needed. However it has to be wisely managed in

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order to maintain the amount of product distributed, within the limits defined by the European regulation.

A useful disease control strategy in organic pear production, taking into account the effect dosage/efficacy, would be that to apply the highest dose (50 g/hl ion Cu++) only when weather conditions are favourable for disease development and disease pressure is high.

With this regard, BSP-Cast model (Llorente et al., 2000) could be very useful to time correctly copper applications. References E.U.Reg. n. 473/2002 European Commission, (2002). Llorente, I., Vilardell, P., Bugiani, R., Gherardi, I. & Montesinos, E. 2000: Evaluation of

BSPcast Disease Warning System in reduced fungicide use programs for management of brown spot of pear. – Plant Disease 84: 631-637.


pp. 123 - 127

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Screening of organically based fungicides for apple scab (Venturia inaequalis) control and a histopathological study of the mode of action of a resistance inducer Marianne Bengtsson, Hans J. Lyngs Jørgensen, Anh Pham, Ednar Wulff, John Hockenhull Section for Plant Pathology, Department of Plant Biology, The Royal Veterinary and Agricultural University (KVL), Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. E-mail: [email protected] Abstract: A range of possible substitutes for copper-based fungicides for control of apple scab (Venturia inaequalis) in organic growing were tested in laboratory and growth chamber experiments in the Danish project StopScab (2002-2004). Eighteen crude plant extracts, 19 commercial plant-based products and 6 miscellaneous compounds were tested for their ability to reduce scab symptoms on apple seedlings. Most of the compounds were also tested for their effect on conidium germination on glass slides. Fourteen of the crude plant extracts, 13 of the commercial plant products and 5 of the miscellaneous compounds showed promising control efficacies when used either preventively or curatively in the plant assay. A histopathological study was carried out on the mode of action of the resistance inducer, acibenzolar-S-methyl (ASM), which reduced scab severity and sporulation on apple seedlings in several plant assays when applied as preventive treatment. The effect of the inducer on key pre- and post-penetration events of V. inaequalis was studied and compared to these events in water-treated control leaves. The histopathological study showed that the inducer had its strongest effect on post-penetration events indicated by delayed infection and reduced stroma development. In addition, a small but significant inhibition of conidial germination and a stimulation of germ tube length were observed. This investigation provides new histopathological evidence for the mode of action of ASM against V. inaequalis and serves as a model for evaluation of the mechanisms by which the organically based fungicides reduce infection of V. inaequalis. Key words: apple scab control, acibenzolar-S-methyl, botanical fungicides, induced resistance, organic growing, plant extracts, histopathological study, screening Introduction Apple scab (Venturia inaequalis (Cke.) Wint.) causes serious losses in quality and yield of organically as well as conventional grown apples. No effective eradicative or curative fungicides for apple scab control are presently available for use by organic growers, and while protective, copper-based fungicides and lime-sulphur are allowed in most European countries, only elemental sulphur is permitted in Denmark. Since sulphur is not very effective against apple scab, and the use of copper fungicides will be phased out in Europe within the next few years, alternative fungicides for apple scab control are increasingly needed.

As a part of the Danish research project StopScab (2002-2004) (Bengtsson & Hocken-hull, in press; Bengtsson et al., 2004) we have been searching for and screening for organi-cally based fungicides to control V. inaequalis in laboratory, growth chamber and green house experiments. Two routine screening systems were used, one testing the effect of materials on conidium germination on glass slides, and one testing the effect of compounds on disease severity on apple seedlings. As a model for evaluation of the mechanisms by which

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organically based fungicides reduce infection by V. inaequalis, a histopathological study was carried out on the mode of action of the resistance inducer, acibenzolar-S-methyl (ASM). Materials and methods Fungal inoculum Conidia of a mono-conidial isolate of V. inaequalis were produced by the bottle wick method modified from Williams (1978). The concentration of harvested conidia was adjusted with sterile water to 1.5 x 105 conidia/ml, and the conidial inoculum was stored at –18°C until use. Plant material Apple seedlings were produced from seeds of Malus x domestica cv. ‘Golden Delicious’ (Eichenberg, Germany) and grown in small pots in a growth chamber at 15-16°C under cycles of 12 h light and 12 h darkness. Preparation of test solutions Water extracts and solutions (w/v or v/v) of test compounds, including crude plant extracts, commercial plant-based materials and miscellaneous commercial compounds were prepared according to protocols supplied by companies or based on literature studies. Water and elemental sulphur (0.27% (w/v); Kumulus S, BASF) were used as standard control treatments. Glass slide test Different concentrations of selected compounds were tested for effect on conidial germination. Inoculum of V. inaequalis was mixed with test solutions and incubated at 18°C. After 24 and 48 hours, the percentage of germinated conidia was assessed using a light microscope and compared to germination in the water control, which was set to 100 %. Plant assay Seedlings with four to six leaves were used and each treatment consisted of eight plants. Test solutions were applied to the adaxial leaf surface with a hand sprayer 1-3 days before inoculation (preventive treatment) or 1 day after inoculation with V. inaequailis (curative treatment). Plants were incubated in darkness under a clear plastic cover for 24 hours after the preventive / curative treatment and for a further 48 hours after inoculation. Disease severity was assessed 14 days after inoculation following a scale 0-7 (Croxall et. al., 1952; Parisi et al., 1993): 1 =: 0% < percentage of scabbed leaf surface (sls)< 1%; 2 = 1%<sls<5 %; 3 = 5%< sls< 10%; 4 = 10< sls< 25%; 5 = 25%<sls<50%; 6 = 50%<sls<75%; 7 = 75%<sls. Histopathological study Apple seedlings were sprayed with 200 ppm acibenzolar-S-methyl, ASM (Bion WG 50 TM, Syngenta Crop Protection) or water. Three days later, the adaxial leaf surfaces were inoculated and plants were incubated as described above. The 2 youngest leaves from each of 4 replications were harvested either 1 or 5 days after inoculation. Clearing was carried out by placing the leaves on absorbent paper saturated with a fixative (96% ethanol, 100 % acetic acid; 24:1 v/v) in closed plastic containers. The cleared leaves were mounted in lactic acid and examined using light microscopy. For each treatment the overall germination rate was determined and the following events were examined for 200 germinated conidia: formation of appressoria, cuticle penetration, formation of primary stroma, runner hypha and secondary stroma. In addition, the length of germ tubes and average length of runner hyphae per stroma were determined. All data were analyzed using SAS, version 8.2 (SAS Institute, Cary, NC). Continuous variables were analyzed by analysis of variance assuming a normal distribution. Discrete variables were analysed by logistic regression, assuming a binomial distribution.

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Table 1. Overview of test results of different materials in the conidial germination test and plant assay screening for apple scab control.

Glass slide test Plant assay

Type of material

No. tested

No. with moderate /

strong effecta

No. tested

No. with no

efficacy

No. with some

efficacyb

No. with promising efficacyc

No. with high

efficacyd

Crude plant extracts 9 6 18 3 1 14 0

Commercial botanical products

14 12 19 4 2 9 4

Other commercial compounds

3 2 6 1 0 3 2

Total 26 20 43 8 3 28 6 a Compounds able to reduce conidial germination or affect germ tube growth at different

concentrations. b Compounds showing a significant protective or curative effect in at least one plant assay. c Compounds showing a highly significant protective or curative effect in at least one plant assay. d Compounds repeatedly showing highly significant effect in several plant assays.

ASM

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Figure 1. Efficacy (%) of ASM (100, 200 and 400 ppm) and sulphur (0.2, 0.4 and 0.8 %) in

reducing germination of conidia in the glass slide test 24 and 48 hours after start of incubation. Bars represent standard deviations of means.

Results and discussion Screening assays Most of the materials tested in the glass slide test reduced conidial germination either moderately or strongly (Table 1). Twenty-eight materials, mainly crude plant extracts and commercial plant-based products, showed promising efficacy to control apple scab in the

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plant assay screenings and four showed high efficacy in this assay (Table 1). Two of the miscellaneous compounds, namely sulphur (Kumulus S) and acibenzolar-S-methyl, ASM, consistently showed repeatedly high efficacy in reducing apple scab severity when applied as protective treatments. Sulphur strongly reduced conidium germination in the glass slide test, while ASM did not (Figure 1). Histopathological study The histopathological observations of key infection events in treated leaves sampled one day after inoculation are presented in Table 2 and the observations made 5 days after inoculation are presented in Table 3. Table 2. Effect of ASM on pre-penetration events of Venturia inaequalis on apple leaves,

determined 1 day after inoculation.

Key events: Control (water) ASM Percent conidia germinated 60.0 45.0*** Average length of germ tubes (µm) 23.1 29.5*

***=significant at P<0.001, and *=significant at P<0.05.

The ASM treatment showed a highly significant inhibition of conidial germination and a positive effect on germ tube length on leaves one day after inoculation (Table 2). Although less pronounced, the inhibitory effect on conidial germination was also found on the fifth day after inoculation (Table 3). This result is in sharp contrast to the results obtained in the conidial germination tests carried out on glass slides, where the inhibitory effect of ASM on spore germination (Figure 1) was very small. Strong effects of ASM were observed on penetration and post-penetration events as the infection process was delayed and development of primary and secondary stroma was reduced. Thus, inhibition of germination on leaves treated with ASM could be interpreted as an early expression of induced resistance. This, together with the observation that ASM did not inhibit conidial germination to any notable extent in the glass slide test, lead us to conclude that ASM mainly acts against the apple scab pathogen by the mechanism of induced resistance (Jørgensen et al., 2004). The developed system can serve as a model for evaluation of the mechanisms by which organically based fungicides reduce infection of V. inaequalis. Table 3. Effects of ASM on pre- and post-penetration events of Venturia inaequalis conidia

on apple leaves, determined 5 days after inoculation.

Key events: Control (water) ASM Germinated conidia (%) 58.0 50.5* Conidia forming appressoria (%)a 94.5 86.0** Conidia attempting penetration (%)a 87.5 62.5*** Appressoria attempting penetration (%) 86.5 76.1*** Appressoria producing primary stromata (%) 59.2 36.2*** Conidia producing runner hyphae (%)a 50.5 27.0*** Number of runner hyphae formed per stroma 5.6 4.3* Conidia forming secondary stromata (%)a 13.0 2.5**

***=significant at P<0.001, **=significant at P<0.01, and *=significant at P<0.05. a: Events based on 200 germinated conidia

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Acknowledgements This work was supported by The Danish Research Centre for Organic Growing (DARCOF II). We thank Karin Olesen and Susanne Kromann Jensen for assisting in inoculum and seedling production and the glass slide test. ASM was kindly supplied by Syngenta Crop Protection. References Bengtsson, M. & Hockenhull, J. 2006: Control of apple scab (Venturia inaequalis) in organic

apple growing. StopScab: a Danish research programme for screening substitutes to copper fungicides. – IOBC /wprs Bulletin 29(1): 1-3.

Bengtsson, M., Wulff, E., Pedersen, H. Lindhard, Paaske, K., Jørgensen, H.J.L. & Hocken-hull, J. 2004: New fungicides for apple scab control in organic growing. – Newsletter from Danish Research Centre for Organic Farming, September, No 3. http://www.darcof.dk/enews/sep04/scab.html.

Croxall, H.E., Gwynne, D.C. & Jenkins, E.E. 1952: The rapid assessment of apple scab on leaves. – Plant Pathology 1: 39-41.

Jørgensen, H.J.L., Bengtsson, M., Wulff, E. & Hockenhull, J. 2004: Control of apple scab by use of the plants own defence mechanisms. Newsletter from Danish Research Centre for Organic Farming, DARCOFenews, June, No. 2. http://www.darcof.dk/enews/june04/defence.html.

Parisi, L., Lespinasse, Y., Guillaumés, J. & Krüger, J. 1993: A new race of Venturia inaequa-lis virulent to apples with resistance due to the Vf gene. – Phytopathology 93: 533-537.

Williams, E.B. 1978: Handling the apple scab organism in laboratory and greenhouse. – Proceedings of Apple and Pear Scab Workshop, Kansas City, Missouri, July 1976. New York State Agricultural Experiment Station, Geneva. Special Report 28: 16-18.


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Development of an integrated pest and disease management system for apples to produce fruit free from pesticide residues – Aspects of disease control Angela Berrie, Jerry Cross East Malling Research, New Road, East Malling, Kent, ME19 6BJ, United Kingdom Abstract: In experiments aimed at improving dormant season disease control in apple orchards, observations on time of leaf fall showed that in Cox this was generally complete by end of November compared to Bramley and Jonagold where leaves could still be present on trees at the end of December. Leaves dipped in captan degraded slowly compared to untreated leaves. Copper oxychloride and pyrifenox also delayed leaf rotting. Other trials showed that powdery mildew overwintering in buds could be reduced or eliminated by spraying a surfactant when the trees were fully dormant. In a large plot replicated orchard experiment, over four seasons, the pest and disease control achieved in managed plots, based on a zero pesticide residue management system was compared with that in plots sprayed conventionally or left untreated. The zero pesticide residue management system (MS/MR) is based on the use of conventional pesticides (excluding organophosphorus insecticides) up to petal fall and after harvest, but using biocontrol for dealing with pests and sulphur or cultural methods to control powdery mildew and storage rots between petal fall and harvest. Dormant season control of scab and powdery mildew is a key component of the system. Over the four seasons the control of scab, powdery mildew and storage rots achieved by the MS /MR system was as good as or better than that in the conventionally treated plots and at lower cost. No residues of pesticides applied to the MS /MR plots were detected in harvested fruit. Key words: Apple scab, powdery mildew, storage rots, fungicide, dormant season treatment, surfactants Introduction UK consumers expect perfect apples, but in recent years this requirement has extended to perfect apples that contain no pesticide residues. The main UK apple varieties Cox, Gala, Jonagold and Bramley are susceptible to all the major pests and diseases and the UK climate ensures that one or other of these problems are significant in most seasons. So to satisfy this consumer requirement is a very high expectation. Most apples in the UK are produced using integrated pest and disease management, and this ensures that pesticide use is targeted and therefore minimised so that residues, if present in the harvested crop, are well below the Maximum Residue Limit (MRL) permitted. However, consumers no longer trust science and to them the scientific facts of MRLs are irrelevant and the presence of any pesticide residue in apples is unacceptable. The challenge is therefore to develop crop protection systems that satisfy the consumer, but that are also profitable and sustainable for the grower. Other researchers have tackled the problem of the high pesticide input into apple crops (Wilcox et al., 1992; Jones et al., 1993). These studies concentrated on disease control only and their approach was to focus on reduced fungicide inputs in the growing season. The approach tackled here is to develop production methods for pest and disease control where use of conventional pesticides is restricted to pre petal fall and after harvest to minimise overwintering inoculum. Cultural, biological and other non chemical methods could then be

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used to control pests and diseases during the apple development period in summer. Such an approach may be achievable since many important pests and diseases on apple such as scab or caterpillars could be controlled during the dormant season and/or pre bloom. There are however, certain pests and diseases such as powdery mildew, storage rots and rosy apple aphid that are mainly active post bloom and non chemical methods of control would need to be developed to effectively control these problems in such a residue-free system. This project was initiated to determine whether this approach to residue-free production was feasible and to identify which pests and diseases were likely to be difficult to control. Preliminary results for pests and diseases have been reported (Berrie & Cross, 2005). This paper will concentrate on disease control in the system, in particular dormant season control of scab and mildew and control of storage rots. Material and methods Dormant season disease control Apple scab: Apple scab overwinters as the sexual state on leaves on the orchard floor and this is probably the major source of inoculum in spring. Elimination of overwintering leaf litter is therefore one of the key factors in the integrated approach to scab control. A spray of 5% urea can be applied post harvest before leaf fall to reduce or eliminate the overwintering inoculum (Burchill et al., 1965). The success of this is dependent on time of leaf fall and speed of leaf degradation which is dependent on apple variety, post harvest treatment and weather conditions during autumn and winter.

In 1998-2000 trees of Cox, Fiesta, Gala, Jonagold and Bramley’s Seedling in orchards at East Malling were monitored weekly between October and December and the stage of leaf fall recorded. Experiments were also established over the same period to examine the effect of variety and chemical treatment on leaf degradation. For each experiment leaves of Cox, Gala, Fiesta, Jonagold and Bramley were collected from apple orchards at East Malling in the first week of November and stored at 4 ºC in a fridge until required. In December the leaves were spread in batches of 30 on the surface of the ground herbage under the trees in a mixed variety apple orchard at East Malling, in a fully replicated randomised block design. Each batch (a plot) was held in place by rigid plastic netting which was secured to the ground by metal pins. The number of leaves remaining under each net was assessed at roughly monthly intervals from the time of treatment and mean values calculated. In a second experiment in December, aqueous solutions were prepared of the test treatments (Table 1). Four replicate nets of 30 leaves of cv Cox or Bramley’s Seedling were dipped into each chemical for half a minute, stirring to ensure thorough mixing. The leaves in their nets were allowed to dry overnight. Four replicate nets of leaves were also left untreated. The day after treatment, the leaves were spread in batches of 30 on the surface of the ground herbage under the trees as described above. The number of leaves remaining under each net was similarly assessed. Powdery mildew: Powdery mildew overwinters as mycelium in fruit buds or vegetative buds that emerge as mildewed blossoms at pink bud or mildewed shoot tips at petal fall. Spores from these infect developing flowers, leaves and shoots and initiate the secondary mildew epidemic. Successful control of powdery mildew is dependent on maintaining the overwintering primary mildew at a low level (<0.5% mildewed blossoms or shoots). Frick & Burchill (1972) and Bent et al. (1977) showed that surfactants applied in the dormant season could eradicate mildew from apple buds and hence eliminate primary mildew from orchards, improving mildew control the following spring and reducing the need for costly fungicide programmes. The research resulted in the development of Dormakill (PPP222 – ICI Ltd), for use in commercial orchards. Unfortunately this product is no longer available. Since the

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original research considerable development has taken place in types of surfactants, particularly in the development of silicone-based surfactants. These may have increased penetration into the plant and may also be active at lower doses and less phytotoxic.

In winter 2004, when the apple trees were fully dormant, three separate small plot trials were established to evaluate the efficacy of surfactants in eradicating powdery mildew overwintering in apple buds. The treatments and results for one of these trials is reported here. On 9 February 2004, single tree plots of Cox on M9 rootstock (orchard CW120/121) were treated with a range of surfactants including non-ionic surfactants (e.g. Agral, Activator 90) and organo silicon surfactants (e.g. Silwett L-77), at concentrations of 1.5 or 3%, at 500L/ha, using a Hardi MRY motorised knapsack sprayer. An untreated control was included. Treatments were replicated four times in a randomised block design. Primary blossom mildew was assessed at pink bud as percentage mildewed blossoms, on the whole tree by recording the total number of blossoms present and the number with powdery mildew. Similarly at petal fall, the percentage vegetative primary mildew was recorded from the total number of vegetative shoots on each tree and the number infected with mildew. In addition the effect of treatments on tree development was recorded by noting the stage of development of 50 fruit buds on each tree plot prior to flowering. The effect of treatments on fruit set was assessed by recording the total number of flowers on two marked branches and then later in June the resulting number of fruits. Any obvious phytotoxicity resulting from treatments, such as leaf distortion, flower distortion or leaf discoloration, were also noted. Disease control post blossom Control of powdery mildew post blossom – alternatives to sulphur: Currently the residue-free apple programme relies on the use of sulphur post blossom for the control of secondary powdery mildew on susceptible varieties such as Cox and Gala and alternative methods of control are required. In 2002 in a mature apple orchard of cv. Cox on M9 rootstock and where the incidence of powdery mildew was known to be high, the efficacy of potassium phosphite (Farmfos) (5l/ha) in controlling powdery mildew on apple shoots was compared to that of potassium bicarbonate (5kg/ha + 0.1%Agral) or benzothiadiazole (Bion) (400g/ha) in small plot (4 tree plots). All products were applied at 500L/ha using a self-propelled small plot orchard sprayer (Solo). Sulphur (5l/ha) and an untreated were included as controls. Treatments were applied at two week intervals from petal fall and the mildew recorded on the youngest five leaves on ten shoots per plot also at two week intervals. The trial was repeated in 2003. Control of storage rots: Cultural control and rot risk assessment were the main methods used to minimise losses in store due to rots. Previous work has shown that cultural methods of control such as mulching the soil surface or selective picking (only fruit >0.5m above the ground harvested for storage) were effective in controlling Phytophthora rot (Phytophthora syringae) in stored Cox apples (Berrie & Luton, 1996). Other cultural methods such as removal of cankers in summer or of brown rotted fruit before harvest, though not effective alone, will contribute to reducing inoculum in an integrated approach to disease control. Rot risk assessment (Berrie, 2000; Cross & Berrie, 2001) was developed to determine the risk of rotting in an orchard prior to harvest to decide on treatment need or on the storage potential of the fruit based on the likely losses in store due to rots. Evaluation zero residue management system Experimental plan: The trial was set up at East Malling where use could be made of existing established plots (planted in 1995) of disease susceptible apples (Cox, Gala, Fiesta)

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and scab resistant apples (Saturn, Ahra,). The variety Discovery was common to both sets of plots. Each plot consisted of 144 trees on M9 rootstock and was separated from adjacent plots by alder windbreaks. In these plots the pest and disease control achieved following a routine conventional pesticide programme was compared to that achieved following the zero pesticide residue management system (MS / MR). Untreated plots of disease susceptible and resistant varieties were included (Table 1). Each treatment was replicated twice in a randomised block design. Only the disease control aspects are reported here. Treatments: Zero residue management system (MS, MR) Scab control in MS plots pre bloom is based on a pre bud burst spray of copper oxychloride and use of conventional fungicides (dithianon, captan, myclobutanil) with ADEM key stage system (Berrie & Xu, 2003) to determine frequency of spraying. The final conventional fungicide spray is applied at the key stage of petal fall. In MR plots scab sprays are applied only at bud burst and petal fall. Post bloom sulphur sprays are used for mildew control in both MS and MR plots. Post harvest (MS and MR plots), sprays of DMI fungicides and 5% urea are applied to limit the development of late scab and the overwintering scab sexual state and encourage leaf rotting.

Pre bloom, control of powdery mildew is based on removal of infected blossoms and shoots promptly at pink bud and petal fall and the use of DMI fungicides to suppress sporulation. Post bloom the mildew incidence is monitored weekly and the information used with ADEM to make decisions on sulphur use. Post harvest, DMI sprays are used to control mildew on late shoot growth. Any mildew overwintering in silvered shoots is removed during winter pruning.

Control of storage rots was achieved by a combination of cultural measures and rot risk assessment as described above (Table 1).

Conventionally treated system (CS, CR) In the conventional system control of scab and mildew was based on a routine fungicide (dithianon, captan, myclobutanil, bupirimate) programme applied at ten day intervals from bud burst to harvest. In CR plots sprays for scab control were applied only at bud burst and petal fall. Captan, applied 28 and 14 days pre harvest was used for control of storage rots. Untreated (US, UR) No pest or disease controls were applied. All treatments received the standard programme for nutrients (including calcium sprays) and for weed control. Assessments Diseases were assessed using standard methods (Cross & Berrie, 1995) at standard IPM timings of green cluster/pink bud, petal fall and at monthly intervals to harvest in order to make decisions on pesticide use and to assess the success of the management systems. At harvest fruit yield and disease incidence were assessed on the fruit from ten trees per variety per plot. Fruit quality (size and russet) were assessed on a random sample of 100 fruit per variety per plot. At harvest, fruit from each treatment (at least one bulk bin of fruit per variety per plot) was stored in a commercial controlled atmosphere (3.5ºC; 1.25% O2; < 1% CO2) store and the incidence of rotting recorded in February or March. During the trial, records were kept of timings for cultural, monitoring and other management inputs such that an economic appraisal of the systems could be made.

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Table 1. Zero pesticide residues in apples: treatments, pesticide programmes and varieties.

Treatment Pesticide use Varieties IPDM programme US Untreated Susceptible

Cox, Gala, Fiesta, Discovery

None

CS Conventional Susceptible Cox, Gala,

Fiesta, Discovery

Routine pesticides Captan (28 & 14 days pre harvest) for

storage rot control MS Managed Susceptible

Cox, Gala, Discovery

Managed pesticides early and after harvest. Biocontrol during fruit development Rot risk, selective picking, inoculum

removal for storage rot control UR Untreated Scab-resistant

Saturn, Ahra, Discovery

None

CR Conventional Scab-resistant Saturn, Ahra,

Discovery

Routine insecticides and mildewicides. Reduced scab fungicide programme

Captan (28 & 14 days pre harvest) for storage rot control

MR Managed Scab-resistant Saturn, Ahra,

Discovery

Managed pesticides early and after harvest. Biocontrol during fruit development Rot risk, selective picking, inoculum

removal for storage rot control Results and discussion Dormant season disease control Apple scab: The results of observations on the time of leaf fall for various apple varieties showed that leaf fall on Cox was generally complete about 2-3 weeks before that on the other cultivars and generally by the end of November. Bramley and Jonagold were usually the last to drop their leaves but this was complete usually by end of December. In experiments established to investigate the effect of variety and chemical treatment on leaf degradation, Cox rotted most rapidly and Bramley and Jonagold were slowest to break down. Cox leaves treated with 5% urea were first to degrade in all years and those treated with captan the slowest. Copper and pyrifenox also delayed leaf rotting, particularly in Cox. However, in 1998/99 season conditions were favourable for degradation and most leaf litter had disappeared before bud burst in March (Table 2). In 1999/2000, similar results were obtained, but leaf degradation was slower and significant numbers of leaves still remained in April after bud burst. Post harvest treatments most often used in UK apple orchards include urea for scab control and copper oxychloride for control of apple canker. Use of copper may delay leaf degradation, particularly in varieties like Bramley or Jonagold where leaf degradation is slow. Careful consideration must therefore be given to choice of fungicide and products such as captan or copper fungicides avoided.

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Powdery mildew: The percentage of mildewed blossoms and shoots was recorded in May. The incidence of primary blossom mildew was significantly reduced compared to the untreated by Activator (Non-ionic wetter), Agral (Non ionic wetter), Mixture B (Non ionic wetter). Designer (organo silicon wetter) and Silwett (organo silicon wetter) at the higher concentration and also by Agral and Silwett at the lower concentration. However, despite the significant effect of these treatments, the incidence of primary blossom mildew was still unacceptably high (3% mildewed blossoms for Silwett at 3% concentration compared to 12.7% mildewed blossoms in untreated). In general use of the surfactants at the higher concentration was more effective in reducing primary blossom mildew. Silwett appeared to be the best surfactant overall, but it was not significantly better than Agral or Mixture B. Similarly the incidence of primary vegetative mildew was significantly reduced compared to the untreated by all. These results have shown that overwintering mildew can be reduced by dormant season sprays of surfactants. Further work is required to examine the effect of including fungicides with the surfactants, which could improve efficacy at lower concentration of surfactant. However, use of surfactants in the dormant season does offer a means of reducing or eliminating primary mildew and avoiding the need for intensive fungicide programmes to control mildew post blossom. Table 2 Effect of chemical treatment applied to detached leaves in December on rate of leaf

degradation in Cox and Bramley in 1998.

Apple variety / Mean % leaves left mid Feb 1999 Chemical treatment Active ingredient

Cox Bramley’s Seedling Untreated – 9 33 Cuprokylt copper oxychloride 42 39 Dorado pyrifenox 18 42 urea urea 0 2 Captan captan 69 84

Disease control post blossom Control of powdery mildew post blossom – alternatives to sulphur: Disease incidence was assessed on six occasions in 2002 and in the untreated control varied from 54-91% mildewed leaves. None of the spray treatments gave complete control of powdery mildew. Sulphur was the most effective with mildew incidence varying from 22-39% mildewed leaves and commercially unacceptable. Mildew incidence in plots treated with Bion, Farmfos or potassium bicarbonate was similar, generally less than in untreated plots, and varied from 32-80% mildewed leaves. None of the test products gave satisfactory control of mildew, although mildew incidence was reduced compared to untreated plots. Similar results were obtained in 2003. Other researchers have reported promising results with these products, but where the mildew risk is high their use alone will be inadequate. Further trials will be conducted using combinations of products with sulphur.

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Table 3. Fungicides applied to managed (MS, MR) and conventional (CS, CR) plots in 2002. Treatments were applied at the recommended dose unless stated.

Treatment / number of sprays (% dose)

Fungicide / Timing CS CR MS MR

Pre Bud Burst copper 0 0 1 1

dithianon 2 1 2 1 myclobutanil +captan 4 0 4 1

myclobutanil 0 2 0 2 captan 0 0 1(50) 0

Bud Burst – Petal Fall

bupirimate 0 3 1(50) 3 myclobutanil + captan 4 1 0 0

myclobutanil 0 0 0 0 bupirimate 4 7 0 0 captan 4 2 0 0

Petal Fall – harvest

sulphur 0 0 9 (30-50) 9(30-50) myclobutanil + captan 0 0 1 1 Post harvest

urea 0 0 1 1 Cost £/ha 384 304 262 217

Evaluation zero residue management system The fungicide programme applied to plots in 2002 (Table 3) is given as an example. Fungicides applied in 2001, 2003 and 2004 were similar, apart from a pre bud burst spray of Cuprokylt (copper oxychloride) applied to managed plots in 2002-2004, but not in 2001. This treatment was applied to control scab overwintering on the tree. Up to ten sprays of sulphur, none at the full dose, were applied to managed plots in all years. These treatments appeared to give adequate control of mildew, as the incidence of primary mildew in these plots the following season (2002-2004) was negligible. Eventually it is hoped that alternative strategies can be developed for mildew control, as described above, to replace or minimise the use of sulphur. The cost of fungicides in the managed programmes was 30 to 40% cheaper than in the conventional systems.

The incidence of scab and powdery mildew in 2002 and 2004 is presented in Table 4. In both 2002 and 2004, scab control in the MS plots was better than that achieved in the routine sprayed plots (CS), with 0.1-2.7% of Gala fruits scabbed at harvest compared to 2.4-5.6% scabbed fruit in CS plots and more than 89% on fruit from untreated plots. The weather in 2002 and 2004 was favourable for scab, with high scab risk at bud burst in March and from mid blossom onwards. In both 2001 and 2003 there was a similar pattern with scab control as good as or better in MS plots than that in CS plots. No scab was recorded on the resistant varieties Saturn and Ahra in the sprayed plots. The incidence of scab in MS plots has consistently remained at negligible incidence, despite the absence of specific sprays for scab control in the post blossom period. The most likely reason for this are the treatments applied post harvest to minimise inoculum carryover.

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Table 4. Incidence of powdery mildew and scab in managed (MS, MR), conventionally treated (CS, CR) and unsprayed (US / UR) plots in 2002 and 2004.

2002 2004

Treatment / cultivar

Primary mildew

May

% mildewed

shoots June

% scab

shoots July

% scab infected

fruit harvest

Primary mildew

May

% mildewed

shoots June

% scab

shoots Sept

% scab infected

fruit harvest

Cox 0 65.0 97.5 43.2 1.8 87.5 100.0 48.1 US Gala 0 95.0 100.0 98.0 0.5 100.0 100.0 89.3 Saturn 0 100.0 0 0 0.4 100.0 0 0 Ahra 3.3 92.5 0 0.5 26.9 100.0 0 0

UR

Discovery 0 20.0 0 1.6 0.1 42.5 1.3 0.3 Cox 0 7.5 2.5 1.4 0 10.0 3.8 0.4 CS Gala 0 12.5 7.5 5.6 0 15.0 5.0 2.4 Saturn 0 7.5 0 0 0 5.0 0 0 Ahra 0.1 0 0 0 0 0 0 0

CR

Discovery 0 0 0 0 0 0 0 0 Cox 0 0 0 0.4 0 12.5 0 0.05 MS Gala 0 17.5 0 2.7 0 10.0 0 0.1 Saturn 0 2.5 0 0 0 7.5 0 0 Ahra 0.8 10.0 0 0 0 10.0 0 0

MR

Discovery 0 0 0 0.1 0 5.0 0 0.5

Mildew control in the MS / MR plots was also similar to that in the CS / CR plots, and did not exceed 20% of shoots mildewed. The primary mildew in managed plots at the start of 2002, 2003 and 2004 was negligible, indicating that the system was not resulting in an increase in overwintering mildew.

Sooty blotch (Gloeodes pomigena) and leaf / fruit spot ( Phoma sp) were also recorded in the trial, mainly in unsprayed plots and mainly on the varieties Saturn and Fiesta. Storage rots In 2001, pre harvest rot risk assessment identified a risk of Phytophthora rot (Phytophthora syringae) in the later harvested varieties (Gala and Saturn). This was effectively controlled by selective picking (only fruit > 0.5m above the ground picked for storage) in the MS / MR plots and a pre harvest captan spray in the CS / CR plots. Up to 4% rotting due to Phytophthora was recorded in Gala and Saturn from untreated plots. Phytophthora incidence was negligible in the earlier harvested varieties (Cox, Fiesta, Ahra), where no risk had been identified. This approach for control of storage rots was continued in subsequent seasons. The results from 2003/2004 for Cox and Gala are shown in Table 5. Weather conditions pre harvest were dry and hence the risk of Phytophthora rot negligible. No Phytophthora rot was recorded in stored fruit (Table 5). Brown rot (Monilinia fructigena) was the main rot identified and this was adequately controlled by selective picking at harvest in the MS / MR plots. Nectria (Nectria galligena) and Gloeosporium rots (Gloeosporium sp.) were also recorded at quite high incidence in the untreated plots and are probably not well controlled by selective picking. Studies are now in place to identify control strategies for these rots based on controls applied during blossom and at petal fall.

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Table 5. Mean % losses due to rots in apples cvs Cox and Gala harvested September 2003 from conventional (CS, CR), managed (MS, MR) and untreated plots (US, UR) and stored in CA ( 1.25% O2 <1% CO2) at 3.5oC until mid March 2004.

Cox Gala Fungal Rot US CS MS US CS MS

Monilinia fructigena 16.0 2.7 1.7 12.6 0.2 0.6

Botrytis 0.3 0.1 0 1.1 0.02 0.06 Phytophthora 0 0 0 0 0 0 Penicillium 1.8 0.4 0.2 1.1 0 0.05 Nectria 2.1 0.9 0.9 8.0 0.2 0.08 Gloeosporium 1.8 0.9 1.1 2.2 0.04 0.21 Other 3.7 1.4 0.9 1.3 0.3 0.3 Mean % loss 25.5 6.2 4.6 26.2 0.7 1.3

The disease control achieved by the zero pesticide residue management system (MS, MR) in this study has been as good as or better than that in the conventionally treated plots (CS, CR) and at lower cost. Dormant season disease control appears to be a key factor in the success of the system. No residues of pesticides applied to the MS and MR plots were detected in harvested fruit. Residues of captan, bupirimate and chlorpyrifos were frequently detected in apples from CS and CR plots, although all residues were below the MRL permitted.

The trial at East Malling will be continued for two further seasons to examine the impact of reduced pesticide programmes on the incidence of non target pests and diseases. The zero residue system will also be evaluated on commercial farms. Acknowledgements We are grateful to the Department for the environment, food and rural affairs for funding this work and to Barbara Ellerker, Karen Lower, Joyce Robinson, Adrian Harris and other staff at East Malling for assistance with the trials. References Berrie, A.M. 2000: Pre-harvest assessment of the risk of storage rots in Cox apples. –

IOBC/WPRS Bulletin 23(12): 159-169. Berrie, A.M. & Cross, J.V. 2005: Development of an integrated pest and disease management

system for apples to produce fruit free from pesticide residues. – In press. Berrie, A.M. & Luton, M.T. 1996: Integrated control of Phytophthora fruit rot (Phytophthora

syringae) in stored Cox apples. Proceedings Brighton Crop Protection Conference. – Pests and Diseases: 925-932.

Berrie, A.M. & Xu, X.-M. 2003: Managing apple scab and powdery mildew using Adem™. – International Journal of Pest Management 49: 243-250.

Bent, K.J., Scott, P.D. & Turner, J.A.W. 1977: Control of apple powdery mildew by dormant-season sprays: Prospects for practical use. – Proceedings 1977 British Crop Protection Conference. Pests and Diseases: 331-339.

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Burchill, R.T., Hutton, K.E., Crosse, J.E. & Garrett, C.M.E. 1965: Inhibition of the perfect stage of Venturia inaequalis by urea. – Nature 205: 520-521.

Cross, J.V. & Berrie, A.M. 1995: Field experimentation on pests and diseases of apples and pears. – Aspects of Applied Biology 43: 229-239.

Cross, J.V. & Berrie, A.M. 2001: Integrated pest and disease management in apple production. – In: The Best Practice Guide for UK Apple Production. Department for Environment, Food & Rural Affairs (DEFRA), Horticulture Research International, Farm Advisory Services Team Ltd, ADAS, Worldwide Fruit/Qualytech: pp 2.1-2.94.

Frick, E.L. & Burchill, R.T. 1972: Eradication of apple powdery mildew from infected buds. – Plant Disease Reporter 56: 770-772.

Jones, A.L., Ehret, G.R., El-Hadidi, M.F., Zabik, M.J., Cash, J.N. & Johnson, J.W. 1993: Potential for zero residue disease control programs for fresh and processed apples using sulphur, fenarimol and myclobutanil. – Plant Disease 77: 1114-1118.

Wilcox, W.F., Wasson, D.L. & Kovach, J. 1992: Development and evaluation of an integrated, reduced-spray program using sterol demethylation inhibitor fungicides for control of primary apple scab. – Plant Disease 76: 669-677.


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Evaluation of alternative treatments to urea to eliminate leaf litter in organic apple production Angela Berrie, Barbara Ellerker, Karen Lower East Malling Research, New Road, East Malling, Kent, ME19 6BJ, United Kingdom Abstract: Elimination of overwintering inoculum is one of the key factors in the integrated approach to scab control. In conventional production a spray of 5% urea is applied post harvest before leaf fall to reduce or eliminate the overwintering inoculum. The urea acts in two ways (1) directly on the scab fungus by interfering with the formation of the sexual state and (2) by encouraging colonisation of the fallen leaves by micro-organisms, which initiate rotting and make the leaves more palatable to earthworms. Unfortunately use of urea is not permitted in organic production. The purpose of this work was therefore to identify alternatives to urea which could be used in organic production.

Five separate experiments, the latter two at each of two sites, were conducted to test alternatives to urea for post harvest treatment to encourage rotting of apple leaves on the surface of the ground in the orchard post harvest. Apple leaves cv Fiesta collected from an organic apple orchard before leaf fall were dipped in solutions of the test treatments in December, then held on the surface of the ground in batches of 30 in the test orchard. The numbers of leaves that disappeared subsequently due to degradation and earthworm activity was assessed at intervals during the dormant period following treatment. Treatments tested included compost tea, Nugro, Maxicrop, Digester, Sea Vigour (fish oil) and Liquid Vinasse with urea as the standard and an untreated control.

None of the treatments evaluated at standard rates were as consistent or as effective in encouraging leaf decay as urea. Sea Vigour (Fish oil) and Nugro (4000 ppm N = ten times normal rate) encouraged leaf rotting compared to the untreated in some seasons and may be worth including as post harvest pre leaf fall treatments. Compost tea (bacterial or fungal) was completely ineffective and may have delayed leaf rotting. Key words: apple, scab, leaf rotting, dormant season control, urea Introduction Apple scab overwinters as the sexual state on leaves on the orchard floor. In spring during rain ascospores are released from the leaf litter to initiate new infections on the developing leaves on the trees. Apple scab can also overwinter on trees as wood scab or as mycelium on the base of shoots or on bud scales but leaf litter on the orchard floor is probably the major source of inoculum in spring. Elimination of overwintering inoculum is one of the key factors in the integrated approach to scab control. In conventional production a spray of 5% urea is applied post harvest before leaf fall to reduce or eliminate the overwintering inoculum. The urea acts in two ways (1) directly on the scab fungus by interfering with the formation of the sexual state and (2) by encouraging colonisation of the fallen leaves by microorganisms, which initiate rotting and make the leaves more palatable to earthworms (Burchill et al., 1965; Burchill & Cook, 1971). Unfortunately use of urea is not permitted in organic production. The purpose of this work was therefore to identify alternatives to urea which could be used in organic production.

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Material and methods Five separate experiments were conducted between 2000 and 2004 to test alternatives to urea for post harvest treatment to encourage leaf rotting. Source of leaf material For each experiment leaves were collected from the organic-managed experimental apple orchard cv Fiesta at Oakwood farm, Robertsbridge, East Sussex in the first week of November and stored at 4 ºC in a fridge until required. In 2000, leaves were also collected and similarly stored from a conventionally sprayed Fiesta orchard at East Malling. Treatments and application In December, aqueous solutions were prepared of the test treatments (Table 1). Four replicate nets of 30 leaves of cv Fiesta were dipped into each solution for half a minute, stirring to ensure thorough mixing. The leaves in their nets were allowed to dry overnight. Four replicate nets of leaves were also left untreated. Table 1. Products evaluated as treatments to encourage leaf rotting in apple orchards. Urea

was included as the standard.

Product Active ingredient

Product rate per litre

Years evaluated

Untreated – – 2000-2004 urea urea 50g 2000-2004 Compost tea microorganisms 1:10 dilution 2000, 2002 Nugro Liquid fertilizer 5ml 2000, 2001, Nugro Liquid fertilizer 312.5ml 2000 Nugro Liquid fertilizer 20ml 2001, 2002, 2004 Nugro Liquid fertilizer 50ml 2003, 2004 Maxicrop original Seaweed extract 1:10 dilution 2000, 2001 Digester Fermentation

extracts 1:10 dilution 2000, 2001, 2002

Nugro + Digester Liquid fertilizer + Fermentation extracts

20ml + 100ml 2001, 2002

Headland Liquid Sulphur 58.8% sulphur 5.6ml 2000

Sea Vigour Fish oil 12.5ml 2001, 2002 Sea Vigour Fish oil 50ml 2001, 2002 Sea Vigour Fish oil 200ml 2003, 2004 Liquid Vinasse Sugar beet waste 50ml 2001, 2002 Compost tea (bacterial) microorganisms Fresh undiluted 2003

Compost tea (fungal) microorganisms Fresh undiluted 2003

Assessment of treatments The day after treatment, the leaves were spread in batches of 30 on the surface of the ground herbage under the trees in the organic-managed apple orchard cv Fiesta located at Oakwood

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farm, in a randomised block design. Each batch (a plot) was held in place by rigid plastic netting which was secured to the ground by metal pins. The number of leaves remaining under each net was assessed at roughly monthly intervals from the time of treatment and mean values calculated. In 2003 and 2004 identical batches of treated leaves were also spread out in a second organic orchard, Village Fields, located at East Malling Research. Results and discussion 2000 – Experiment 1 In the first experiment, the number of untreated leaves from the organic orchard (Oakwood) remaining declined steadily between January and March (Table 2). Those from the non-organic orchard (EM) appeared to decay at a greater rate. The reduced rate of decay of the organic leaves may have been due to a lower N content in the leaves or because they were contaminated with sulphur, applied during the growing season for control of scab and mildew. The urea and the Nugro - high concentration (at the same concentration N as the urea of 25,000 ppm) treatments increased the rate of leaf decay, indicating that the nitrogen concentration was the key factor in encouraging leaf rotting. Leaves treated with Maxicrop, Nugro (recommended rate) and Digester decayed at a faster rate than the untreated but the compost tea and sulphur appeared to be ineffective. Sulphur is a general biocide and therefore may have inhibited the colonisation of leaves by microorganisms and hence delayed leaf rotting. 2001-2003 – Experiments 2 and 3 In the second experiment, mild, wet weather conditions were very favourable for leaf rotting during the 2001/2002 winter. For all of the treatments almost half the leaves had rotted by the end of the first month after treatment. By the next assessment in February almost all the leaf material had rotted and it was not possible to distinguish between the effects of treatments as any effects were masked by the rapid natural rate of rotting. Thus, the relative effects of the treatments could not be determined. Table 2. Number (maximum=30) of leaves remaining following treatment with various

chemicals in December 2000 in the leaf rotting experiment Oakwood Farm in winter 2000/2001.

Treatment Concentration (amount/litre

16 Jan 2001

16 Feb 2001

28 Mar 2001

Untreated (Oakwood) – 26.0 14.0 0.4 Untreated (EM) – 25.8 8.1 0.3 Urea 50 g 21.3 1.9 0 Compost Tea 1:10 dilution 27.9 12.3 0.5 Nugro (400ppm N) 5 ml 25.6 7.8 0.1 Nugro (25000ppm N) 312.5 ml 11.9 0.6 0 Maxicrop original 1:10 dilution 25.6 10.9 0.4 Digester 1:10 dilution 27.4 8.1 0 Sulphur 5.6 ml 27.4 12.6 0.4

Leaf rotting was also very rapid in the third experiment, even on the untreated, such that on most treatments almost all the leaves had disappeared by the first assessment in late

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January. Liquid Vinasse (sugar beet waste) and Nugro (1600ppm N) appeared to delay rotting. By the second assessment in late February most of the leaves had disappeared and there were no differences between treatments. Leaf rotting was too rapid in this experiment to distinguish treatment effects 2003 – Experiment 4 In 2003 experiments were conducted at two organic sites to increase the chances of obtaining differences between treatments. Experiment one had demonstrated that the concentration of nitrogen was the key factor in urea and Nugro (25,000ppm N rate) that resulted in rapid leaf rotting. Previous studies by Burchill and Cook (1971) demonstrated that the high nitrogen encouraged microorganisms to colonise the leaves and accelerate rotting. An alternative approach to the use of high nitrogen products would be to apply microorganisms directly by the use of compost tea. These products (bacterial and fungal) were therefore included in experiments in 2003. At the Oakwood site after one month, most of the leaves treated with urea had rotted (Table 3). Over half the leaves treated with Nugro (4000ppm N) or Fish oil had rotted, whereas very little rotting had occurred in leaves treated with compost tea or left untreated. By the second assessment in February almost all leaves had rotted in all treatments apart from compost tea where around 25% of leaves were left. The compost tea treatments appeared to delay rotting, but at this site natural decay of leaves over the winter was sufficient to result in minimal leaf litter remaining at bud burst in March. By contrast at the East Malling site (EM), after one month little rotting had occurred in any of the treatments, including urea (Table 3). After two months some rotting had occurred in leaves treated with urea or Nugro, but practically all leaves remained in the untreated plots or those treated with Fish oil or compost tea. No further assessments were carried out, but observations in March and April indicated that substantial numbers of leaves remained in all plots. Reasons for the poor leaf rotting at this site were not clear. Earth worm casts were abundant in the orchard, indicating that earth worms were active, but appeared to show no interest in the treated leaves. Table 3. Number (maximum=30) of leaves remaining following treatment with various

chemicals in December 2003 in the leaf rotting experiment Oakwood Farm in winter 2003/2004.

Treatment Concentration (amount/litre

6-7 Jan 2004

3-4 Feb 2004

Oakwood Farm Untreated – 21.3 2.6 Urea 50 g 5.5 0.8 Nugro (4000ppm N) 50 ml 11.3 1.1 Fish oil 200 ml 13.3 1.8 Compost Tea (bacterial) Undiluted 24.3 8.0 Compost Tea (fungal) undiluted 22.0 6.0 Village Field (EM) Untreated – 29.9 28.8 Urea 50 g 28.4 20.1 Nugro (4000ppm N) 50 ml 27.6 18.0 Fish oil 200 ml 30.0 29.5 Compost Tea (bacterial) Undiluted 29.4 28.2 Compost Tea (fungal) Undiluted 29.4 28.0

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2004 – Experiment 5 In 2004 the experiment were repeated at the two sites. Compost tea was omitted, and Nugro at two rates (4000ppm N and 1600ppm N) included. Autumn /winter 2004 / 2005 was relatively dry and leaf rotting slower than in previous years. At the Oakwood site, after approximately six weeks almost all leaves had rotted in the urea, Nugro (4000ppm N) and Fish oil treated plot (Table 4), compared to the untreated and Nugro (1600ppm N) treated plots. By mid March (bud burst) only a few leaves remained in the untreated and Nugro (1600ppm N) plots. Leaf rotting was much slower at the East Malling site, particularly in the untreated leaves, where over 50% of the leaves remained in mid March. Rotting was most rapid in plots treated with urea or Fish oil. In contrast to the Oakwood site, there was no difference in leaf rotting between the two rates of Nugro. Table 4. Number (maximum=30) of leaves remaining following treatment with various

chemicals in December 2004 in the leaf rotting experiment at Oakwood Farm and EM in winter 2004/2005.

Treatment Concentration

(amount/litre 10-11 Feb

2005 15-16 March

2005 Oakwood Farm Untreated – 12.8 5.1 Urea 50 g 0.1 0 Nugro (4000ppm N) 50 ml 2.5 0.4 Nugro (1600ppm) 20 ml 12.3 4.6 Fish oil 200 ml 5.0 1.4 Village Field (EM) Untreated – 25.5 17.3 Urea 50 g 6.0 3.1 Nugro (4000ppm N) 50 ml 13.0 6.1 Nugro (1600ppm) 20 ml 13.5 6.5 Fish oil 200 ml 4.4 1.9

Discussion It is clear from these results that the rate of leaf decay varies greatly between seasons, depending on temperature and more important rainfall during the dormant period, and also between sites. In mild wet winters as in 2001/2002 and 2002/2003, at the Oakwood site leaf rotting was relatively rapid in the untreated plots such that most leaves had disappeared by bud burst. Thus in most seasons here treatments to encourage leaf rotting, other than mowing to shred leaves may not be necessary. By contrast, at East Malling, rotting in the untreated plots was considerably slower than in treated plots, such that more than half the leaves remained at bud burst. Treatments to encourage leaf rotting would be of great benefit in minimising scab inoculum for the next season. None of the treatments evaluated at their normal rates were as effective or consistent as urea in encouraging leaf rotting. Applying Nugro, which has the highest N content of organic approved foliar feeds in the UK, at 62 times its normal rate to achieve an equivalent N content to urea at 5% (25,000ppm) resulted in accelerated leaf decay similar to urea, indicating that N content is the critical factor. Such a

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rate of use however, would be excessively expensive. Nugro at ten times the normal dose and Fish oil did encourage leaf rotting, although not as consistently as urea, and maybe worth considering as treatments especially if combined with mechanical methods such as leaf shredding. Urea also has the additional effect of preventing formation of the scab pseudothecia during the dormant period such that ascospore dose is considerably reduced on any leaves remaining in spring. The effect of the alternative treatments on the scab sexual state was not investigated in this study. Acknowledgements The authors would like to thank DEFRA and Horticulture LINK for funding this work and Mr Matthew Wilson for providing the experimental site at Oakwood Farm. References Burchill, R.T., Hutton, K.E., Crosse, J.E. & Garrett, C.M.E. 1965: Inhibition of the perfect

stage of Venturia inaequalis by urea. – Nature 205: 520-521. Burchill, R.T. & Cook, R.T.A. 1971: The interaction of urea and micro-organisms in

suppressing the development of perithecia of Venturia inaequalis. – In: Ecology of Leaf Surface Micro-organisms, eds. Preece, T.F. and Dickinson, C.H., Academic Press, London: 471-483.


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Heterogeneity in apple scab: implication for management Odile Carisse, C. Meloche, Tristan Jobin, D. Rolland Agriculture and Agri-Food Canada, Horticultural Research and Development Centre 430 Gouin, Saint-Jean-sur-Richelieu, Québec, Canada, J3B 3E6. Abstract: Scab is an important disease of apple and its control depends almost exclusively on frequent use of fungicides. In north-eastern North America, the strategy to manage apple scab is mainly based on a good control of primary infections, in order to avoid epidemic build-up caused by secondary infections and subsequent fungicide applications during the summer months. During the primary infection period, fungicides are applied on a calendar basis, or based on risk of scab development. In the latter case, the risk for primary infections is estimated mainly from the stage of ascospore maturation and climatic conditions associated with risk of infection.

Disease measurement is essential in every epidemiological study and can be used to evaluate various management tactics. "Without quantification of disease no studies in epidemiology, no assessment of crop losses and no plant disease surveys and their applications would be possible" (Kranz, 1988). To accurately measure disease (incidence or severity, and inoculum level) knowledge on spatial and temporal distribution is crucial. Most of the discussion has focused on temporal distribution and spatial distribution has received far less attention. The objective of this work was to study spatial heterogeneity in apple scab epidemiological components, including primary inoculum, disease expression and host growth.

Heterogeneity in ascospore production was studied at various scales (leaf, tree, and orchard). Regardless of the scale used, high level of heterogeneity was observed among leaves, within tree canopy and among trees within an orchard. This high level of heterogeneity makes ascospore inoculum difficult to measure. Spatial heterogeneity in disease expression (primary lesions) was studied at the orchard level. Overall, spatial distribution of primary lesions was homogeneous. Consequently, primary lesions could be measure accurately using proper sampling plan. Finally, heterogeneity in host growth (leaf emergence and growth) was studied at the orchard level. At the beginning of the season, leaf emergence was homogenous among trees and between various cultivars. From the information on apple scab heterogeneity, it was concluded that primary inoculum is highly heterogeneous and primary lesion as well as growth were homogenous. From these results a different approach of scab management was designed based on detecting first ascospore ejection, rate of leaf emergence and scab development at the end of the primary infection period. Key words: epidemiology, spatial distribution, Venturia inaequalis Introduction Most control strategies for apple scab aimed at controlling the primary infections to avoid epidemic build-up caused by secondary cycles. In northeastern U.S. and eastern Canada, the primary infections caused by ascospores occur from bud break to mid June when almost all ascospores were ejected (Gadoury & MacHardy, 1982). Several models were developed for the estimation of ascospore maturity (Gadoury & MacHardy, 1982; St-Arnaud & Neumann, 1990; Rossi et al., 2000). These models, based on degree-day accumulation, are generally used to predict the beginning and the end of the ascospore ejection period. However, they tend to be regionally based and generally are not accurate enough to provide the time at which the first and the last fungicide applications should be done. Fungicide timing is seldom based on inoculum assessment (McHardy, 2000; Mills, 1944; Aylor, 1998). The potential ascospore

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dose (PAD) is a useful tool for predicting the total amount of inoculum in an orchard and has been shown to effectively improve apple scab management (McHardy et al., 1993). Despite the obvious interest of classifying orchards based on PAD, there are limits to the use of this criterion. PAD provides an overall estimation of the inoculum potential and is based on the assumption that the primary inoculum (ascospores per m2) is uniform within the orchard as it provides one risk value per orchard. Monitoring of airborne ascospores has been suggested as one mean to more precisely estimate scab risk for a specific infection period (Aylor & Kiyomoto, 1993; Charest et al., 2002). Nevertheless, all prediction or simulation models for apple scab assume that primary inoculum is uniformly distributed within an orchard and within the tree canopy. Results and discussion Heterogeneity in primary inoculum at the orchard level The spatial distribution of V. inaequalis ascospores was studied in a commercial apple orchard of 0.43 ha. Airborne ascospore concentration was studied in space and in time during two different years (1999 and 2000) using a 5 x 8 lattice of adjacent quadrats of 13.5 × 8 m each. Each quadrat was monitored during the major rain events of the spring 1999 and 2000 using spore samplers. The variance to mean ratio for the PAD and for most of the AAC sampling dates were >1, indicating an aggregated pattern of distribution. None of the frequency distributions of the most important ascospore ejection events followed the Poisson probability distribution, indicating that the pattern of distribution was not random. For all events, AAC had an aggregated pattern of distribution as suggested by the negative binomial distribution. The PAD followed neither the Poisson nor the negative binomial distribution. Geostatistical analyses confirmed the aggregated pattern of distribution. The cultivars had an effect on the PAD and AAC distribution pattern, but both PAD and AAC were not uniformly distributed within a block of the same cultivar. Therefore, the number, location and height of samplers required to estimate AAC in orchards need to be investigated before using information on AAC as decision making tool. Heterogeneity in primary inoculum at the tree level Initial scab infection assumes several steps: ascospore maturation, liberation of ascospores that become airborne, deposition on susceptible tissues, and infection. However, the spatial heterogeneity of ascospores within the tree canopy is unknown. Aerial concentration of ascospore (ACA), ascospore concentration in rain water (ACR) and ascospore deposition (AD) were therefore measured at 6 heights (20 to 257 cm from the ground) with rotating-arm air samplers, funnels, and greased glass slides, respectively, during 5 rain events in both 2001 and 2002. In addition, ACR, AD and scab lesions were measured at eight locations within tree canopy at 196 cm height. A similar experimental design was used in 2003 to study the spatial heterogeneity of both AD and primary scab lesions. ACA and AD decreased with increasing height, while ACR increased with increasing height. Based on both variance to mean ratio and the power law, both years, rate of ACR was heterogeneous, while rate of AD was heterogeneous only during the peaks of ascospore release. Rate of ACR was significantly higher at the center of the trees and the rate of AD was significantly higher at the center and at the western edge of the trees. Only cumulative rate of AD was significantly correlated with apple scab lesions at the same location (r=0.83). In 2003, a similar pattern of spatial heterogeneity within the tree canopy was observed for AD and primary scab lesion counts and there was a linear relationship (R2=0.84) between these two variables. It was concluded that ACR and AD within the tree canopy are not randomly distributed at least during peaks of

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ascospore release and that AD is a good estimate of apple scab lesion development. As a consequence, spatial distribution should be considered when estimating ascospore deposition using mathematical models or when quantifying ascosporic inoculum using spore samplers.

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Figure 1. Apple leaf emergence measured in 2003, 2004, and 2005 on six apple cultivars and

at two sites (Frelighsburg and St-Paul d’Abbotsford). Heterogeneity in disease expression at the orchard level Heterogeneity of primary apple scab incidence and severity was studied in commercial orchards located in two apple growing areas of south west and south east of the province of Quebec, Canada. Leaf scab on terminal shoots was assessed weekly from May to July or once in July depending on sites. Leaf scab on clusters was assessed at the end of the primary scab period. Disease assessments were made on one tree located in the center of each quadrat. Assessments on terminal shoots and clusters were made on five shoots or clusters per tree selected at random within each tree. All leaves per shoot or clusters were examined for scab lesions and the number of lesions noted. The variance to mean ratio and the Lloyds index of patchiness for scab incidence were < 1 at all sampling dates for all sites indicating a regular pattern of the scabbed leaves distribution. Similarly, the variance to mean ratio and the

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Lloyd’s index of patchiness of scab severity were also < 1 for all sampling dates and all sites indicating a regular pattern of distribution of the primary scab lesions. They were a significant relationship between the logs of the sample variance and the logs of the sample mean except at one site. The estimates of the parameter log(b) were significantly smaller than 1 (P< 0.05) for both scab incidence and severity suggesting an absence of heterogeneity.

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in 2003 and 2004 for the cultivar McIntosh at the site of Frelighsburg. Heterogeneity in host growth To evaluate the influence of rootstocks on apple leaf emergence, a first set of experiment was conducted during the spring of 2003 and 2004 at a commercial orchard located in St-Gregoire, Quebec, Canada. Apple leaf emergence was monitored on trees of the cultivar Summerland McIntosh grafted on rootstocks M.9, M.26, and M.111. Early in the season, before bud break, three trees per rootstock were selected at random and 10 to 15 terminal shoots per tree were tagged. The number of unfolded leaves per shoot was counted every two to three days from

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April 30 to June 6 and from April 22 to June 16 in 2003 and 2004, respectively. Unfolded leaf was defined as a leaf with an angle of at least 45o between upper surface edges. To study leaf emergence during the apple scab period, a second set of experiment was conducted during the spring of 2003, 2004 and 2005 at the Agriculture Canada Experimental Farm, in Frelighsburg and at a commercial farm in St-Paul d’Abbotsford, Québec, Canada. At each sites, leaf emergence was monitored on six apple cultivars: Cortland, Empire, Lobo, McIntosh, Paulared and Spartan. At the beginning of the spring before bud break, three trees per cultivar were selected at random within the orchard blocks. For each tree, 10, 15 and 15 shoots were selected at random in 2003, 2004, and 2005, respectively. On each trees, care was taken for selecting 50% of the shoots as terminals and 50% as cluster even though this proportion changed slightly later in the season because of the difficulty to distinguish between terminal and cluster shoots early in the spring. The number of unfolded leaves per shoot was counted every 2 to 3 days from April 30 to June 19, from April 21 to July 1, and from April 18 to July 6, in 2003, 2004, and 2005, respectively.

The effect of rootstock and cultivars on leaf emergence and leaf expansion was analyzed with a repeated measures analysis of variance (ANOVA). Leaf emergence was also expressed as rate of leaf emergence as the number of new leaves per. The trend in leaf emergence on apple trees grafted on M.111, M.26, and M.9 rootstocks was similar in 2003 and 2004 and there was no significant effect of rootstock or cultivar on leaf emergence (P<0.05). On both terminal and cluster shoots, the pattern of leaf emergence was sigmoid with a period of slow leaf emergence early in the season followed by a period of rapid increase (Figure 1).

From the results of these studies it was proposed that risk of primary apple scab be based on rate of leaf emergence and the length of the ascospore ejection period both predicted from degree day accumulation and conditions for infection (Mills, 1944) (Figure 2). References Aylor, D.E. & Kiyomoto, R.K. 1993: Relationship between aerial concentration of Venturia

inaequalis ascospores and development of apple scab. – Agriculture and Forest Meteorology 63: 133-147.

Aylor, D.E. 1998: The aerobiology of apple scab. – Plant Dis. 82: 838-849. Charest, J., Dewdney, M., Paulitz, T., Philion, V. & Carisse, O. 2002: Spatial distribution of

Venturia inaequalis airborne ascospores in orchards. – Phytopathology 92: 769-779 Gadoury, D.M. & MacHardy, W.E. 1982: A model to estimate the maturity of ascospores of

Venturia inaequalis. – Phytopathology 72: 901-904. Kranz, J. 1988: Measuring plant disease. – In Experimental Techniques in Plant Disease

Epidemiology, ed. J Kranz, J Rotem, pp. 35-50. Berlin: Springer-Verlag MacHardy, W.E., Gadoury, D.M. & Rosenberger, D.A. 1993: Delaying the onset of fungicide

programs for control of apple scab in orchards with low potential ascospore dose of Venturia inaequalis. – Plant Dis. 77: 372-375.

MacHardy, W.E. 1999: A review of apple scab research presented at “IOBC Integrated Control of Pome Fruit Diseases” workshops, 1987-1999. – IOBC/WPRS Bulletin 23(12): 171-182.

Mills, W.D. 1944: Efficient use of sulfur dusts and sprays during rain to control apple scab. – Cornell Ext. Bull. 630: 1-4.

Rossi, V., Ponti, I, Marinelli, M, Giosuè, S. & Bugiani, R. 2000: A new model estimating the seasonal pattern of air-borne ascospores of Venturia inaequalis (cooke) wint. in relation to weather conditions. – J. Plant Pathol. 82: 111-118.

St-Arnaud, M. & Neumann, P. 1990: Évaluation au Québec d'un modèle de prédiction de la fin de la période annuelle d'éjection des ascospores du Venturia inaequalis. – Phyto-protection 71: 17-23.


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Field and in vitro sensitivity of Valsa ceratosperma (Cytospora vitis) to fungicides Marina Collina, Elena Cicognani, Benedetta Galletti, Agostino Brunelli Dipartimento di Protezione e Valorizzazione Agroalimentare, Università di Bologna, Viale G. Fanin, 46 – 40127 Bologna, Italy Abstract: The occurrence of Valsa ceratosperma (Tode ex Fr.) Maire, causal agent of Valsa canker, was reported for the first time as a pathogen on pear in the Emilia Romagna region (Italy) in 2001 and since then the disease incidence has greatly increased. Valsa canker is one of the most important diseases of apple orchards in China, Japan and Korea where preventative good farming and chemical treatments (especially with SBIs and benzimidazoles) are the best way to try to control the disease on pear. In order to investigate the possibility of chemical disease control, tests in vitro (mycelial growth on PDA) and a field trial were carried out. SBIs, benzimidazoles, strobilurins, dicarboximides, tolylfluanid, fluazinam, fludioxonil, cyprodinil, dithianon, mancozeb and captan were tested in vitro assays. Some of the formulated compounds used in in vitro assays were applied in the field trial. All fungicides tested in vitro were more or less active; SBIs, benzimidazoles, fludioxonil, fluazinam and iprodione showed the best effectiveness. No activity was shown by any of the products in the field trial, probably because they are not able to penetrate through the bark and reach the pathogen in the cortical tissues and phloem. Key words: Valsa ceratosperma, Cytospora vitis, fungicide sensitivity, control Introduction The Ascomycete Valsa ceratosperma (Tode ex Fr.) Maire [anamorph Cytospora sacculus (Schwein.) Gvritischvili = C. vitis] is a new causal agent of bark canker recently reported in pear growing areas in the Emilia Romagna (Italy) (Montuschi, 2003; Montuschi & Collina, 2003). This is the first report of Valsa ceratosperma on pear in Europe while Valsa canker is one of the most important diseases of apple orchards in China, Japan and Korea; in these countries the fungus was only occasionally found on pear and quince (Agrios, 1997).

In the Far East good farming practices are suggested in order to prevent Valsa disease. Keeping trees in good vigour, avoiding wounding and severe pruning, removing and burning infected tissues are the practices firstly recommended. Chemical control of Valsa ceratosperma is reported by several authors, first of all with the protection of bark injuries through sprays of fungicides (i.e. Lime sulphur) in late autumn after harvest and in early spring after pruning (Washio et al., 1977). Fungicides in paste can be applied on trunk or scaffold limbs where cankers have been surgically removed (i.e. thiophanate methyl with polyvinyl alcohol or pine oil, benomyl). High effectiveness was shown by benzimidazoles, SBIs and oxide-copper in spray applications throughout the year (Washio et al., 1977) and Neozin solution (ammonium salt ferric methyl arsenic acid) applied directly on cankers (Uhm & Sohn, 1991). Other authors affirm instead that curative fungicides are not available and the disease often recurs after applications (Sakuma, 1990).

In this study laboratory and field assays were carried out in order to achieve more information about the efficacy of many fungicides towards V. ceratosperma and to outline a first control strategy.

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Materials and methods In vitro tests In vitro tests were carried out on PDA (39 g l-1, Becton Dickinson) amended with several fungicides at different concentrations. The amended medium was poured into 9 cm Petri dishes which were inoculated with an inverted mycelium plug (5 mm diameter) taken from the edge of 7 day old colonies. Three replicates were prepared for each concentration and all assays were repeated twice. The mycelial growth was evaluated after 7 and 10 days of incubations at 20°C and 12 hours of photoperiod, measuring and averaging the two perpendicular diameters of fungal colonies. EC50 and Minimum Inhibitor Concentration (MIC) were determined by probit analysis. Several formulated compounds and technical grades were tested (Table 1).

Table 1. Formulated compounds used in vitro tests and in field trial (with field dose rate).

Active Ingredient Formulated compound

Field dose rate (g o ml formulate/litre)

Bitertanol PROCLAIM SC (Bayer) n.a.f. Difenoconazole SCORE 25 EC (Syngenta) 3 ml Difenoconazole + Pine oil

SCORE 25 EC (Syngenta) +VAPORGARD (Intrachem)

3 ml + 15 ml

Tebuconazole FOLICUR WG (Bayer) n.a.f. Benomyl BENLATE WG (Du Pont) n.a.f. Thiophanate methyl ENOVIT METIL FL (Sipcam) 10 ml Thiophanate methyl + Pine oil

ENOVIT METIL FL (Sipcam) + VAPORGARD (Intrachem)

10 ml + 15 ml

Azoxystrobin QUADRIS (Syngenta) 10 ml Kresoxim-methyl STROBY (Basf) n.a.f. Trifloxystrobin FLINT (Bayer) 1.5 g Iprodione ROVRAL 50WP (Bayer) 15 g Procymidone SUMISCLEX 50 WG (Basf) n.a.f. Captan MERPAN 80 WDG (Cheminova Agro Italia) n.a.f. Copper chelate KELAL KUBIG (BMS Micro Nutrition Italia) 100 ml Dithianon DELAN WG (Basf) n.a.f. Fluazinam OHAYO (Syngenta) 10 ml Fludioxonil CELEST (Syngenta) n.a.f. Fludioxonil + Cyprodinil SWITCH (Syngenta) 8 g

Lime sulphur POLISOLFURO DI CALCIO (Chemia) Not diluted Mancozeb DITHANE DG (Caffaro) n.a.f. Pine oil VAPORGARD (Intrachem) 15 ml Tolylfluanid EUPAREN M 50 WG (Bayer) n.a.f.

(n.a.f.= not applied in field)

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In a preliminary test formulated compounds of SBIs (difenoconazole and bitertanol), benzimidazoles (thiophanate-methyl, benomyl, carbendazim), strobilurins (azoxystrobin, trifloxystrobin, kresoxim-methyl), dicarboximides (procymidone, iprodione), tolylfluanid, fluazinam, fludioxonil, cyprodinil, dithianon, mancozeb and captan were tested (Table 1) at 0, 10, 100 and 1000 mg a.i. l-1. Only MIC values were evaluated in this assay.

Some of these fungicides were subsequently used both as formulated compounds and as technical grades. Formulated compounds of tebuconazole (Folicur WG), difenoconazole (Score 25 EC), bitertanol (Proclaim SC), thiophanate methyl (Enovit Metil FL), iprodione (Rovral 50WP) and fluazinam (Ohayo) were tested at 0, 0.01, 0.1, 0.5, 1 and 10 mg l-1 of active ingredients. Technical grades of bitertanol and tebuconazole (supplied by Bayer CropScience), difenoconazole and fludioxonil (supplied by Syngenta CropProtection) and thiophanate methyl (purchased from Sigma) were tested at concentrations 0, 0.001, 0.01, 0.05, 0.1, 0.5 and 1 mg a.i. l-1. The assay with thiophanate methyl was repeated also at concentra-tions of 0, 1, 2.5, 5, 7.5, 10, 15 and 20 mg a.i. l-1. Stock solutions were prepared for each active ingredient in acetone; the solvent concentration never exceeded 1% (v/v). EC50 and MIC were evaluated. Field trial A field trial was carried out in a pear orchard seriously injured by Valsa ceratosperma, located close to Bologna. It was carried out by swabbing the formulated compounds directly on cankers on the trunk and on the main crotch (the canker borders were delimitated by water- resistant marker pen).

Three treatments were applied (the 13th of October and the 14th of December 2004, the 15th of April 2005) using formulated compounds at concentrations of 10 times those reported on the label for other diseases. The active ingredients were difenoconazole and thiophanate-methyl alone and tank mixed with pine oil, azoxystrobin, trifloxystrobin, fluazinam, iprodione, fludioxonil mixed with cyprodinil, lime sulphur, copper chelate and pine oil (Table 1). Four pear trees were chosen for each fungicide. The evaluation was carried out on the 5th of August 2005 considering the tree vegetative conditions and the growth of the treated cankers.

Results and discussion In vitro tests The SBIs difenoconazole, bitertanol and tebuconazole showed the best activity against mycelial growth of Valsa ceratosperma, with the lowest EC50 and MIC values both as formulated compounds and as technical grades. Benzimidazoles were all effective (mycelial growth was completely inhibited at concentrations lower than 10 mg l-1 of active ingredient) but only thiophanate methyl was included in Annex 1 and it could thus be possible to be used again in the future. Fluazinam, fludioxonil and iprodione also demonstrated a good activity, while other products were all much less effective starting from strobilurins, procymidone, captan, cyprodinil, dithianon, mancozeb and tolylfluanid with MIC values over 1000 mg l-1 of a.i. for most cases (Table 2). Field trial Field trial results were obtained observing both vegetative conditions of plants and Valsa canker area development but no differences were observed between treated and untreated pear trees. These results could be explained by taking into account that fungicides are not able to penetrate through the bark and reach the pathogen in the cortical tissues and phloem.

Therefore, at the present time, there are no fungicides able to have a direct effect on V. ceratosperma cankers already evident in field, and good cultural practices are the only way to

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prevent this disease. In the future, studies on SBIs, thiophanate methyl, fludioxonil, iprodione and fluazinam activities in vitro and in field on conidia will be carried out.

Table 2. In vitro mycelial sensitivity to fungicides tested as formulated compounds and/or technical grades (EC50 and MIC in mg a.i. l-1).

Formulated compound

Technical grade

Active ingredient

EC50 MIC EC50 MIC

Bitertanol 0.02 0.5-1 0.03 0.5-1 Difenoconazole 0.01 0.5-1 0.01 < 0.5 Tebuconazole 0.02 0.1-0.5 0.05 < 0.5 Benomyl – < 10 – – Carbendazim – < 10 – – Thiophanate methyl 0.56 < 10 1.25 5 ÷7.5 Azoxystrobin – > 100 – – Kresoxim-methyl – > 1000 – – Trifloxystrobin – > 1000 – – Iprodione 1.32 > 10 – – Procymidone – > 100 – – Captan – > 1000 – – Cyprodinil – > 1000 – – Dithianon – > 1000 – – Fluazinam 0.05 > 1 – – Fludioxonil – > 1 0.09 > 1 Mancozeb – > 100 – – Tolylfluanid – > 1000 – –

References Agrios, G.N. 1997: Valsa, or Cytospora, canker and dieback. – In: Plant Pathology. Academic

Press, 4° ed.: 374-378. Montuschi, C. 2003: Il “cancro da Valsa”, nuova malattia del pero. – Agricoltura 2: 66-68. Montuschi, C. & Collina, M. 2003: Prima segnalazione in Italia di Valsa ceratosperma su

pero. – Informatore agrario 50: 55-57. Sakuma, T. 1990: Valsa canker. – In Compendium of Apple and Pear Diseases, eds. Jones

A.L. & Aldwinckle H.S., APS Press, St. Paul, U.S.A, 39-40. Uhm, J.Y. & Sohn H.R. 1991: Neozin solution, a possible control agent of apple Valsa

canker. – Ann. Phytopath. Soc. Japan 57: 577-581. Washio, S., Sasaki, M., Tamakawa, K., Nakagawara, I. & Takahashi, M. 1977: Some factors

affecting the occurrence of Japanese apple canker and its control. – Bulletin of the Aomori field crops and horticultural experiment station 2: 1-43.


pp. 155 - 161

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Sensitivity in vitro of Stemphylium vesicarium to fungicides Marina Collina, Giulia Alberoni, Agostino Brunelli Dipartimento di Protezione e Valorizzazione Agroalimentare - Università di Bologna, Viale G. Fanin 46, 40127 Bologna, Italy Abstract: Brown spot, caused by Stemphylium vesicarium, is the most important pear fungal disease in Northern Italy and its management relies on frequent preventative applications of fungicides. Dicarboximides (mainly procymidone) are the fungicides most effective against this pathogen but the occurrence of field resistant isolates in the early 1990s led to a reconsideration of other products. S. vesicarium sensitivity to SBIs, dithiocarbamates, anilinopyrimidines, strobilurins, phenylpyrroles and sulphamides was evaluated through inhibition tests of conidial germination, mycelial growth and hyphal elongation. Fenbuconazole, penconazole, ziram and strobilurins showed good efficacy on conidial germination also in isolates resistant to dicarboximides, while the best activity on mycelial growth was obtained with anilinopyrimidines, tebuconazole, flutriafol, difenoconazole and propi-conazole. The best hyphal growth inhibition was observed with ziram and fenbuconazole. Key words: Stemphylium vesicarium, fungicides, sensitivity, resistance, pear Introduction Stemphylium vesicarium (Wallr) Simm. is the causal agent of brown spot, the main fungal disease of pear in Italy (Po valley) since the late 1970s, but it is also present in other European countries such as Spain, France, The Netherlands and Belgium. It is a Mitosporic fungus and its teleomorphic form is the Ascomycetes Pleospora allii (Rab.) Ces. & de N. (Simmons, 1969).

Symptoms consist of necrotic areas (brown spots) on leaves and fruits that can rot before, during or after harvest. Plants may be affected throughout the entire season and yield loss can be up to 100%. Commercial damage is caused during the early stages of conidial germination by the release of two host specific toxins (SV-toxin I and II) which act on the plasma membranes of susceptible cells (Singh et al., 1999, 2000; Ponti & Cavanni, 1983; Cavanni & Ponti, 1994). Pear varieties have, in fact, a different susceptibility towards pear brown spot: Abbé Fétel and Conference are considered highly susceptible, Doyenne, Passe Crassane, Kaiser and Packam’s Triumph are susceptible and Williams, Max Red Bartlett, Santa Maria, Spadona, Blanquilla, Butirra Hardy, Luis Bonne, Grand Champion and Highland are not susceptible (Ponti et al., 1980; Montesinos et al., 1995; Blancard et al., 1989). Because of these characteristics S. vesicarium is not an easy disease to control.

Besides some cultural practices, scheduled preventative applications of fungicides from petal fall to fruit ripening are the only way to control the brown spot. Since the appearance of the disease many fungicides have been applied in field, as dithiocarbamates (mainly thiram), dichlofluanid, captan and dicarboximides. Among them, procymidone was the most widely used, iprodione was less applied because of its phytotoxicity towards some pear varieties, while vinclozolin and chlozolinate were not used. In the late 1990s new products were introduced to control pear brown spot, such as tebuconazole among SBIs, strobilurins kresoxim-methyl and trifloxystrobin, the ready mixture fludioxonil+cyprodinil and tolylfluanid (Ponti et al., 1996; Brunelli et al., 2004).

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In the early 1990s, problems in brown spot control were reported in some areas of Northern Italy with schedules based on procymidone (Brunelli et al., 1997). A sensitivity study of S. vesicarium to dicarboximides started in 1995 showed a high resistance level towards procymidone, with a cross resistance of lower level towards the other fungicides of this group (Collina et al., 2002; Alberoni et al., 2005).

The present study was undertaken to evaluate S. vesicarium sensitivity towards a wide range of fungicides other than dicarboximides. Materials and methods Fungicides Dicarboximides (procymidone and iprodione), dithiocarbamates (thiram and ziram), pyrimethanil, fenbuconazole, flusilazole, flutriafol and nuarimol were used as formulated compounds. Table 1. Fungicides tested in vitro against Stemphylium vesicarium.

Fungicide group Active ingredients Tested compounds Solvent

procymidone Sialex 50% WG, Siapa water Dicarboximides iprodione Rovral 50% WP, Aventis water thiram Tetrasar 50% WP, Isagro water Dithiocarbamates ziram Pomarsol 81%WDG, Bayer water

Sulphamides dichlofluanid Pure a.i. methanol

bitertanol Baycor 25%WP, Bayer Pure a.i.

water isopropanol

difenoconazole Score 25% EC, Syngenta Pure a.i.

water acetone

fenbuconazole Indar 3% EW, Dow Agrosciences water flusilazole Nustar 40% EC, Du Pont water flutriafol Impact 12.5% SC, Cheminova water

hexaconazole Anvil 2.9% SC, Syngenta Pure a.i.

water acetone

myclobutanil Systhane 6.2% FL, Dow Agrosciences Pure a.i.

water methanol

penconazole Topas 10.5% EC, Syngenta Pure a.i.

water hexane

propiconazole Pure a.i. hexane

tebuconazole Folicur 25% WDG, Bayer Pure a.i.

water isopropanol

prochloraz Sportak 40% EC, Basf Pure a.i.

water acetone

fenarimol Rubigan 6% WP, Dow Agrosciences Pure a.i.

water methanol

nuarimol Bloc 6% WP, Dow Agrosciences water

SBIs

fenpropimorph Pure a.i. acetone cyprodinil Pure a.i. acetone Anilinopyrimidines pyrimethanil Scala 37.4% SC, Basf water

Phenylpyrroles fludioxonil Pure a.i. acetone kresoxim-methyl Pure a.i. acetone Strobilurins trifloxystrobin Pure a.i. acetone

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Dichlofluanid, fludioxonil, propiconazole, fenpropimorph, cyprodinil and strobilurins (kresoxim-methyl and trifloxystrobin) were used as pure active ingredients.

Some fungicides (bitertanol, difenoconazole, hexaconazole, myclobutanil, penconazole, tebuconazole, prochloraz and fenarimol) were tested both as formulated compounds and as active ingredients. All pure active ingredients were purchased from Sigma Aldrich (Table 1).

Formulated compounds were suspended in distilled water whereas stock solutions were prepared for active ingredients in different solvents (Table 1). Solvent concentration never exceeded 1% (v/v). Fungal isolates In this study six isolates were considered. They were selected from our S. vesicarium collection on the base of their different sensitivity to dicarboximides: 2 sensitive (S I, S II) and 4 resistant (R I, R II, R III, RIV) isolates. Collection was carried out between 1995 and 2004 by fungal isolations from symptomatic pear fruits of different cultivars (mainly Abbé Fétel, Conference but also Doyenne, Kaiser and Passe Crassane) collected in Po Valley.

Isolates were cultured on V8 Agar made of 20% V8 (vegetable juice, Campbell’s Grocery Ltd), 1.5% technical agar (Agar Grade A, Becton Dickinson), 0.4% Calcium Carbonate (Fluka) in dH2O amended with 50 mg l-1 of streptomycin sulphate. They were incubated at 23°C under fluorescent light (12 hours of photoperiod). Sensitivity tests The six isolates were tested in vitro for fungicide sensitivity through assay on mycelium towards most of the fungicides but through conidial assay towards strobilurins, because of their known poor activity on mycelial growth.

Assays on mycelium. Mycelial disks of pathogens (5mm in diameter) removed from the margins of a 7 day old culture were transferred to V8 media amended with fungicides. SBIs were tested at 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20 mg a.i. l-1. Cyprodinil and fludioxonyl were tested at 0.5, 1, 2.5, 5, 7.5, 10, 25, 50, 100 mg a.i. l-1; dichlofluanid and pyri-methanil at 1, 2.5, 5, 10, 25, 50, 100 mg a.i. l-1; thiram at 1, 5, 10, 25, 50, 75, 100 mg a.i. l-1; dicarboximides from 1 up to 5000 mg a.i. l-1.

Three replicates were used per treatment. For each active ingredient and concentration, inhibition of radial growth compared with untreated control was calculated after 3 days of incubation at 23°C under fluorescent light (12 hours photoperiod). Results were expressed as effective concentration EC50 (the concentration which reduces mycelial growth by 50%) determined by probit analysis. Resistance Factor RF was calculated as ratio between the average EC50 of resistant isolates and the average EC50 of sensitive ones.

Assays on conidia. Spore suspensions were prepared for each isolate from 8 day old V8 agar plates adding a few ml of water and gently scratching the surface with a spatula; they were then filtered through a 100 µm filter. Spore concentration was adjusted to about 105 conidia ml-1. One drop (20 µl) of each suspension was placed onto the surface of 1.5% water agar plates amended with different concentrations of fungicides (0.01, 0.02, 0.03, 0.05, 0.5 mg a.i. l-1) and 100 mg l-1 of SHAM (Salicylhydroxamic acid), which blocks the Alternative Oxidase. SHAM was prepared as stock solution of 10,000 mg l-1 in methanol.

The inoculated agar plates were then incubated at 23°C under fluorescent light. Germi-nated conidia (100 spores for each drop) were evaluated after 3, 5 and 24 hours. Percentage of conidia germinated after 5 hours were processed through probit analysis to obtain EC50 (the concentration which reduces conidial germination by 50%), EC95 (the concentration which reduces conidial germination by 95%), MIC (Minimal Inhibitory Concentration) and RF (Resistance Factor).

After this overview on the six strain sensitivities, obtained by comparing EC50 and RF values, fungicide inhibition effect on mycelial growth, conidial germination and hyphal

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elongation were compared in an isolate resistant to procymidone, to obtain a preliminary picture of the behaviour in vitro of dicarboximide resistant isolates towards other fungicides.

Tebuconazole, hexaconazole, fenbuconazole, myclobutanil, flutriafol, penconazole, bitertanol, difenoconazole, flusilazole, fenarimol, nuarimol, prochloraz, procymidone and ziram were tested as formulated compounds at 4, 20, 62.5, 100, 125, 250, 500 mg a.i. l-1 against mycelial growth and at 62.5, 125, 250, 500 mg a.i. l-1 against conidial germination and hyphal elongation.

Mycelial disks of pathogens (5mm in diameter) removed from the margins of a 7 day old culture were transferred to PDA media (39 g l-1, Becton Dickinson) amended with fungicides at tested concentrations. Three replicates were prepared for each concentration and all the trials were repeated three times. Mycelial growth was evaluated after 2, 4, 6, 8, 10, 12 and 14 days of incubation at 23°C and 12 hours of photoperiod (fluorescent light), measuring and averaging the two perpendicular diameters of each fungal colony. Results were expressed as relative efficacy: (control diameter – treated diameter)/control diameter ×100.

The effects of fungicides upon spore germination and germ tube elongation of S. vesicarium isolates were tested as follows. Spore suspension (5×104 conidia ml-1) was prepared in distilled sterile water from 7-10 day old cultures. Four drops of 10 µl of spore suspension were then put onto the surface of PDA plates supplemented with fungicides. The percentage of spore germination (100 spores for each drop) and the length of germ tubes (10 germinated spores for each drop) were estimated under a microscope, after 1, 2 ,3 ,4 ,5, 6, 7, 8, 24, 48 hours of incubation at 23°C, in the dark. A spore was considered as having germinated if the germ tube length was equal to at least once that of conidium. All assays were repeated three times. Results were expressed as percentage of germination and as germ tube growth rate both in relation to an unamended control. Results Sensitivity tests on mycelial growth showed the lowest EC50 values for cyprodinil and pyrimethanil (anilinopyrimidines), difenoconazole, propiconazole, flutriafol and tebuconazole (SBIs). They ranged from 0.29 to 1.4 mg a.i. l-1 for all tested isolates. These values were comparable with procymidone and iprodione EC50 values in the case of sensitive strains (from 0.62 to 0.98 mg a.i. l-1). Thiram and dichlofluanid showed very high EC50 values, they ranged from 21.2 to 83.7 mg a.i. l-1 for all tested isolates (Table 2).

Strobilurins showed very low EC50 values in sensitivity tests on conidial germination with a slightly good activity of trifloxystrobin (from 0.01 to 0.03 mg a.i. l-1) compared to kresoxim-methyl (from 0.02 to 0.04 mg a.i. l-1). EC95 values were also low: from 0.09 to 0.13 mg a.i. l-1 for kresoxim-methyl and from 0.05 to 0.12 mg a.i. l-1 for trifloxystrobin. MIC was always below 0.5 mg a.i. l-1 up to 24h (Table 3).

Resistance factors all had a value of around one, except obviously for procymidone (>5000) and iprodione (17.63) confirming the partially cross-resistance only between this two dicarboximides (Table 2 and Table 3).

In further tests carried out with a dicarboximide resistant isolate to better observe fungicide efficacy, the good activity against mycelial growth of tebuconazole, flutriafol and difenoconazole was confirmed during all the evaluations up to 14 days at the highest concentration (Table 4 and Table 5). In this test prochloraz was as active as tebuconazole, flutriafol and difenoconazole against mycelial growth.

The best activity against hyphal elongation was shown by ziram and fenbuconazole. They were able to reduce hyphal growth rate respectively up to 99.904 and 98.71 % compared to an unamended control even at the lowest concentration (Table 4).

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Table 2. Fungicide sensitivity on radial mycelial growth (EC50 and RF on fungicide-amended V8 agar for 3 days at 23°C) of Stemphylium vesicarium resistant (R I, R II, R III, R IV ) and sensitive (S I, S II) isolates to dicarboximide fungicides.

Isolate EC50 (mg a.i. l-1)

Group name Active ingredient S I S II S

average R I R II R III R IV R average

Resistance Factor

procymidone 0.93 0.98 0.96 >5000 >5000 >5000 >5000 >5000 >5235 Dicarboximides iprodione 0.77 0.62 0.70 8.00 13.5 18.0 9.50 12.3 17.6

Dithiocarbamates thiram 45.5 39.5 42.5 40.2 51.2 21.2 31.8 36.1 0.85 Sulphamides dichlofluanid 61.0 83.7 72.4 75.0 67.8 22.8 25.0 47.7 0.66

bitertanol 3.99 4.30 4.15 9.30 4.10 1.25 2.15 4.20 1.01 difenoconazole 0.61 0.89 0.75 1.20 0.49 0.53 0.29 0.63 0.84 flutriafol 1.10 1.02 1.06 1.25 0.97 1.20 1.00 1.11 1.04 hexaconazole 0.78 0.84 0.81 1.61 1.04 1.32 1.67 1.41 1.74 myclobutanil 5.81 5.73 5.77 4.06 4.06 0.30 2.58 2.75 0.48 penconazole 0.88 2.64 1.76 0.94 0.98 0.50 2.05 1.12 0.63 propiconazole 0.54 1.15 0.85 0.43 0.48 0.75 0.43 0.52 0.62 tebuconazole 0.29 0.37 0.33 0.82 0.8 1.40 0.54 0.89 2.70 prochloraz 1.34 1.75 1.55 1.34 1.53 3.43 2.42 2.18 1.41 fenarimol 2.14 2.2 2.17 1.76 1.39 1.06 1.75 1.49 0.69

SBIs

fenpropimorph 1.95 1.68 1.82 1.9 1.92 2.45 3.95 2.56 1.41 cyprodinil 0.63 0.98 0.81 0.75 0.78 0.68 0.74 0.74 0.92 Anilinopyrimidines pyrimethanil 0.50 0.54 0.52 0.53 0.54 0.55 0.54 0.54 1.04

Phenylpyrroles fludioxonil 2.20 0.70 1.45 2.18 3.75 2.33 2.20 2.62 1.80 Table 3. Strobilurin sensitivity on conidial germination (EC50, EC95, MIC and RF on

fungicide-amended water agar for 5 hours at 23°C) of Stemphylium vesicarium resistant (R I, R II, R III, R IV ) and sensitive (S I, S II) isolates to dicarboximide fungicides.

Only fenbuconazole, penconazole and ziram besides strobilurins showed very good inhibition of conidial germination (Table 4) which was stable up to 8, 24 and 48 hours respectively at the highest concentration (Table 5).

Discussion

Sensitivity studies carried out in vitro on mycelial growth showed good activity of anilinopyrimidines; many SBIs appeared also effective with better results by tebuconazole, flutriafol, difenoconazole and propiconazole. Good activity of both strobilurins (kresoxim- methyl and trifloxystrobin) and ziram was shown by the conidial germination assays while

Active ingredient

Sensitivity parameter

(mg l-1) S I S II R I R II R III R IV Resistance

Factor

kresoxim-methyl EC50 EC95 MIC

0.02 0.09 0.05-0.5

0.04 0.13 0.05-0.5

0.02 0.08 0.05-0.5

0.03 0.13 0.05-0.5

0.04 0.13 0.05-0.5

0.03 0.1 0.05-0.5

1

trifloxystrobin EC50 EC95 MIC

0.01 0.05 0.05-0.5

0.03 0.12 0.05-0.5

0.01 0.05 0.05-0.5

0.02 0.08 0.05-0.5

0.02 0.09 0.05-0.5

0.01 0.07 0.05-0.5

0.75

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Table 4. In vitro efficacy of fungicides on mycelial growth, conidial germination and hyphal elongation of a S. vesicarium isolate resistant to dicarboximides.

Active ingredients

Mycelial growth (% relative efficacy after

14 days of incubation)

Conidial germination (referred as % of

unamended control) after 3 h of incubation

Hyphal elongation (hyphal growth rate

referred as % of unamended control)

Concentrations (mg a.i. l-1) 4 20 62.5 100 125 250 500 62.5 125 250 500 62.5 125 250 500

procymidone 55 70 84 85 94 95 97 71.54 67.59 64.82 51.38 5.64 4.37 4.40 3.86ziram 42 65 75 79 85 87 98 0 0 0 0 0.096 0.103 0.038 0 bitertanol 51.8 62.6 63 63.5 64.5 65 69 98.26 93.73 91.29 87.11 55.01 51.15 45.68 42.73difenoconazole 79 93 99 99 100 100 100 33.33 28.87 1.72 0 3.67 3.32 2.96 2.77flutriafol 57 80 95.5 98.5 99.5 99.5 100 97.95 96.25 94.54 91.47 7.29 7.03 6.73 6.11hexaconazole 72 90 91 92 92.5 92.5 92.5 98.29 97.61 93.86 89.42 3.41 3.03 2.63 2.16myclobutanil 15 51 85 86 90 94 97 76.79 65.53 47.78 15.36 7.08 6.05 5.54 5.41penconazole 21 58 87 90 93 95 97 0 0 0 0 4.47 4.09 1.58 0.70tebuconazole 64 84 98 99 99 100 100 95.88 93.81 90.38 44.67 7.37 7.38 6.85 6.46prochloraz 78 96 98 99 100 100 100 84.12 78.04 72.97 52.36 4.49 4.68 4.86 4.61fenbuconazole 27 64 87 89 92 97 99 0 0 0 0 1.29 1.11 0.72 0.46flusilazole 43 79 90 95 96 96 97 71.08 62.02 50.52 26.13 7.48 7.50 7.14 4.49fenarimol 88 91 94 95 96 98 99 65.88 61.82 45.61 18.24 12.47 10.90 9.78 8.37nuarimol 89 91 91 91 93 93 95 7.39 7.00 5.06 3.11 10.47 7.88 6.22 4.21 Table 5. In vitro efficacy of fungicides on S. vesicarium: period of inhibition of conidial

germination and radial mycelial growth of an isolate resistant to dicarboximides.

among SBIs fenbuconazole and penconazole were the most active. Ziram confirmed its effectiveness also on hyphal elongation while fenbuconazole performed the best activity among SBIs. Difenoconazole, prochloraz and flutriafol had the longest period of mycelial growth inhibition (14 days) among SBIs, while penconazole and ziram demonstrated the best inhibition activity on conidial germination (24 and 48 hours respectively).

Active ingredients Inhibition of conidial germination (hours)

Inhibition of radial mycelial growth (days)

Concentrations (mg a.i.l-1) 500 250 125 62.5 500 250 125 100 62.5 20 procymidone 2 1 1 1 11 11 11 10 8 7 ziram 48 24 8 6 5 5 5 4 3 2 bitertanol 1 1 1 1 1 1 1 1 1 1 difenoconazole 4 1 1 1 14 14 14 13 13 7 flutriafol 0 0 0 0 14 13 13 13 11 5 hexaconazole 1 0 0 0 8 8 8 8 8 8 myclobutanil 2 2 1 1 11 11 11 7 7 1 penconazole 24 8 8 8 10 10 10 10 10 1 tebuconazole 1 1 1 1 14 14 13 11 11 3 prochloraz 2 1 1 1 14 14 14 13 13 11 fenbuconazole 8 8 8 8 13 13 13 11 7 3 flusilazole 1 1 1 1 12 12 12 12 12 5 fenarimol 2 1 1 1 7 7 7 7 7 6 nuarimol 1 1 1 1 6 6 6 6 6 5

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These results demonstrate that, as well as dicarboximides and dithiocarbamates, many fungicides are effective in vitro on Stemphylium vesicarium and none of them shows a cross-resistance towards dicarboximides. Actually only few of them have been developed in Italy for field control of brown spot (strobilurins, the mixture cyprodinil+fludioxonil, only tebuconazole among SBIs).

In particular the lack of SBIs among available fungicides to control the disease may be due, besides other factors, to the low dose rates applied, which are very effective in field towards other pathogens such as V. inaequalis and U. necator but not adequate to control S. vesicarium. The application rate of tebuconazole much higher than those of the other SBIs should strengthen this hypothesis.

References Alberoni, G., Collina, M., Pancaldi, D. & Brunelli, A. 2005: Resistance to dicarboximide

fungicides in Stemphylium vesicarium of Italian pear orchards. – European Journal of plant pathology. In press.

Blancard, D., Allard, E. & Brest, P. 1989: La Stemphyliose du poirier ou « macules brunes ». – Phytoma 406: 37-39.

Brunelli, A., Gherardi, I. & Adani, N. 1997: Reduced sensitivity of Stemphylium vesicarium, causal agent of pear brown spot, to dicarboximide fungicides. – Informatore Fitopatologico 47: 44-48.

Brunelli, A., Gianati, P., Berardi, R., Flori, P., Alberoni, G. & Pancaldi, D. 2004: Activity of strobilurin fungicides on pear brown spot (Stemphylium vesicarium). – Atti Giornate Fitopatologiche 2: 109-114.

Cavanni, P. & Ponti, I. 1994: Maculatura bruna del pero: una micopatia sempre d’attualità. – Rivista di Frutticoltura 12: 37-42.

Collina, M., Gherardi, I. & Brunelli, A. 2002: Acquired resistance of Stemphylium vesicarium to procymidone on pear in Italy. – Acta Horticulturae 596: 547-549.

Montesinos, E., Moragrega, C., Llorente, I., Vilardell, P. 1995: Susceptibility of selected European pear cultivars to infection by Stemphylium vesicarium and influence of leaf and fruit age. – Plant Disease 79 (5): 471-473.

Ponti, I. & Cavanni, P. 1983: Indagine preliminare sulla fitotossicità di filtrati colturali di Stemphylium vesicarium, agente della “maculatura bruna” del pero. – Informatore Fito-patologico 9: 55-57.

Ponti, I., Brunelli, A. & Flori, P. 1980: L’alternariosi delle pere: una grave minaccia per la frutticoltura padana. – Informatore Agrario 5: 8939-8944.

Ponti, I., Brunelli, A., Tosi, C., Cavallini, G. & Mazzini, F. 1996: Chemical control trials on pear brown spot (Stemphylium vesicarium). – Atti Giornate Fitopatologiche 2: 165-172.

Simmons, E.G. 1969: Perfect states of Stemphylium. – Mycologia 61: 1-26. Singh, P., Bugiani, R., Cavanni, P., Nakajima, H., Kodama, M., Otani, H. & Kohmoto, K.

1999: Purification and biological characterization of host-specific SV-toxins from Stem-phylium vesicarium causing brown spot of European pear. – Phytopathology 89: 947-953.

Singh, P., Park, P., Bugiani, R., Cavanni, P., Nakajima, H., Kodama, M., Otani, H. & Kohmoto, K. 2000: Effects of host-selective SV-toxin from Stemphylium vesicarium, the cause of brown spot of European pear plants, on ultrastructure of leaf cells. – Journal of Phytopathology 148: 87-93.


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Relationship between biological agent populations and biocontrol of Monilinia spp. in peaches Antonieta De Cal1, Inmaculada Larena1, Belén Guijarro1, Rosario Torres2, Mar Liñan1, Pietro Domenichini3, Alberto Bellini3, Xavier Ochoa de Eribe4, Josep Usall2, Paloma Melgarejo1 1Departamento de Protección Vegetal, INIA, Crtra. De la Coruña km. 7, 28040 Madrid, Spain; 2Postharvest Unit, CeRTA, Centre UdL-IRTA, 191 Rovira Roure Ave. 25198 Lleida, Catalonia, Spain; 3SIPCAM S.p.a., Via Sempione 195, 20016 Pero (Milano), Italy; 4SIPCAM INAGRA S.A., Prof. Báguena 5, 46009 Valencia, Spain Abstract: Nine field experiments were carried out in commercial peach orchards located in Spain and Italy from 2002 to 2004. Epicoccum nigrum and Penicillium frequentans treatments were applied at flowering and preharvest in a standard schedule to control brown at a concentration of approx. 106 or 107 conidia ml-1, respectively. Populations of both fungi were estimated on peach surfaces (flowers and fruits) before and after applications as 1) the number of fungal conidia per flower or fruit and 2) the colony forming units (cfu) of each fungi per flower or fruit. The number of E. nigrum and P. frequentans conidia was 1000 to 10-fold higher respectively than the cfu estimated at the same time. A consistent population of E.nigrum (ranging from 103 to 105 conidia per flower or fruit) and P. frequentans (ranging from 104 to 106 conidia per flower or fruit) were obtained. Our results suggest that fresh conidia were more susceptible to environmental stress than conidial formulations. Key words: biocontrol, brown rot, Epicoccum nigrum, Monilinia spp., Penicillium frequentans Introduction Brown rot of peaches (Prunus persica (L.) Batch) is caused in the European Mediterranean areas by the fungi Monilinia laxa (Aderh et Rulh) Honey and Monilinia fructigena Honey in Whetzel (De Cal & Melgarejo, 1999), being a serious disease that result in significant losses (Hong et al., 1998). European brown rot is usually initiated in the spring as blossom blight from inoculum derived from overwintered mummified fruits, fruit stalks, scars and buds, as well as in cankerous lesions (De Cal & Melgarejo, 2000). Under favourable environmental conditions, blossom blight can progress to twig blight and branch canker, which can serve as additional sources of secondary inoculum, and may eventually lead to latent infection of immature green fruit, and pre- and postharvest brown rot of mature stone fruits. Postharvest losses are typically more severe than preharvest losses and routinely occur during storage and transport (Ogawa & English, 1991). Fungicides (pre- and postharvest) are generally applied to reduce disease (Osorio et al., 1993). Development of pathogen resistance to fungicides (Elmer & Gaunt, 1993), strong public opinion against the use of synthetic fungicides, and health risks involved in their application make other solutions for control of the brown rot disease more desirable.

The potential of two antagonistic fungi Epicoccum nigrum Link:Fr., and Penicillium frequentans Westling, components of the resident mycoflora of peach twigs and flowers (Melgarejo et al., 1985), for biological control of peach twig blight caused by Monilinia laxa has been demonstrated in field experimental orchards under artificial inoculation of the pathogen (De Cal et al., 1990; Madrigal et al., 1994).

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The phyllosphere is a relatively harsh environment for demonstrating consistent disease control (Kinkel, 1997). Biocontrol strategies may be possible alternatives to other control methods, but understanding the ecological mechanisms involved with disease control, and the population dynamics of biocontrol agents in the environment is required for better disease control (Collins et al., 2003). Effective colonization and high population size and viability of biocontrol agents on plant surfaces have been considered to be important factors in the successful control of plant diseases (Bull et al., 1991).

This paper describes attempts to relate populations of the fungal biocontrol agents in the surface of fruits with incidence of disease. Materials and methods Isolate and inoculum preparation Epicoccum nigrum isolate 282, and P. frequentans (ATCC number 66108) obtained from healthy peach twigs in Madrid, Spain, and shown to be antagonistic to Monilinia spp. (De Cal et al., 1990, Madrigal et al., 1994) were used in all experiments. Fungi were stored on homemade potato dextrose agar (PDA) slants at 4 ºC and grown at 20 to 25 ºC for 7 days on PDA in Petri dishes for conidial inoculum production. Fresh conidia of E. nigrum and P. frequentans were produced in a solid-state fermentation system described in De Cal et al. (2002), and Larena et al. (2003). Fresh conidia of P. frequentans were resuspended in sterile distilled water and filtered through 1µm filter paper in a Büchner funnel for obtaining FOR-Pf1. Formulations FOR-EPI, and FOR-Pf2 were obtained as follows. Fresh conidia of E. nigrum and P. frequentans were resuspended in 2.5% methyl cellulose (FOR-EPI), or 10% glycerol + 2% carboximethylcellulose (FOR-Pf2), respectively, for 10 min; then, silica gel was added to FOR-EPI, and FOR-Pf2 for obtaining a conidial paste. Each conidial paste was introduced into a fluidized bed-dryer at the highest air flow rate and at 40ºC for obtaining a conidial moisture content < 15% as described in Larena et al. (2004) and Guijarro et al. (in press). Field trials Nine field experiments were carried out in commercial peach orchards located in Spain and Italy from 2002 to 2004. Trees were selected at random in each orchard. Three trees were used as the sample unit and every treatment was repeated four times. Two guard trees were used to separate sample units to avoid spray drift. E. nigrum and P. frequentans treatments were applied at flowering and preharvest in a standard schedule to control brown at a concentration of approx. 106 or 107 conidia ml-1, respectively, with a backpack sprayer operating at a pressure of 10 bar, with hollow cone nozzle of 1 mm. Orchards received the cultural and crop protection practices usual in each location. To compare E. nigrum and P. frequentans populations on peach surfaces, 10 flowers or 5 fruits per replicate and treatment were sampled 9 to 10 times in each year, respectively, in each orchard. Sampling was carried out just after treatments and also in dates not coinciding with applications. Samples were suspended in sterile distilled water (SDW), and shaken for 30 min at 150 rpm. The liquid was then concentrated by centrifugation for 10 min at 14,040x g and the pellet resuspended in 5 ml SDW. Populations of E. nigrum and P. frequentans were estimated before and after applications as 1) the number of fungal conidia per flower or fruit and 2) the colony forming units (cfu) of each fungi per flower or fruit. Numbers of conidia were counted in a haematocytometer under a light microscope (x100) in the resuspended pellet. Cfus were estimated on Petri plates containing potato dextrose agar amended with 0.5 g l-1 streptomycin sulphate (PDAs). One hundred aliquots from undiluted and diluted concentrate were spread onto PDAs. Three replicate dishes were used for each replicate and dilution. Petri dishes were maintained at 20-25 ºC for 5 to 7 days and the colonies were counted (Lacey et al., 1980). Data of number of conidia and cfu in each orchard were subjected to analysis of variance (Snedecor & Cochran, 1980). The number of conidia and cfu per flower

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or fruit were log10x and log10(x+1) transformed, respectively, before analysis to improve homogeneity of variances. When the F-test was significant at P = 0.05, treatment means were compared by Student-Newman-Keul´s test (Snedecor & Cochran, 1980). The Area Under the Number of Conidia Progress Curve (AUNCPC) and the Area Under Colony Forming Units Progress Curve (AUcfuPC) were calculated as described for Area Under Disease Progress Curve (AUDPC) (Campbell & Madden, 1990). Results and discussion The number of E. nigrum and P. frequentans conidia estimated on stone fruit surfaces (flowers and fruits) in each sampling date was 1000 to 10-fold higher respectively than the colony forming units (cfu) estimated at the same time (Fig.1). In marine environments, large differences between bacterial numbers determined by plate count method and those determined by direct microscopic methods have been observed (Jannasch & Jones, 1992). The magnitude of differences between the direct count and the plate count can be more or less in relation with the genus and the species (Wilson & Lindow, 1992). Population of Vibrio vulnificus estimated by the plate count method dropped to undetectable levels while those estimated by the direct counting still indicated populations of 104 to 105 cfu ml-1. (Linder & Oliver, 1989).

A consistent population of E.nigrum (ranging from 103 to 105 conidia per flower or fruit) and P. frequentans (ranging from 104 to 106 conidia per flower or fruit) were obtained on peach trees sprayed with E. nigrum and P. frequentans respectively following a standard schedule against brown rot (Fig.1). The log number of E. nigrum conidia estimated per flower or fruit surfaces varied from 3.9 to 4.9 for fresh conidia, and from 3.8 to 4.9 for FOR-EPI. The log number of P. frequentans conidia estimated per flower or fruit surfaces varied from 4.9 to 6.1 for FOR-Pf1, and from 4.4 to 5.9 for FOR-Pf2. Non significant differences were observed between numbers of conidia recovered on peach trees sprayed with fresh conidia of E. nigrum and FOR-EPI or between formulations of P. frequentans. Colonization of peach surfaces by both fungi appears to follow a general pattern: 1) a higher colonization in preharvest than in flowering, 2) a high population after treatments, specially after preharvest treatments, and 3) a slight decline between treatments. Studies on population dynamics in time and space have two basic goals: identification of recurring patterns in the dynamics of the population, and knowledge of the mechanisms that generate those patterns (Kinkel, 1997). Patterns in space to fungal populations were associated with peach surfaces where the biological agent was isolated. E. nigrum and P. frequentans were isolated more frequently from fruit than from flowers, as was also observed for fungi isolated from cranberry fruits vs leaves (Jeffers, 1991). There were substantial differences in the three-dimensional structure of peach flowers and fruits. In particular, peach surfaces were markedly rougher than flowers. An increase in surface roughness may alter the efficiency of immigration or emigration (Kinkel, 1997). Pattern variations of fungal populations in time were frequently correlated with specific environmental events. E. nigrum and P. frequentans were isolated more frequently in summer than in spring. The dynamics of individual populations within the epiphytic community are determined by rates of inmigration, emigration, growth and death (Wilson & Lindow, 1992). Both immigration and emigration are strongly influenced by the physical environment, including wind, rainfall and solar radiation (Jacobs & Sundin, 2001). No significant differences (P = 0.05) were observed between the AUNCPC of fresh and formulated cells for each fungi (Fig. 2).

The log (cfu+1) of E. nigrum conidia estimated per flower or fruit surfaces varied from 0.2 to 1.4 for fresh conidia, and from 0.5 to 1.6 for FOR-EPI (Fig. 1). The log (cfu+1) of P.

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Figure 1. Population dynamics of Epicoccum nigrum (EPI) and Penicillium frequentans (Pf) as log (nº of conidia) (___) and log [colony forming units (cfu)+1] (- -) recovered from flower or fruit, in Alfarras (Lleida-Spain) (fresh conidia (•,○) or formulations (■,□). Numbers of conidia were counted in a haematocytometer under a light microscope (x100). Cfu of were estimated on potato dextrose agar amended with 0.5 g l-1 streptomycin (PDAs). Data are the mean of four replicates, with 10 flowers or 5 fruits per replicate.

frequentans conidia estimated per flower or fruit surfaces varied from 2.1 to 5.7 for FOR-Pf1, and from 2.0 to 4.8 for FOR-Pf2 (Fig. 1). Fungal colony forming unit pattern estimated on peach surfaces after applications of fresh conidia of E. nigrum were different to that estimated after application of FOR-EPI (Fig.2). Higher colony forming units were recorded on peach fruits than on flower surfaces after applications of fresh conidia, while colony forming units were maintained throughout the season on peach surfaces treated with FOR-EPI. Furthermore, the area under colony forming units curve (AUcfuPC) of FOR-EPI were significantly higher than those of fresh conidia (P = 0.05). Our results suggest that fresh conidia were more susceptible to environmental stress than conidial formulations. A rapid colonization of blossom tissues by E. nigrum and P. frequentans and their ability to survive and colonize peach surfaces under field conditions would be a important prerequisite for effective biological control of brown rot.

EPI

Pf

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Figure 2. Effect of different treatments of Epicoccum nigrum (A) and Penicillium frequentans (B) on area under the number of conidia progress curve (AUNCPC) (▒) and area under colony forming units progress curve (AUcfuPC) (█). Data are the mean of four replicates.

Acknowledgements This work has been carried out with financial support from the European Commission project QLK-1999-01065 and from AGL2002-4396-CO2 (Plan Nacional de I+D+I, Ministerio de Educación y Ciencia, Spain). We wish to thank to A. Barrienuevo, MT. Clemente and Y. Herrainz for technical support, and growers for support and collaboration. References Bull, C.T., Weller, D.M. & Thomashow, L.S. 1991: Relationship between root colonization

and suppression of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens strain 2-79. – Phytopathology 81: 954-959.

Campbell, C.L. & Madden, L.V. 1990: Introduction to Plant Disease Epidemiology. – New YorkWiley-Interscience.

Collins, D.P., Jacobsen, B.J. & Maxwell, B. 2003: Spatial and temporal population dynamics of a phyllosphere colonizing Bacillus subtilis biological control agent of sugar beet cercospora leaf spot. – Biol. Control 26: 224-232.

De Cal, A. & Melgarejo, P. 1999: Effects of long-wave light on Monilinia growth and identification of species. – Plant Dis. 83: 62-65.

A

B

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De Cal, A. & Melgarejo, P. 2000: Momificado de los frutales de hueso. – In: eds. Montesinos, Melgarejo, Cambra and Pinochet, Ediciones Mundi-Prensa, Madrid, Spain: 66-67.

De Cal, A., M-Sagasta E. & Melgarejo P. 1990: Biological control of peach twig blight (Monilinia laxa) with Penicillium frequentans. – Plant Pathol. 39: 612-618.

De Cal, A., Larena, I., Guijarro, B. & Melgarejo, P. 2002: Solid state fermentation to produce conidia of Penicillium frequentans, a biocontrol agent against brown rot on stone fruits. – Biocontr. Sci. Technol. 12: 715-725.

Elmer, P.A.G. & Gaunt, R.E. 1993: Effect of frequency of dicarboximide applications on resistant populations of Monilinia fructicola and brown rot in New Zealand orchards. – Crop Prot. 12: 83-88.

Guijarro, B., Larena, I., Melgarejo, P. & De Cal, A. 2005: Effect of drying on viability of Penicillium frequentans, a biological control agent against brown rot disease caused by Monilinia spp. – Biocontr. Sci. Technol. in press.

Hong, C., Michailides, T.J. & Holtz, B.A. 1998: Effects of wounding, inoculum density, and biological control agents on postharvest brown rot of stone fruits. – Plant Dis. 82: 1210-1216.

Jacobs, J.L. & Sundin, G.W. 2001: Effect of solar UV-B radiation on a phyllosphere bacterial community. – Appl. Environ. Microbiol. 67: 5488-5496.

Jannasch, H.W. & Jones, G.E. 1992: Bacterial populations in seawater as determined by different methods of enumeration. – Limnol. Oceanogr. 4: 128-139.

Jeffers, S.N. 1991: Seasonal incidence of fungi in symptomless cranberry leaves and fruit treated with fungicides during bloom. – Phytopathology 81: 636-644.

Kinkel, L.L. 1997: Microbial population dynamics on leaves. – Ann. Rev. Phytopathol. 35: 327-347.

Lacey, J., Hill, S.T. & Edwards, M.A. 1980: Microorganisms in stored grains: their enumeration and significance. – Trop. Stor. Prod. Inf. 39: 19-33.

Larena, I., De Cal, A., Liñán, M. & Melgarejo, P. 2003: Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease. – J. Appl. Microbiol. 94: 508-514.

Larena, I., De Cal, A.& Melgarejo, P. 2004: Solid substrate production of Epicoccum nigrum conidia for biological control of brown rot on stone fruit. – Int. Food Microbiol. 94: 161-167.

Linder, K. & Oliver, J.D: 1989. Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. – Appl. Environ. Microbiol. 55: 2837-2842.

Madrigal, C., Pascual, S., & Melgarejo, P. 1994: Biological control of peach twig blight (Monilinia laxa) with Epicoccum nigrum. – Plant Pathol. 43: 554-561.

Melgarejo, P., Carrillo, R. & M.-Sagasta, E. 1985: Mycoflora of peach twigs and flowers and its possible significance in biological control of Monilinia laxa. – Trans. Brit. Mycol. Soc. 85: 313-317.

Ogawa, J.W. & English, H. 1991: Diseases of temperate zone tree fruit and nut crops. – Univ. Calif. Div. Agric. Nat. Res. Oakland. Publ. 3345.

Osorio, J.M., Adaskaveg, J.E. & Ogawa, J.W. 1993: Comparative efficacy and systemic activity of iprodione and the experimental anilide E-0858 for control of brown rot on peach fruit. – Plant Dis. 77: 1140-1143.

Snedecor, G.W. & Cochran, W.G. 1980: Statistical Methods. 17th Edn. – Ames, Iowa, USA: The Iowa State University Press.

Wilson, M. & Lindow, S.E. 1992: Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae. – Appl. Environ. Microbiol. 58: 3908-3913.


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Modelling dynamics of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot of pear Simona Giosuè1, Vittorio Rossi1, Riccardo Bugiani2, Chiara Mazzoni2 1Istituto di Entomologia e Patologia vegetale, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, I-29100 Piacenza, Italy; 2Servizio Fitosanitario, Regione Emilia-Romagna, Via di Saliceto 81, I-40128 Bologna, Italy Abstract: The BSPcast model has been validated in the pear-growing areas of the Emilia-Romagna region (North Italy) and it gave satisfactory results in identifying infection periods of brown spot, so that it is now in use for advising farmers. Nevertheless, this model produces unjustified alarms when the airborne inoculum of S. vesicarium is absent or at low densities. To improve the accuracy of BSPcast, a model (BSPspor) able to simulate the dynamic of inoculum density (i.e. airborne spores) was elaborated. This model allows daily estimation of the potential for inoculum availability, using meteorological data. The model was elaborated using data collected from volumetric spore samplers in several epidemiological conditions (years and locations) and from laboratory experiments. BSPspor calculates an index, cumulated over a 3-day period, based on favourable conditions of temperature and moisture, and corrected by an index of seasonality. To reduce early unjustified alarm, BSPcast outputs can be considered for warnings only when the BSPspor model in the previous days had signalled a peak of inoculum. Key words: Pyrus communis, environmental conditions, spore sampling, simulation model, disease management Introduction Brown spot, caused by Stemphylium vesicarium (Wallr.) Simmons, is one of the most important pear diseases in the Emilia-Romagna region (northern Italy) (Ponti et al., 1982) as in other European pear-growing areas, including Spain (Vilardell, 1988), France (Blancard et al., 1989), The Netherlands (Van Dijke, 2002), Portugal (Llorente et al., 2003), and Belgium (Van Laer & Creemers, 2005). The disease causes severe yield losses that can reach 80-90% of affected fruits. Control measures aimed at reducing inoculum density and producing unfavourable conditions for disease development have almost no effect. Therefore, intensive applications of fungicides (with a time span of 7-14 days) are necessary in the period between the end of flowering and harvesting (Ponti et al., 1996).

Following this approach, 15 to 20 fungicide sprays are needed to control brown spot; therefore, strategies are necessary to rationalize fungicide treatments. For this reason epidemiological models have been elaborated to understand when environmental conditions are favourable for brown spot infections to occur. A model elaborated by Montesinos et al. (1995), has been validated in the pear-growing areas of the Emilia-Romagna region and it gave satisfactory results in identifying infection periods of brown spot (Bugiani & Gherardi, 1998; Bugiani et al., 2004). As a consequence, this model, named BSPcast is now in use for advising farmers (www.regione.emilia-romagna.it/fitosanitar). Nevertheless, this model produces some unjustified alarm when S. vesicarium inoculum is absent or at low densities. To complement model outputs spore traps are used to monitor the airborne inoculum, but they are expensive and time-consuming (Bugiani et al., 2004).

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To improve the accuracy of BSPcast, a model able to simulate the dynamic of inoculum availability (i.e. airborne spores) as a function of meteorological variables was elaborated. Materials and methods Seven-day volumetric spore samplers (Lanzoni, VPPS-2000, Bologna, Italy) were installed in pear orchards in northern Italy, between 1993 and 2002, and operated continuously during the development of brown spot epidemics (mid April to mid August). Hourly values of temperature (T), relative humidity (RH), rainfall (R), wetness duration (W), average and maximum wind speed were recorded from automatic weather stations placed near the pear orchards during the whole period of spore sampling. Daily averages or summations of the previous variables were calculated starting from hourly values; vapour pressure deficit (VPD) was calculated as a function of temperature and relative humidity.

A previous analysis of these data (Rossi et al., 2005) showed that the dynamic of airborne spores during the season had a regular pattern over time: in the early season conidia are nearly absent, and later they increase until they reach a peak; thereafter, spores are constantly present and periods with high number of spores alternate with low densities. Differences between years concerned the date of the first spore peak, the number of peaks and total spore concentrations. There was a significant correlation between spore peaks (>30 conidia·m-3·day-1) and days with favourable weather conditions, defined as days with air temperature between 15° and 25°C and high humidity (RH≥80%, R>0, W>10h, VPD≤5). Occurrence of one or more consecutive days with favourable weather conditions determined an increase in the airborne concentration of conidia, which usually lasted some days and then decreased. Model structure The model calculates an index, named BSPspor (acronym of brown spot prediction for sporulation), as:

BSPspori = µi·[ τi + f(Ti)]·ζi where: i = is the day of the spore sampling season;

µ = index of the presence of favourable conditions of moisture; f(T) = rate of spore production calculated as a function of air temperature; τ = index of the presence of favourable conditions of temperature; ζ = index of seasonality in spore trappings.

The final BSPspor index is the accumulation of the daily values over a 3-day period, as the BSPcast risk index does: BSPspori + BSPspori-1 + BSPspori-2.

The µ index is calculated daily as: µi = rhi + ri + wi + vpdi where: if RHi ≥80%, rhi =1, otherwise rhi = 0;

if Ri >0 mm, ri =1, otherwise ri = 0; if Wi >10 h, wi =1, otherwise wi = 0; if VPDi ≥5 hPa, vpdi =1, otherwise vpdi = 0.

Then µ ranges between 0 (none condition is verified) and 4 (all conditions are verified). The τ index is calculated daily as: τi = ti where: if 15≤Ti ≤25°C, ti =1, otherwise ti = 0. The value f(T) is calculated daily as:

f(Ti) = [7.765*(Teqi) 2.38*(1-Teqi)] 6.824 where Teq is an equivalent of T, calculated as: (Ti-3)/30.

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This equation was obtained by fitting experimental data on spore production. Six strains of S. vesicarium were cultured on V8 agar, with a photoperiod of 12 hours of fluorescent light and 12 h of darkness, at different T regimes (5 to 37°C). Conidia produced after 31 days of incubation were enumerated microscopically and relative sporulation was calculated for

each strain by dividing each value for the maximum observed. Average relative sporu-lation was calculated over the 6 fungal strains and these data were fitted to a Bete function using the non-linear regression procedure of SPSS ver. 11.5 (SPSS Inc., Chicago, IL). The index f(T) therefore accounts for the effect of T on the production of conidia on the inoculum sources (Fig. 1).

The ζ index is an index of seasonality in spore samplings; it is calculated according to the

graph of Fig. 2. Numbers of conidia trapped over a 10-year period (1993 to 2002) were standardized by dividing each daily value by the total number of conidia trapped each year, so that the standardized value represents the contribution of a day to the seasonal conidia. The average of these standardized values was then calculated for each day over the 10-year period; finally the 7-day moving averages of these daily averages were calculated: ζ was empirically determined taking into account maximum values of the 7-day moving averages.

0.00

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Inde

xof

seas

onal

ityζ

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Inde

xof

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onal

ityζ

1 10 19 28 37 46 55 64 73 82 91 100 109 118Days from April 15

1 10 19 28 37 46 55 64 73 82 91 100 109 118Days from April 15

Figure 2. Dynamic of the index of seasonality ζ in airborne S. vesicarium conidia (─), in comparison with the 7-day moving averages of the standardized spore numbers caught with a volumetric spore sampler from the pear orchard air over a 10-year period (1993 to 2002) (---).

0.0

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e pr

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tion

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Spor

e pr

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Figure 1. Influence of temperature (T) on relative production of conidia by S. vesicarium.

172

Model validation The model was operated using the meteorological data recorded by automatic weather stations placed near the pear orchards considered for the monitoring of S. vesicarium conidia, between 1993 and 2002. Daily data of T, RH, R, W, and VPD were used as input variables to calculate daily and cumulated values of BSPspor, between 15 April and 15 August.

Model outputs were then compared with the daily numbers of conidia caught by the volumetric spore samplers installed in the pear orchards. Details on spore catches have been previously published (Rossi et al., 2005). Results and discussion Model outputs were obviously influenced by weather conditions, because the index of seasonality ζ depends only on the day of the sampling season (Fig. 2). Effects of these conditions on the moisture index µ and the spore production index f(T) are shown in Fig. 3 for two representative years: 1997 (Fig. 3A and 3C) and 2002 (Fig. 3B and 3D).

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Day (15 April to 15 August) Figure 3. Dynamics of the model indexes of moisture (µ) and spore production (f(T))

calculated using meteorological data collected in 1997 (A and C, respectively) and 2002 (B and D, respectively).

The µ index accounts for the presence of favourable conditions of moisture; it ranges between 0 and 4, when the weather is very moist. The examples of Fig. 3A and 3B show the dynamics of µ in two years of spore samplings: in 1997 (A) favourable conditions of humidity are less frequent than in 2002 (B) in the period between the beginning of May and the first half of June, while in the following period, favourable conditions are frequent in both cases.

The effect of air temperature is accounted for the indexes τ and f(T). In 1997 the period with favourable conditions of T started on 25 April while in 2002 it began earlier (mid-April) (not shown). The index of spore production (f(T)) had similar dynamics in the two years, with the exception of a period between the end of May and the beginning of June: in 2002 high

173

peaks of spore production were observed (Fig. 3C), while in 1997 spores were produced at lower rates (Fig. 3D).

In most cases there was a close relationship between outputs of the BSPspor model and airborne concentrations of S. vesicarium conidia, with accurate simulations of both the first seasonal peak of conidia and dynamic of airborne spores during the sampling season (Fig. 4).

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0

30

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90

1200

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60

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AA

CC

BB

DD

Figure 4. Comparison between the dynamic of S. vesicarium conidia sampled from pear

orchard air (▲) and outputs of the BSPSpor model (—) in 4 years: A, 1996; B, 1997; C, 1998; D, 2002.

However, the model showed some discrepancies from actual data, especially in the late season (Fig. 5). In 1995 (Fig. 5B) and 1999 (Fig. 5C) the model overestimated actual spore concentration after mid July, while in 1994 (Fig. 5A) and 2001 (Fig. 5D) it underestimated it. On the contrary, the dynamic of S. vesicarium conidia in the early season was estimated correctly, with the only exception of the year 1995: in this year the model did not simulate an early period of spore peak at the end of April and also underestimated spore concentrations in May (Fig. 5B).

Validation of the BSPspor model showed a good agreement between model simulations and dynamics of airborne S. vesicarium conidia in pear orchard affected by brown spot. In general, model accuracy was higher early in the season than later, when the presence of airborne spore is more erratic. The model was very accurate in determining the period of first spore peaks, that are of great interest in disease management because they define the time when there is an actual risk for infection taking place.

Therefore, it can be concluded that the BSPspor model can potentially improve BSPcast simulations: in order to reduce early unjustified alarm, BSPcast risks should be considered only when BSPspor signals the presence of significant airborne inoculum.

174

30

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)A

irbor

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)

Day (15 April to 15 August) Figure 5. Comparison between the dynamic of S. vesicarium conidia sampled from pear

orchard air (▲) and outputs of the BSPSpor model (—) in 4 years: A, 1994; B, 1995; C, 1999; D, 2001.

In the example of Fig. 6, the BSPcast index repeatedly overcame the band of action

threshold (0.3 to 0.6) starting from early April, with two high peaks between the end of April and the beginning of May (Fig. 6B): these peaks would suggest an early application of fungicides to prevent infection. The BSPspor index was very low in this period, showing a low airborne inoculum, so that these fungicide applications should be saved (Fig. 6A). Actually the spore sampler installed in the pear orchard confirmed the low presence of S. vesicarium conidia in the air (Fig. 6A), and no brown spot symptoms appeared on the unsprayed trees as a consequence of these possible infection events (Fig. 6C).

According to BSPspor outputs the first peak of spores was expected, and actually observed, between the end of May and the first days of June (Fig. 6A), when favourable conditions for infections were also signalled by BSPcast (Fig. 6B). These conditions actually favoured infection, and disease symptoms appeared in early June (Fig. 6C). Four more periods of risk were defined based on outputs of the two models, the first one, mid June, with very favourable conditions for both inoculum and infection, that resulted in a severe increase of the disease (Fig. 6C). In this example, the use of BSPspor saved 3 unjustified fungicide treatments at the beginning of the season, with respect to the use of BSPcast only.

In conclusion, BSPspor model can be considered a useful tool for rationalizing fungicide treatments against brown spot on pear, when associated with the BSPcast model.

Acknowledgements This work was funded by the Emilia-Romagna Region and co-ordinated by CRPV.

175

BSPc

ast(

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20

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cted

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ast(

---)

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22/4

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50

100

150

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Airb

orne

spor

es /m

3ai

r (

)

C

10

20

30

40

0

Affe

cted

fruits

(%)

Day (1 April to 30 August) Figure 6. Example of the use of BSPspor and BSPcast models for defining the periods of risk

for brown spot infections on pear. A) Comparison between dynamics of airborne spore samplings (▲)and BSPSpor outputs (⎯) (dotted line define spore peaks higher than 30 conidia/m3 air day); B) BSPcast risk index (---) and its thresholds at 0.3 and 0.6 (⎯); C) risk periods for S. vesicarium infection based on both the risk indexes (■) and progress of pear fuits affected by brown spot in unsprayed ‘Conference’ trees (•) (---) indicates the fitting of disease incidence data).

References Blancard, D., Allard, E. & Brest, P. 1989: La Stemphyliose du poirier ou ‘macules brunes’. –

Phytoma 406: 37-38. Bugiani, R. & Gherardi, I. 1998: Nuovi indirizzi per la razionalizzazione della difesa del pero

dalla maculatura bruna. – Informatore Fitopatologico 48 (6): 65-70. Bugiani, R., Giosuè, S., Rossi, V. & Spada, G. 2004: I modelli previsionali per la lotta alla

maculatura bruna del pero. – www.phytomagazine.com. 3(6): 43-50. Llorente, I., Vilardell, P., Moragrega, C., Bonaterra, A. & Montesinos, E. 2003: Biology,

epidemiology and integrated control of Stemphylium vesicarium on pear, an emerging

176

disease of economic impact in Europe. – In: 8th International Congress of Plant Pathology, Christ Curch, New Zealand.

Montesinos, E., Moragrega, C., Llorente, I., Vilardell, P., Bonaterra, A., Ponti, I., Bugiani, R., Cavanni, P. & Brunelli, A. 1995: Development and evaluation of an infection model for Stemphylium vesicarium on pear based on temperature and wetness duration. – Phytopathology 85: 586-592.

Ponti, I., Cavanni, P. & Bugiani, R. 1982: Maculatura bruna delle pere: eziologia e difesa. –Informatore Agrario 32(3): 35-40.

Ponti, I., Brunelli, A., Tosi, C., Cavallini, G. & Mazzini, F. 1996: Aggiornamenti sull’attività dei fungicidi contro la maculatura bruna del pero. – Atti Giornate Fitopatologiche 2: 165-172.

Rossi, V., Bugiani, R., Giosuè, S. & Natali, P. 2005: Patterns of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot disease of pear, in relation to weather conditions. – Aerobiologia, in press.

Van Dijke, J.F. 2002: Incidence of pear fruit spot can increase explosively. – Fruitteelt, Den Haag 92: 8-9.

Van Laer, S. & Creemers, P. 2005: State of the art in Belgium. – 1st European Brown Spot Workshop, Leusden, The Netherlands.

Vilardell, P. 1988: Stemphylium vesicarium en plantaciones de peral. – Fruticultura Profesio-nal 18: 51-55.


pp. 177 - 180

177

Fungicide resistance in apple scab in the province of Québec: an overview of the problem and its implications for disease management Tristan Jobin, Odile Carisse Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin, St-Jean-sur-Richelieu, Qc, Canada, J3B 3E6. Abstract: Since no study has been published on fungicide resistance in the province of Québec for over ten years, we conducted a large survey of the incidence of resistance in isolates of Venturia inaequalis in commercial orchards. Based on criteria set in the literature, we found that 70 % of the isolates departed from baseline for myclobutanil and 34 % for dodine. Over 60 % of the isolates did not respond to high doses of thiophanate-methyl. No cross-resistance has been observed between the different classes of fungicides. For now, no adverse affect on disease control in commercial orchards has been related to this general loss of sensitivity to post-infection fungicides. Key words: fungicide resistance, apple scab, Venturia Introduction In several apple growing areas throughout the world, fungicide resistance in apple scab is a concern for growers. In the province of Québec, Canada, the same situation applies and the loss of good, reliable molecules usable in post-infection strategies and not prone to resistance could make disease control harder for several growers. For over 10 years now, no information has been published on fungicide resistance in Québec. The only survey for fungicide resistance was conducted in 1994 and it targeted only the fungicide fenarimol (Carisse & Pelletier, 1994). Considering that only few fungicides with post-infection activity are available, growers cannot afford to lose these fungicides because of resistance. A two fold research program was initiated in 2003 on 1) assessment of actual status of fungicide resistance throughout the province; 2) determination of implications of fungicide resistance for scab management. Materials and methods In 2003, orchards with various incidence of apple scab were sampled in all apple producing regions in the province. Scab lesions were collected and monoconidial culture produced. More than 200 monoconidial isolates of Venturia inaequalis were tested in vitro for their sensitivity to myclobutanil, kresoxym-methyl, dodine and thiophanate-methyl by measuring radial mycelial growth after 4 weeks on PDA amended with increasing doses of fungicide. Results and discussion Among all isolates tested, 70% of the isolates had ED50 exceeding criteria set by Köller et al. (1991) and were considered as resistant to myclobutanil (Figure 1).

Considering that most of the post-infection sprays in orchards in Québec are made with sterol demethylation inhibitors (DMIs) and that cross-resistance in this family has already

178

Figure 1. Distribution of isolate sensitivities to the DMI fungicide myclobutanil in

commercial orchard in the province of Québec. The arrow indicates threshold of resistance according to Köller et al. (1991).

Figure 2. Distribution of isolate sensitivities to the fungicide dodine in commercial orchard in

the province of Québec. The arrow indicates threshold of resistance according to Köller & Wilcox (1999).

ED50 Myclobutanil0.0105-0.0189

0.0189-0.034

0.034-0.0612

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3.7481-6.7466

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Nb

Isol

ates

0

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20

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40

50

60

n = 289 % Res. Isolates : 70 %

ED50 Dodine

0.0058-0.0105

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21.859+

Nb

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ates

0

10

20

30

40

50

n = 164 % Res. Isolates : 34 %

179

been demonstrated, this high frequency of resistant isolates calls for a rapid change in spraying strategies. Based on criteria set by Köller & Wilcox (1999), 34% of the isolates were resistant to dodine (Figure 2). This product has been recently re-introduced in Canadian orchards after several cases of disease control failures in the United States leaded to its discontinuation. But resistance is still a concern since history of dodine use in Québec is merely known and the actual recommended dose is lower than in the past, leading to many questions regarding the resurgence of resistance. More than 60% of the isolates did not show growth inhibition to high doses of thiophanate-methyl (data not shown). Although benzimidazoles are not nearly as used as before, these results should discourage any growers to rely on them for adequate control. Radial growth of 13% of the isolates was not affected by high doses of kresoxym-methyl unless SHAM was added to the medium, meaning that the resistance displayed by those isolates comes from alternative respiration, which has no adverse effect on disease control in the orchard and that strobilurins might be, for now, a good option for a spray program based on molecule rotation. A recent paper (Köller et al., 2004) suggested that this type of resistance might be leading to qualitative resistance, which leads to disease control failure but no evidence has been raised yet. No positive correlation for cross-resistance has been found for any combinations among myclobutanil, kresoxym-methyl and dodine. Data for myclobutanil and kresoxim-methyl interaction is presented in Figure 3.

Figure 3. Correlation between myclobutanil and kresoxim-methyl ED50 for 192 isolates tested

in vitro.

Despite apparent high levels of fungicide resistance in vitro, no obvious relationships between level of resistance detected in vitro and disease control have been shown yet. The molecules tested in vitro are currently tested in vivo using in-vitro-produced apple trees. This will establish the reliability of trials conducted in vitro and provide information on efficacy of these fungicides in orchard with various level of resistance. The impact of fungicide resistance on aggressiveness is also being investigated by inoculating young rooted trees with sensitive and insensitive isolates. From the results, anti-resistance strategy will be put in place to predict and slow down the development of resistance in commercial orchards.

ED50 Myclobutanil0 2 4 6 8 10 12

ED

50 K

reso

xim

-met

hyl

0

2

4

6

8

10

12

n = 192r = 0.09

180

References Carisse, O. & Pelletier, J.R.. 1994: Tolerance of Venturia inaequalis to fenarimol: Baseline

sensitivity and sensitivity distribution. – Phytoprotection 75: 35-43. Köller, W., Parker, D. & Reynolds, K. 1991: Baseline sensitivities of Venturia inaequalis to

sterol demethylation inhibitors. – Plant Dis. 75: 726-728. Köller, W. & Wilcox, W. F. 1999: Quantification, persistence and status of dodine resistance

in New York and Michigan orchard populations of Venturia inaequalis. – Plant Dis. 83: 66-70.

Köller, W., Parker, D.M, Turechek, W.W., Avila-Adame, C. & Cronshaw, K. 2004: A Two-Phase Resistance Response of Venturia inaequalis Populations to the Qoi Fungicides Kresoxim-methyl and Trifloxystrobin. – Plant Dis. 88: 537-544.


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New strategies to improve the efficacy of BSPcast for control of Stemphylium vesicarium on pear Isidre Llorente, A. Vilardell, Pere Vilardell, Emilio Montesinos Institute of Food and Agricultural Technology-CeRTA. University of Girona. Campus de Montilivi. 17071 Girona (Spain). [email protected] Abstract: Control of brown spot of pear requires fungicide treatments during the growing season. Scheduling fungicide sprays with BSPcast provides fungicide savings ranging from 20 to 70%, but without increasing the disease control levels achieved with the fixed spray schedule. New strategies have been tested in order to increase the efficacy of control using the BSPcast. A BSPcast modified was used and compared to the non modified BSPcast. The BSPcast modified included the new knowledge on the effect of RH during interrupted wetness and used the daily risk (R=0.2) as action threshold instead of the 3-day cumulative risk (CR). Trials were performed during two years in an experimental pear orchard in Spain. We can conclude that the use of a daily risk as action threshold does not improve the efficacy on control. On the other hand biological, chemical and mechanical methods were evaluated in field for reduction of ascospore amount. Mechanical methods consisting of leaf shredding or removal were the most effective. Biological control methods based on the application of Thichoderma sp. formulates were partially effective. Chemical methods based on copper and urea treatments were ineffective. Introduction Brown spot of pear (Pyrus communis L.) is a disease caused by the fungus Stemphylium vesicarium (Wallr.) E. Simmons that produces important economic losses in several fruit tree growing areas of Europe including Spain, Italy, France, Holland, Portugal and Belgium (Montesinos et al., 1995a; Ponti et al., 1982; Rossi et al., 2005). The control of brown spot of pear requires high number of fungicide sprays during the pear growing season that can be applied according to a fixed schedule (every 7 to 15 days) or timed according to the BSPcast forecasting system (Llorente et al., 2000, Montesinos et al. 1995b). BSPcast guided schedule of fungicide sprays provides with average savings in fungicide ranging from 20 to 70%. However, the use of BSPcast does not increased disease control levels compared to the fixed spray schedule. The BSPcast is based on an algorithm which computes the cumulated hours of wetness during 24 h intervals, and does not take into account the effect of interruptions of wetness and the RH during the interruption. Several studies have determined the effect of duration and RH of dry periods interrupting wetness periods (Llorente and Montesinos, 2002). They have demonstrated that no modification in the model predictions is necessary if RH during wetness interruptions is high (≥ 96%), but that wetness periods must be considered as interrupted if the length of the interruption is ≥ 3 h at low RH. On the other hand, the BSPcast uses as action threshold the cumulative risk (CR) obtained totalizing the daily risk (R) for 3 days. As the efficacy of fungicides depends on their action mechanisms in the infection process, and the most effective fungicides, as thiram, are preventive and inhibit the conidia germination, we considered that the use of a daily risk index instead of the 3-day cumulative risk (CR), would probably increase the model accuracy, since fungicides would be applied when the infection starts and it is expected than they will be more effective.

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Under conditions with high disease pressure the efficacy of control measures is not sufficient. To improve disease control complementary methods oriented to decrease inoculum potential are needed. The disease cycle of brown spot of pear is characterised by two different phases. The pathogen overwinters in dead leaves on the ground as pseudothecia (Pleospora allii Rabenth. Ces&DeNot) (Llorente & Montesinos, 2004). In spring the ascospores are released and produce the primary infection. Later, if environmental conditions are favourable, the conidia provoke secondary infections during summer. It is expected that a reduction in the amount of the overwintering inoculum, would decrease the levels of disease incidence in the subsequent year.

The objectives of this work were 1) to evaluate modifications on the BSPcast model in order to improve its efficacy in disease control and 2) to evaluate control methods to decrease the overwintering inoculum and reduce the disease pressure. Materials and methods Evaluation of BSPcast modifications to improve its performance on control of Stemphylium vesicarium on pear A BSPcast modified was compared to the BSPcast model in the control of brown spot of pear. The modifications consisted of the use of the daily risk (R) as action threshold instead of the 3-days cumulative risk (CR). However, the effect of duration of wetness interruption and the RH during the interruption were also incorporated. The dynamics of wetness was analyzed every 24 hours, and if the wetness period was continuous the daily risk (R) was calculated according to the BSPcast, but if wetness was interrupted a detailed analysis of wetness and RH was done. In cases in which the RH during wetness interruptions was low (<96%) wetness periods were considered as interrupted if the length of the interruption was ≥ 3 h, in the other cases the wetness period was considered continuous, then a daily risk (R) was calculated. The action threshold evaluated was R≥0.2. Trials were performed during two years in an experimental pear orchard (cv Passe Crasse) in Spain. The experimental design consisted of randomized blocks with 3 blocks and 5 trees per block. Treatments tested were applications of fungicide according to BSPcast using as action threshold CR≥0.4 or using R≥0.2 and finally the non-treated control. The fungicide used was thiram. Evaluation of different methods to decrease the overwintering inoculum of Pleospora allii With the purpose of decreasing the overwintering inoculum and consequently decreasing the disease pressure, different methods were evaluated. Naturally infected pear leaves showing brown spot lesions caused by S. vesicarium were collected at the end of October and placed into a rectangular device covered with a net. Biological, chemical and cultural methods for control of P. allii were evaluated. In all trials field plot treatments were arranged in a completely randomized block design with three repetitions.

For biological control a commercial formulate of fungus Trichoderma sp. (Trichomic from Trichodex-AMC Chemical, Sevilla, Spain) was used. The Trichoderma treatments were sprayed when the first mature ascospore was observed and repeated few weeks later. The doses of application were 100 ml of a concentration of 1 ml/l or 100 ml/l depending on the trial.

For chemical control Bordeaux mixture (2 g a.i./l) or copper oxychloride (5 g a.i./l) were used spraying 300 ml per repetition. Urea (5 or 10% solution (wt/v)) was also tested. In some trials urea was applied directly to the trees before leaf fall or after the leaves were collected.

Mechanical methods consisted of shredding the leaves with a vortex to obtain small pieces or completely removing leaves from soil.

Ascospore traps were installed in each treatment repetition. Traps consisted of glass slides with the down surface painted with a silicon solution. These slides were positioned

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above the leaves. Glass slides were removed and replaced every 7 to 10 days and transported to the laboratory. The amount of P. allii trapped ascospores was determined by microscope evaluation of two longitudinal traverses for each slide.

Five trails are presented in this work. All trials were performed during three years in different experimental or commercial pear orchards in Spain. Results and discussion Evaluation of BSPcast modifications to improve its performance on control of Stemphylium vesicarium on pear A total of 10 and 15 fungicide applications were performed using the BSPcast (CR≥0.4) in the two trials, whereas using the BSPcast modified (R≥0.2) a total of 11 and 13 applications were done, respectively. Therefore no reduction in the number of fungicide applications was obtained using the BSPcast modified. On the other hand, a similar disease level was obtained using BSPcast and BSPcast modified, and the reduction was significant in comparison to the non-treated controls (Fig. 1 and 2). Modifications in the BSPcast were introduced in order to improve its efficacy in the control of the disease, but we have shown that these modifications are not sufficient. These results can be explained because the daily and cumulative risk dynamics are very similar for the two strategies, and the days in which the risk was higher than the action threshold were the same in the two strategies. Consistently, in most cases, the fungicides were applied on the same days in both strategies.

We can conclude that the use of a daily risk as action threshold did not improve the efficacy on control. Neither the savings in fungicide applications nor the level of disease control were significantly different from those obtained according to the BSPcast. Therefore the use of BSPcast using as action threshold the cumulative daily risk is a useful tool, but an alternative can be to schedule the treatments according to a daily risk (R≥0.2).

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treated with thiram according to BSPcast (threshold CR=0.4) or BSPcast modified (threshold R=0.2). Data correspond to trial 2. Values of disease incidence and severity are the mean of three repetitions of 5 trees per repetition. Mean standard errors are presented.

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treated with thiram according to BSPcast (threshold CR=0.4 ) or BSPcast modified (threshold R=0.2) in trials 1 (A, C) and 2 (B, D). Bars correspond to the mean of three replicates. Values followed by the same letter are not significantly different (P=0.05) according to Fisher’s Least Significant Difference. The standard errors of the mean are presented.

Evaluation of different methods to decrease the overwintering inoculum of Pleospora allii The release of ascospores of P. allii was produced between March and May in all trials. The results obtained in different trials are presented in Figure 3.

Biological method using a commercial Trichoderma sp. reduced significantly the number of trapped ascospores in trial 1 but this reduction was not significant in trials 2, 4 and 5. In spite of this, the reduction on trapped ascospores compared with the no-treated control was between 57 to 96% in all trials.

The use of urea at two doses (5 and 10%) and strategies of application (on trees or on leaf debris) did not show significant effect on reduction of ascospores levels. Copper derivative treatments did not produce a reduction in trapped ascospores compared to the non-treated control.

Mechanical methods based on leaf shredding or removal showed higher efficacy in reduction of inoculum that the other methods. The treatments based on leaf crushing performed in trials 1, 2 and 3 decreased the amount of trapped ascospores in comparison to the non-treated control (82 to 93%) but the reduction was only significant in trail 1. The removal of leaf and fruit debris from the soil (trials 4 and 5) was the most effective method since practically no ascospores were captured.

From these results we can conclude that the recommendations to control P. allii are the sanitation practices consisting of removing the remains of leaves and fruits in the orchard in autumn, and reinforcing this action with the application of Trichoderma sp. (Trichomic), two o more times starting the applications when the first mature pseudothecia are observed or at fixed data for example in middle February, and with a repetition 15 days later.

The objective of a sanitation practice, at commercial scale, is to reduce a high proportion of the primary inoculum. The remaining proportion is probably enough to cause an unacceptable incidence and sanitation practices are unlikely to replace foliar applications of

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fungicides during spring and summer, but they can at least reduce the disease pressure. This would increase the efficacy in the disease control of current fungicides applied according to the BSPcast model.

Figure 3. Effect of different treatments on the release of Pleospora allii ascospores on pear leaf debris in five trials performed in experimental and commercial orchards. Bars correspond to the mean of three replicates. Values followed by the same letter are not significantly different (P=0.05) according to Fisher’s Least Significant Difference. The standard errors of the mean are presented.

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Acknowledgments

This research was supported in part by grants from Instituto Nacional de Investigaciones Agrarias (SC99-055 and RTA03-056), Subdirección General de Cooperación Internacional (Hi2003-0358) of Spain, and Comissió Interdepartamental de Recerca i Tecnologia of the Generalitat de Catalunya.

References

Llorente, I., & Montesinos, E. 2002: Effect of relative humidity and interrupted wetness

periods on brown spot severity of pear caused by Stemphylium vesicarium. – Phytopathology 92: 99-104.

Llorente, I., & Montesinos, E. 2004: Development and field evaluation of a model to estimate the maturity of pseudothecia of Pleospora allii on pear. – Plant Dis. 88: 215-219.

Llorente, I., Vilardell, P., Bugiani, R., Gherardi, I., & Montesinos, E. 2000: Evaluation of BSPcast disease warning system in reduced fungicide use programs for management of brown spot of pear. – Plant Dis. 84: 631-637.

Montesinos, E., Moragrega, C., Llorente, I., & Vilardell, P. 1995: Susceptibility of selected European pear cultivars to infection by Stemphylium vesicarium and influence of leaf and fruit age. – Plant Dis. 79: 471-473.

Montesinos, E., Moragrega, C., Llorente, I., Vilardell, P., Bonaterra, A., Ponti, I., Bugiani, R., Cavanni, P., & Brunelli, A. 1995: Development and evaluation of an infection model for Stemphylium vesicarium on pear based on temperature and wetness duration. – Phytopathology 85: 586-592.

Ponti, I., Cavanni, P., & Brunelli, A. 1982: "Maculatura bruna" delle pere: eziologia e difesa. – Inf. Fitopatologico 3: 35-40.

Rossi, V., Pattori, E., Giosue, S., & Bugiani, R. 2005: Growth and sporulation of Stemphylium vesicarium, the causal agent of brown spot of pear, on herb plants of orchard lawns. – Eur. J. Plant Pathol. 111: 361-370.


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Preliminary studies on biology and epidemiology of Valsa ceratosperma (Cytospora vitis), the causal agent of bark canker on pear in Italy Carla Montuschi1, Marina Collina2, Loredana Antoniacci1, Elena Cicognani2, Silvia Rimondi1, Roberta Trapella1, Eva Baruzzi1, Chiara Mazzoni1, Mirco Iotti2, Agostino Brunelli2 1Servizio Fitosanitario, Regione Emilia-Romagna, Via di Corticella 133, 40128 Bologna, Italy; 2Dipartimento di Protezione e Valorizzazione Agroalimentare, Università di Bologna - Viale G. Fanin 46, 40127 Bologna, Italy Abstract: The Ascomycete Valsa ceratosperma (Tode:Fr.) Maire [anamorph Cytospora sacculus (Schwein.) Gvritischvili = C. vitis] is a new causal agent of bark canker recently reported on pear growing areas in the Emilia Romagna (Italy). The Regional Plant Protection Service isolated the fungus in 2001 and since then reports have greatly increased in all pear growing areas of the region. This is the first occurrence of Valsa ceratosperma on pear in Europe while Valsa canker is one of the most important diseases of apple orchards in China, Japan and Korea; in these countries the fungus was only occasionally found on pear and quince. The poor knowledge about the disease on pear led us to undertake various studies on the epidemiological and biological aspects. Observations of the disease evolution were carried out in affected orchards, and laboratory and greenhouse assays made it possible to clarify the preferential ways of fungus penetration and the optimal climatic conditions for spore release. It was also investigated whether phloridzin (a dominant component distributed in leaves, stems, fruits and roots of apple tree) plays an important role in Valsa canker on pear as well as that reported on apple in Japanese studies. Preliminary results confirmed that the fungus infects pear trees through wounds such as the pruning ends; the pathogen can be isolated for a distance of about 5 cm beyond visible lesions and cankers develop rapidly in spring and early summer. Cankers with pycnidia appear in a short time only by the inclusion of a small piece of mycelium in a wound created in the trunk of pear in pot. The studies also showed that pycnidia release spores from February to November, conidia ooze gradually from the different pycnidia inside the cankers and the most important factor for fungus sporulation is relative humidity rather than temperature. Neither perithecia formation nor ascospore release were observed on Valsa cankers. Key words: Valsa ceratosperma, Cytospora vitis, pear, etiology, biology, epidemiology Introduction Italy is the first country in Europe and the second in the world producing pear fruits with about one million tons per year. Of these, 62% is produced in the Emilia Romagna region and the main cultivar is Abbé Fétel.

In 2001 a new canker disease on pear trees caused by Valsa ceratosperma (Tode:Fr) Maire, anamorph Cytospora sacculus (Schwein.) Gvritischvili [syn. C. vitis] was observed in this region. The Plant Protection Service of Emilia Romagna reported the first cases of the disease with positive results in pathogenicity assay on pear plant in pot (Montuschi, 2003). Cankers were firstly found in 3 orchards (2 near Ferrara and 1 near Bologna) on a limited number of 30-40 year old pear trees. In the following years reports greatly increased in all pear growing areas of the region and the disease was also found on young pear trees (from 8

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years old). The most affected cultivar was Abbé Fétel but other cultivars such as William, Doyenne, Kaiser, Passe Crassane, General Leclerc, Santa Maria, Packam’s Triumph and Conference proved to be susceptible (Montuschi & Collina, 2003). Valsa ceratosperma has consequently been added to the EPPO Alert List (EPPO Reporting Service, 2004/052). In 2004 the disease was also reported in Lombardy (Mantova province) in a pear orchard on cultivar Abbé Fétel (EPPO Reporting Service, 2004/173; Tantardini et al., 2004).

V. ceratosperma is known all over the world as saprophyte on dead or dying twigs and branches of numerous woody Angiosperms (IMI, 1998), while it has been reported as a pathogen only in China, Japan and Korea on apple and occasionally on pear and quince (Sakuma, 1990; Agrios, 1997).

Valsa canker can be easily confused with other pathogens such as Nectria galligena, Phomopsis mali, Sphaeropsis malorum and Erwinia amylovora. Symptoms of the disease are elongated cankers on trunk and branches with cracked edges and clear edges between healthy and diseased tissues. Under humid conditions the affected bark becomes swollen, water soaked and pinkish, and small dark pycnidia develop on the bark. Pycnidia are produced in a black stroma containing several locules around a central ostiole and during rainy periods conidia ooze from pycnidia in yellowish droplets or tendrils causing new infections.

Our knowledge about the V. ceratosperma biological cycle as a pathogen comes from literature and refers to apple trees. The fungus overwinters in infected wood and plant debris and may invade tissue through fruit scars, mechanical and weather injuries (Agrios, 1997). Most new lesions appear between March and late April and cankers develop rapidly in spring and early summer, slowly in late summer and in winter (Tamura & Saito, 1982). The fungus is active beyond 5 cm of the visible canker limits (Sakuma, 1990). Pycnidia develop on cankers from late May to September and conidia ooze from the pycnidia during rainy periods. Perithecia develop in canker during the autumn (Tamura et al., 1975). The disease may remain latent for 1 to 3 years (Liu et al., 1979).

Studies carried out in Japan show that the breakdown products of phloridzin in the apple bark seem to be associated with the symptom development of V. ceratosperma. Phloridzin is in fact a dominant component distributed in leaves, stems, fruits and roots of apple (Natsume et al., 1982) suggesting that it could be involved in the specific host-pathogen relationship (Okuno et al., 1986; Koganezawa & Sakuma, 1982).

Since all information about Valsa canker regards apple cultivation in Asia, the aim of this study was to investigate biology and epidemiology of Valsa ceratosperma on pear trees in Mediterranean climatic conditions. Materials and methods The study was carried out in field, laboratory and greenhouse. Field observations Observations were made in a pear orchard cultivar Abbé Fétel, rootstock BA 29 and Spindle training system. Orchard was planted in 1995 and the first Valsa cankers were reported in 2003 on the low part of the trunk. Since 2004 various observations have been carried out to study the disease development in the orchard: evolution of cankers on diseased plants and appearance of new symptoms; longitudinal growing of the cankers throughout the seasons; fungal presence on tissues beyond the limit of visible cankers.

In summer 2004 all trees in the orchard were classified in three classes: plants without symptoms, diseased and dead plants. In the following surveys (in spring and summer 2005) the vigour changes of the plants and the appearance of new cankers were noted. In addition, in March 2005, twenty cankers (on 20 pear trees) in the orchard were selected and their extent

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was marked with a water-resistant marker pen. In May and August 2005 the longitudinal growth of each canker was measured.

Since the literature reports the presence of fungus for a distance of about 5 cm beyond visible canker, more observations were carried out in the field to confirm this information on pear tree. Twelve cankers were selected in the orchard (on 12 pear trees) and small pieces of bark from canker limit and each 5 cm up to 20 cm were taken in March, May and August. Isolations from these tissues were made on agar media to verify the presence of fungus. Laboratory and greenhouse tests The following tests were carried out in laboratory and greenhouse: molecular identification of the fungus, evaluation of the climatic conditions influencing spore release, identification of preferential ways of fungus penetration and incubation period and the role of phloridzin on canker appearance.

Molecular identification of fungus (PCR on ribosomal DNA). Molecular identification of the isolated mycelium was performed using sequence data of the ITS regions of the nuclear ribosomal DNA. Total genomic DNA was isolated from one-week-old cultures by DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. ITS-1,5.8S and ITS-2 regions were amplified using the primer pair ITS1-ITS4 (White et al., 1990). PCR was performed by a T gradient Thermal Cycler (BIOMETRA, Göttingen, Germany) in a 50 µl volume reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM for each dNTP, 300 µM for each primer, 1.5 units of TaKaRa Taq DNA polymerase (Takara, Otsu, Japan) and 1-10 ng of genomic DNA. The amplicon was purified by Gene Clean II kit (BIO 101, Vista, CA, USA) and sequenced using the two primers mentioned above. Sequence reactions were run in an ABI PRISM 3700 DNA Analyzer (Applied Biosystem, Foster City, CA, USA) with Big Dye Terminator v3.1 chemistry. The ITS sequence was compared to those available in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) using the BLASTN search (Altschul et al., 1997) and deposited in GenBank with the accession number DQ241769.

Evaluation of climatic conditions influencing spore release. The aim of this study was to investigate the effect of relative humidity and temperature on V. ceratosperma spore release.

Different relative humidity conditions (100%, 75% and 56%) were obtained by salt saturated solutions of NaCl and Ca(NO3)2.4H2O (as reported in Winston & Bates, 1960). In a pear orchard affected by Valsa cankers, small pieces of bark (2-3 cm2 each) with pycnidia were cut from symptomatic trees with a sterile knife. In laboratory the pieces of barks were put into hermetic boxes in the different salt solutions and incubated at the temperature of 5, 15 and 25°C. Bark pieces with pycnidia were checked every two days under a binocular microscope to count the number of pycnidia spores released.

Identification of preferential ways of fungus penetration and incubation period. A greenhouse test on plants in pot of William and Abbé Fétel pear trees, Imperatore and Fuji apple trees was carried out to understand which are the preferential penetration ways of this fungus.

The plants were inoculated with mycelium or conidial suspension sprayed upon wounds caused by detaching leaves and fruits, lopping cuts and tappings along the stems. A humid chamber was created around wounds for 24 h, made with cotton steeped in water and cellophane. Suspensions were obtained from mycelial grown on oat meal-agar (72.5%, Becton Dickinson). Small pieces of mycelium were also applied in deep carvings on stems of other pear and apple plants and a humid chamber was created for 24 h also around these wounds. The pieces of mycelium were then removed.

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Role of phloridzin on canker appearance. To investigate the presence and amount of phloridzin in bark tissue of pear trees (as demonstrated in Japanese studies on apple), a Thin Layer Cromatography analysis was prepared. The active ingredients of breakdown products of phloridzin (Hidroxybenzoic acid, Protocatechuic acid, Propionic acid, Phloroglucinol, Hydroxyacetophenone) and Phloridzin were spotted on TLC silica gel plates at different concentrations (0.5 and 0.1 mg spotting drops of 10 µl; 0.05, 0.01, 0.005 and 0.001 mg in drops of 1 µl). Chromatography was carried out in an ascending manner with ethanol/ammonium hydrate/water (8:1:1 v/v) and continued until the solvent front had migrated 10 cm. After drying, plates were observed under UV irradiation (283 nm). Results and discussion Field observations In the first survey in summer 2004, 25% of plants in the pear orchard were affected by Valsa canker, while 2% were dead because of the fungus. After six months (spring 2005) the number of trees with cankers increased to 36% and in summer 2005 plants with symptoms and dead ones almost doubled (respectively 46% and 4%) in comparison to the first survey (Figure 1). In this first year the disease development was quite rapid although the evolution of symptoms seemed to be strongly influenced by plant vigour and cultivar. In particular V. ceratosperma appears to progress very quickly on weak plants and on cultivar Abbé Fétel.

In the same orchard spore release was also observed from February to November, a longer period than that reported in literature. Moreover no perithecia formation and ascospores were detected in this orchard or in others.

Figure 1. Observations on the disease development.

In May 2005, two months after delimitation of the 20 cankers, 70% of these developed further. The longest longitudinal growth was 15 cm and the average growth was 4 cm. In August 2005, 95% of cankers increased with the greatest growth of 56 cm and the average value of 21 cm. According to the literature this study showed that cankers develop rapidly in spring and early summer. Surveys are currently in progress in order to discover whether the fungus really decreases its growth rapidity in autumn and winter.

The laboratory analysis carried out on pieces of bark taken each 5 cm up to 20 cm on the canker limit in March, May and August 2005 showed that V. ceratosperma was active up to

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5 cm in the asymptomatic tissues. This observation confirmed what is cited in the literature and showed the ability of mycelium to invade the cortical tissue before symptoms occur on apple and pear trees. This information is important in recommending appropriate pruning to control the disease spread. Laboratory and greenhouse tests Molecular identification of fungus. The amplicon resulting from ITS1/ITS4 amplifica-tion showed a size of 620 bp. The ITS1-5.8S-ITS2 sequence obtained from the isolated mycelium showed a high level of similarity (98%) with an isolate of V. ceratosperma (accession number AF192326) described by Adams et al. (2002). Sequence variation between these two sequences was found to be low. In particular they differed by only 4 bp in the ITS-1 and by 4 bp in the ITS-2.

Evaluation of climatic conditions influencing spore release. The observa-tions on diseased barks incubated at different temperature and Relative Humidity showed that the most important factor in spore release was R.H. rather than temperature. In fact at 100% RH the maximum number of spore droplets or tendrils was observed irrespective of temperature. At 75% and 56% RH only few pycnidia released spores (Figure 2). Moreover conidia oozed gradually from the pycnidia in the same piece of bark probably because of a gradual pycnidia ripening inside canker.

Figure 2. Temperature and R.H. influencing spore release.

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Identification of preferential ways of fungus penetration and incubation period. No cankers appeared upon pear and apple plants inoculated with the mycelial and conidial suspensions. One month after inoculation, isolations carried out from the edge of all wounds showed the presence of fungus on pear and apple plants. Further tissue isolations carried out during the following months at different distances from the edge of wounds (2, 4, 6 mm) showed the presence of the fungus only at 2 mm. On the contrary cankers appeared upon pear stems only twenty days after inoculation with a piece of mycelium while no symptoms were observed on apple stems. Further assays are carrying out.

Role of phloridzin in canker appearance. Active ingredients of breakdown products of phloridzin were evident in TLC analysis at the concentration used. This technique is being experimented on the ground wood extracts of pear trees to check for any presence of phloridzin. Furthermore studies on the probable differences of the phloridzin amount among several pear tree varieties are being carried out. In fact pear varieties in field seem to have a different susceptibility towards Valsa canker.

References Adams, G.C., Surve-Iyer, R.S. & Iezzoni, A.F. 2002: Ribosomal DNA sequence divergence

and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees. – Mycologia 94 (6): 947-967.

Agrios, G.N. 1997: Valsa, or Cytospora, canker and dieback. Plant Pathology, – Academic Press, 4° ed.: 374-378.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W. & Lipman, D.J. 1997: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. – Nucleic Acids Res. 25: 3389-3402.

EPPO Reporting Service, 2004: New canker disease of pear in Italy: addition of Valsa ceratosperma to the EPPO Alert List. 2004/052.

EPPO Reporting Service, 2004: Valsa ceratosperma found in Lombardia (IT). 2004/173. IMI 1998: Description of Fungi and Bacteria No.1366. Valsa ceratosperma. – CABI,

Wallingford, UK. Koganezawa, H & Sakuma, T. 1982: Possible role of breakdown products of phloridzin in

symptom development by Valsa ceratosperma. – Ann. Phytopath. Soc. Japan, 48: 521-528.

Liu, F.C., Chen, C., Shi, X.Q., Guo J.G., Xing Z.F., Zhang X.W. & Chen Y.X. 1979: Studies on latent infection of the causal organism of Valsa canker of apple. – Acta Phytophyla-cica Sinica 6 (3): 1-8

Montuschi, C. 2003: Il “cancro da Valsa”, nuova malattia del pero. – Agricoltura 2: 66-68. Montuschi, C. & Collina, M. 2003: Prima segnalazione in Italia di Valsa ceratosperma su

pero. – L’informatore agrario 50: 1-3. Natsume, H, Seto, H. & Otake, N. 1982: Studies on apple canker disease. The necrotic toxins

produced by Valsa ceratosperma. – Agric. Biol. Chem. 46 (8): 2101-2106. Okuno, T., Oikawa, S., Goto, T., Sawai, K., Shirahama, H. & Matsumoto, T. 1986: Structures

and phytotoxicity of metabolites from Valsa ceratosperma. – Agric. Biol. Chem. 50 (4): 997-1001.

Sakuma, T. 1990: Valsa canker. – In: Compendium of Apple and Pear Diseases, eds. Jones and Aldwinckle, APS Press, St. Paul, U.S.A.: 39-40.

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Tamura, O. & Saito, I. 1982: Histopathological changes of apple bark infected by Valsa ceratosperma (Tode ex Fr.) Maire during dormant and growing periods. – Ann. Phytopath. Soc. Japan, 48: 490-498.

Tamura, O., Saito, I., Takakuwa, M. & Baba, T. 1975: Seasonal fluctuation of spore produc-tion and dispersal in Valsa ceratosperma (= V. mali), the causal fungus of Japanese apple canker. – Bull. of Hokkaide Pref. Agr. Exp. Stn 31: 34-42.

Tantardini, A., Calvi, M. & Cavagna, B. 2004: First report of Valsa ceratosperma in pear in Lombardy. – Journal of Plant Pathology 86: 335.

White, T.J., Bruns, T.D., Lee, S.B. & Taylor, J.W. 1990: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. – In: PCR protocol: a guide to methods and applications, eds. Innis, Gelfand, Sninsky and White, Academic Press, New York: 315-322.

Winston, P.W. & Bates, D.H. 1960: Saturated solutions for control of humidity in biological research. – Ecology 41: 232.


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Virulence of Stemphylium vesicarium isolates from pear and other host species Elisabetta Pattori1, Vittorio Rossi1, Riccardo Bugiani2, Simona Giosuè1

Istituto di Entomologia e Patologia vegetale, Università Cattolica S. Cuore,Via E. Parmense 84, I-29100 Piacenza, Italy; 2Servizio Fitosanitario, Regione Emilia-Romagna, Via di Saliceto 81, I-40128 Bologna, Italy Abstract: Brown spot, caused by Stemphylium vesicarium, is one the most important pear disease in Europe. The disease is caused by fungal strains producing host-specific toxins which are responsible for the disease symptoms on some pear varieties. It is known that there is a high degree of differentiation in host specificity among the different isolates of S. vesicarium. Pathogenicity and virulence of 78 S. vesicarium strains obtained from pear and other host species were studied by a leaf necrosis assay on 3 pear varieties showing different susceptibility to natural brown spot epidemics. The bioassay was performed using conidial suspensions and autoclaved fungal culture filtrates. Strains of S. vesicarium showed high variability for both progress of necrotic spot appearance and final disease incidence. Four virulence groups were defined using a multivariate data analysis. Group I included 49 strains from pear, which caused severe necrosis on all the varieties. Group II included only 5 strains isolated from pear which caused severe necrosis on ‘Abate Fétel’ and ‘Conference’, as the strains of group I did, but symptoms on ‘William’ were very light. In group III there were 19 strains from pear which showed less severe symptoms on all the varieties. Finally, group IV was formed by the S. vesicarium strains isolated from asparagus, pea, and onion, as well as the un-inoculated test. These fungal strains showed only small sporadic necrosis at the end of incubation. Key words: brown spot disease, fungal strain, pathogenicity, virulence Introduction Brown spot of pear (Pyrus communis), caused by Stemphylium vesicarium (Wallr.) Simmons, is a disease of economic importance in fruit-growing areas in Italy, Spain, France, Portugal, The Netherlands, and Belgium (Ponti et al., 1982; Vilardell, 1988; Blancard et al., 1989; Realise et al., 2002; Polfliet, 2002; Van Dijke, 2002; Llorente & Montesinos, 2002).

Disease symptoms consist of extended necrotic areas on leaves and shoots; fruits show small necrotic spots that progressively enlarge and deepen in round-shaped brown areas that can rot. The fungus produces high economic losses when weather conditions are favourable for infections, with incidences of 80-90% of fruits affected (Ponti & Laffi, 1993).

Despite the importance of this disease, little information on the mode of pathogenesis is available. Most fungi belonging to the genus Stemphylium, including S. vesicarium, are saprophytes growing on dead plants and cellulose materials (Simmons, 1969; Ellis, 1971; Onions et al., 1981), and are also able to grow as endophytes in the living leaves of various plants (Larran et al., 2000; Sultanova et al., 2002). S. vesicarium is also known to affect a number of host plants in addition to pear, such as garlic and onion (Rao & Pavgi, 1975), tomato (Porta-Puglia, 1981), asparagus (Lacy, 1982), aster (Ichikawa & Sato, 1994), Lucerne (Irwin & Bray, 1991), and mango (Johnson et al., 1990).

Notwithstanding the wide host range of this fungus, there is a high degree of differentiation in host specificity among the different isolates of S. vesicarium (Singh et al.,

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1999), for both pathogenicity and virulence (Bansal et al., 1991; Montesinos et al., 1995; Basallote-Ureba et al., 1999; Singh et al., 1999; Köhl et al., 2005). Throughout this paper, pathogenicity is the ability of an isolate to cause disease in a given host species, while virulence is such an ability in a host genotype, i.e a pear variety (Vanderplank, 1963, 1968; Andrivon, 1993, 1995).

Both pathogenicity and virulence of S. vesicarium on pear are due to the action of host-specific toxins (HSTs) (Singh et al., 1997). Two HSTs, SV-toxins I and II, are produced by the fungus in both culture filtrates and host tissue, which have an early effect on plasma membranes of susceptible leaves (Singh et al., 1999).

In this work pathogenicity and virulence of S. vesicarium strains obtained from pear and other host species were studied by a leaf necrosis assay on 3 pear varieties showing different susceptibility to natural brown spot epidemics. The bioassay was performed using conidial suspensions. To avoid possible errors due to a possible ‘between strain’ variability for spore germination and mycelium growth under the specific conditions of the bioassay, autoclaved fungal culture filtrates were also used on a sub-set of strains. In cultural filtrates both conidia and hyphae were killed by high temperature and ability in causing leaf necrosis should be due to the presence of HSTs produced by the fungus, that are stable over high temperature (Singh et al., 1999). Material and methods A total of 78 monosporic isolates of S. vesicarium were used: 73 of them were obtained from pear leaves or fruits showing typical brown spot symptoms collected in pear orchards of the Po Valley (northern Italy) between 1993 and 2000, while 5 were isolated from onion, peas and asparagus. Fungal strains and preparation of conidial suspensions Fruit or leaf segment were washed in running tap water for 20 min, sterilized with ethyl alcohol (70%) for 15 s and in sodium hypochlorite (2% of available chlorine) for 2.5 min, rinsed 3 times in sterile water, and dried on absorbent paper under a sterile air flow. Segments were placed in Petri dishes containing water agar (1.5 %) adjusted to pH 5.2, and incubated for 6-8 days at 25°C. Fungal colonies growing from segments were transferred to potato dextrose agar (PDA) and incubated at 25°C, with 12 h day-length. S. vesicarium colonies were identified according to Ellis (1971). All isolates were purified by single-spore isolation and stored in 20% glycerol at -80°C until use.

To prepare the conidial suspensions the fungi were subcultured on V8 agar (100 g of V8 juice, 3 g CaCo3, 20 g of agar per l of distilled water), pH adjusted to 7, at 20°C, with a daily photoperiod of 12 h of fluorescent light and 12 h of darkness. After 14 days of incubation the fungal colonies were dispersed in 10 ml of distilled water using a spatula and the resulting suspensions filtered through a double layer of cheesecloth; suspensions were then adjusted to 1 x 105 conidia per ml. Fungal culture filtrates Eighteen representative strains were selected. They were cultured on potato dextrose agar (PDA) medium for 10 days and used as initial inoculum. Three inoculum disks (0.5 cm diameter) were cut with a cork borer and transferred to 250-ml flasks containing 100 ml of Barash liquid medium (Barash et al., 1975) and incubated at 25°C, 12 h light photoperiod, in still conditions, for 15 days. The broth was filtered through filter paper no. 595 1/2 and then through 0.2 µm membrane filter (Schleicher & Schuell, Dassel, Germany), and autoclaved at 120°C for 20 minutes. Culture filtrates were used at a dilution of 1:1.

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Pear leaf necrosis assay Ability of the S. vesicarium strains to cause leaf necrosis was determined using a bioassay on detached pear leaves (Kohmoto, 1992) of 3 varieties showing different susceptibility to brown spot under orchard conditions: ‘Abate Fétel’, highly susceptible; ‘Conference’, susceptible; ‘William’, partially resistant.

Fresh young leaves were detached from 2-year old potted plants grown in a glasshouse at 20 ± 2°C, washed in running tap water for 30 min, dried under a sterile air flow and placed in sterilised plastic boxes (20x30 cm) over blotting paper wetted with sterile water, the upper surface down. Six 10 µl drops of the previously described either conidial suspension or culture filtrates were placed on the lower leaf surface. Control leaves were inoculated with water. Boxes were enclosed in plastic bags and incubated at 25°C, 12 h day-length, for 30 days. Leaves were inspected daily to determine the number of inoculated drops causing necrosis of the leaf tissue. Leaves of the same age were used for each experiment. Each assay was repeated twice. Disease incidence was then calculated as a percentage of total inoculations showing necrosis over the total inoculations. In some cases, lesion area was recorded by tracing the necrotic area from the leaf to graph paper, and measured in mm2 (Singh et al., 1999). Data analysis An exploratory data analysis was performed by: (i) plotting the average incidence of necrotic spots over time of incubation for each fungal strain x pear variety combination; (ii) calculating the area under the disease progress curve (AUDPC) (Campbell & Madden,1990) for each combination; (ii) drawing box plots for the distribution of the UDPC data over the 3 pear varieties.

A multivariate cluster analysis was used to group the 78 fungal strains and the un-inoculated test according their AUDPC on the 3 pear varieties. Clustering was based on the Euclidean quadratic distance between groups, using the centroid as grouping method.

Groups from this analysis were used as grouping variable in a discriminant analysis: the AUDPC values for the 3 pear varieties where used as independent variables for elaborating the discriminat functions defining the belongings of each fungal strain to its group. All the statistical analyses were performed using SPSS ver. 11.5 (SPSS Inc., Chicago, IL).

Results and discussion Pathogenicity and virulence of S. vesicarium strains Strains of S. vesicarium isolated from pear were always pathogenic on pear leaves, while isolates from the other hosts show only few sporadic and late necroses.

Strains from pear showed high variability for both progress of necrotic spot appearance and final disease incidence (Fig. 1). Some strains showed a fast disease progress with final incidence of about 100% in all the pear varieties, as in the example of Fig. 1A; other strains had a similar behaviour on ‘Abate Fétel’ and ‘Conference’, but showed a reduced incidence on ‘William’, as in the examples of Fig. 1A and 1B. In other cases, the disease progressed slowly also on ‘Abate Fétel’ and ‘Conference’ and reached 100% of incidence also on the former variety (Fig. 1D to 1E). In some other strains the disease was less severe in all the pear varieties (Fig. 1F). The leaf area showing necrosis around each inoculation point was in total agreement with the above mentioned differences (not shown).

The box plot analysis (Fig. 2) showed that variability in response to infection, the latter measured as AUDPC, was higher in ‘William’ than in ‘Conference’ and especially in ‘Abate Fétel’. In ‘William’ AUDPC values of the 73 S. vesicarium strains from pear were regularly distributed between 0.3 and 12.1, with median 6.5. In the other varieties distribution of data

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Days of incubation Figure 1. Incidence of necrotic spots on leaves of three pear varieties (Abate, Conference and

William) inoculated with conidial suspensions of 6 S. vesicarium strains that have been isolated from pear. Incidence is expressed as a proportion of inoculation sites that show disease symptoms.

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Figure 2. Box Plot showing variability between S. vesicarium strains for the capacity of causing leaf necrosis on 3 pear varieties, expressed as AUDPC (Area Under the Disease Progress Curve). The boxes include 50% of the data (between 25° and 75° quartiles), the dotted line is the median, whiskers extend to minimum and maximum values, points are outliers (1.5 to 3 times the interquartile range) ( ) or extreme values (>3 times) ( ).

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that the 3 pear varieties used in the present work account for the variability existing in virulence of this pathogen.

The cluster analysis performed on the AUDPC values obtained for the 3 pear varieties produced four different groups (named I to IV) at a rescaled distance of about 5 (Fig. 3). Group IV grouped with the others at great distance.

Group I included 49 S. vesicarium strains, all isolated from pear fruits collected in orchards located in the most important pear-growing area of the Emilia-Romagna region, that includes the districts of Bologna, Modena, and Ferrara. These fungal strains caused severe necrosis on the leaf tissue of all the varieties (Tab. 1).

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and from other host species based on the values of AUDPC (Area Under Disease Progress Curve) obtained by inoculating them on leaves of 3 pear varieties; the dendrogram includes an un-inoculated test. Numbers I to IV indicate the 4 virulence groups.

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Table 1. Numbers of S. vesicarium strains belonging to 4 virulence groups (I to IV) and average values of the AUDPC (Area Under the Disease Progress Curve) obtained by inoculating fungal strains on leaves of 3 pear varieties (standard deviation is between brackets).

Pear variety Virulence

group Number of strains Abate Fétel Conference William

I 49 12.0 (0.96) 11.1 (1.11) 7.3 (1.99)

II 5 12.2 (0.34) 11.8 (0.48) 1.3 (0.64)

III 19 8.3 (2.79) 5.0 (2.70) 1.9 (1.34)

IV 5 0.8 (0.60) 0.4 (0.73) 0.7 (0.32)

Group II included only 5 strains isolated from pear fruits collected in an orchard located in the district of Bologna. These strains caused severe necrosis on leaves of ‘Abate Fétel’ and ‘Conference’, as the strains of group I did, but symptoms on ‘William’ were very light (Tab. 1). Some strains coming from the same orchards were included in group I.

In group III there were 19 strains, all isolated from pear leaves or fruits collected in orchards located in Emilia and Lombardy. They showed less severe symptoms than strains belonging to groups I and II on all the pear varieties (Tab. 1).

Group IV was formed by the S. vesicarium strains isolated from asparagus, pea, and onion, as well as the un-inoculated test. These fungal strains showed only small sporadic necrosis at the end of the incubation period.

Since the 4 groups formed by the cluster analysis showed a consistent behaviour for their ability to cause leaf necrosis on the 3 pear varieties, they were considered as virulence groups: S. vesicarium strains belonging to group I cause high infection levels on all varieties; those in group II cause severe symptoms only on the 2 susceptible varieties, while strains of group III cause lower symptoms on all varieties. In group IV there are strains of S. vesicarium that are not pathogenic for pear.

Results obtained from the present work demonstrated that S. vesicarium strains isolated from pear tissue showing brown spot symptoms are pathogenic on pear leaves but not on the other hosts tested.

This result is consistent with previous findings. Singh et al. (1999) found that S. vesicarium isolates from pear showed a high degree of host specificity, being not able to cause disease on tomato, onion, apple, rose, and Japanese pear. Köhl et al. (2005) showed that the majority of S. vesicarium isolates obtained from lesions of pear fruits were pathogenic on pear, while isolates obtained from asparagus or onion leaves, or living as endophytes of apple leaves did not infect pear. Differences in pathogenicity among strains were also found in the interaction Stemphylium strain x Asparagus taxa (Bansal et al., 1991), while isolates from garlic, onion and asparagus caused disease in all 3 hosts (Basallote-Ureba et al., 1999).

The existence of a wide range in susceptibility among pear genotypes inoculated with one pathogenic strain of S. vesicarium was previously demonstrated (Ponti & Cavanni, 1983; Blancard et al., 1989; Montesinos et al., 1995). The present work demonstrated that fungal strains vary in virulence, being able to infect different pear varieties with different incidences, and that it is possible to separate groups of fungal strains showing different virulence characters.

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Consistency between inoculations with S. vesicarium conidia and culture filtrates Leaf necrosis produced by inoculating fungal culture filtrate mimicked typical Stemphylium brown spot symptoms. Results obtained in the bioassays made using culture filtrates were consistent with those obtained by inoculating spore suspensions.

The analysis of variance showed that both virulence groups and pear varieties significantly (P<0.0001) influenced the incidence of leaf necrosis, as well as their interaction.

After 15 days of incubation, S. vesicarium strains belonging to virulence groups I and II caused very high incidence of leaf necrosis when inoculated on ‘Abate Fétel’ and ‘Conference’, but only the former group caused some necroses on ‘William’. Strains of the group III caused less frequent necrosis on leaves of the 2 susceptible varieties, and they did not affect leaves of the resistant one. Finally no necrosis was observed when strains of group IV were inoculated on any variety (Tab. 2).

Table 2. Incidence (%) of leaf portions showing necrosis after inoculation of culture filtrates obtained from colonies of 18 S. vesicarium strains belonging to the 4 virulence groups on leaves of 3 pear varieties. Averages followed by the same letter are not significantly different using the Tukey test at P≤0.05.

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I 10 100.0 A 83.8 B 2.8 D

II 2 95.5 A 80.3 B 0.0 E

III 2 67.7 C 70.8 C 0.0 E

IV 4 0.0 E 0.0 E 0.0 E

The susceptibility to the culture filtrate was correlated with the behaviour of the pear varieties in both the bioassay with conidia and in natural epidemics under orchard conditions. Similar correlation was also obtained when the host and non-host plant species were tested for the pathogen and its culture filtrate.

Differences in both pathogenicity and virulence have been attributed to differences in the ability to produce HSTs (Wolpert et al., 2002). The S. vesicarium toxin I induced necrosis on leaves of highly susceptible pears at a concentration of 0.01 to 0.1 µg/ml, and at 100 µg/ml on moderately resistant ones. The same toxin was not able to induce damages on resistant genotypes even at 104-fold increase in concentration (Singh et al., 1999). Similar findings were also reported for a biotype of S. botryosum on alfalfa (Heiny & Gilchrist, 1989) and for S. solani on cotton (Mehta & Brogin, 2000). Assigning S. vesicarium strains to virulence groups The discriminant analysis made it possible to assign any strain of S. vesicarium to the right group of virulence.

This analysis was able to correctly classify the un-inoculated test and 76 fungal strains out of 78 (97.5% of total cases) (Tab. 3). Wrong classification occurred only for group III, one strain being classified as belonging to group I and another to group IV; in both cases, probability associated with wrong classification was 67% versus 33% for a correct classification.

202

Table 3. Comparison between original virulence groups of S. vesicarium strains (I to IV) and groups estimated by discriminant analysis: in aggregate 97.5% of strains were correctly classified.

Estimated

Original I II III IV

I 49 (100%) 0 0 0 II 0 5 (100%) 0 0 III 1 (5.3%) 0 17 (89.5%) 1 (5.3%) IV 0 0 0 6 (100%)

Discriminat analysis produced 3 discriminat functions (DF1 to DF3). These functions had the following form: FDn = a + b x1 + c x2+ d x3, where x1, x2, and x3 are the AUDPC values obtained by inoculating any fungal strain on leaves of ‘Abate Fétel’, ‘Conference’, and ‘William’, respectively. DF1 and DF2 accounted for 82.2% and for 13.4% of total variability, respectively (Tab. 4); DF3 had a little contribution (4.4%) and therefore it was considered to have little influence on the classification of fungal strains.

A territorial map was drawn using DF1 and DF2 as axis coordinates (Fig. 4). Solution of these discriminant functions using the AUDPC values obtained by the bioassay for any strain to be classified produces the coordinates of that strain on the two axes, and therefore its position on the map and finally its virulence group.

Table 4. Parameters of the 3 discriminant functions (DF1 to DF3) explaining the belonging of

S. vesicarium strains to the 4 virulence groups: FDn = a + b x1 + c x2+ d x3, where x1, x2, and x3 are the AUDPC values obtained by inoculating any fungal strain on detached leaves of ‘Abate Fétel’, ‘Conference’, and ‘William’ pear varieties, respectively.

Discriminant Functions Function parameters

DF1 DF2 DF3 a -6.880 1.194 -1.422 b 0.289 -0.285 0.592 c 0.369 -0.137 -0.614 d 0.124 0.578 0.151

Variance (%) 82.2 13.4 4.4

203

Solution of the two discriminant functions using the AUDPC (Area Under the Disese

Progress Curve) values obtained by inoculating any fungal strain on detached leaves of ‘Abate Fétel’, ‘Conference’, and ‘William’ pear varieties produces the coordinates of that strain on the two axes, and therefore its position on the map and its virulence group.

In conclusion, the results from the present work confirmed on a wide collection of S. vesicariurm strains the previous results obtained with one or few strains: S. vesicarium strains causing the brown spot disease have a high host-specificity for both pathogenicity and virulence. Four virulence groups can be outlined using a simple bioassay based on artificial inoculation of conidia on detached leaves of 3 pear varieties, which are consistent with the toxicity of their culture filtrates.

Further works will be aimed at characterizing virulence groups based on kind and quantity of the phytotoxins they produce.

Acknowledgements This work is part of research promoted and supported by the Emilia-Romagna Region (Italy) in collaboration with the Centro Ricerche Produzioni Vegetali (CRPV). References Andrivon, D. 1993: Nomenclature for pathogenicithy and virulence: the need for precision. –

Phytopathology 83: 889-890. Andrivon, D. 1995: Nomenclature for pathogenicity and virulence: precision vs tradition. –

Phytopathology 85: 518-519. Bansal, R.K., Menzies, S.A. & Broadhurst, P.G. 1991: Pathogenic variation among strains of

Stemphylium vesicarium causing leaf spot of asparagus. – N. Z. J. Crop Hortic. Sc. 19: 69-71.

Barash, I., Karr, L. & Strobel, A. 1975: Isolation and Characterization of Stemphylium, a Chromone Glucoside from Stemphylium botryosum. – Plant Physiol. 55: 646-651.

-12 -8 -4 0 4 8 12-12

-8

-4

4

12

0

8

Discriminant function 1

Disc

rimin

antf

unct

ion

2

IIV

III II

-12 -8 -4 0 4 8 12-12

-8

-4

4

12

0

8

Discriminant function 1

Disc

rimin

antf

unct

ion

2

IIV

III II Figure 4. Territorial map drawn using the first two discriminant functions listed in Tab. 2: polygons characterize the 4 virulence groups for the S. vesicariumstrains (I to IV), while dots indicate the centroid of each group.

204

Basallote-Ureba, M.J., Prados-Ligero, A.M. & Melero-Vara, J.M. 1999: Aetiology of leaf spot of garlic and onion caused by Stemphylium vesicarium in Spain. – Plant Pathol. 48: 139-145.

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Heiny, D.K. & Gilchrist, D.G. 1989: Isolation and partial characterization of polypeptides associated with phytotoxin in cultures of the cool-temperature biotype of Stemphylium botryosum pathogenic on alfalfa. – Physiol. Mol. Plant Pathol. 34: 483-505.

Ichikawa, K. & Sato, T. 1994: Leaf and stem spot of aster caused by Stemphylium vesicarium. – Ann. Phytopathol. Soc. Jpn. 60: 523-526.

Irwin, J.A.G. & Bray, R.A. 1991: Varation in virulence withing the cool temperature biotype of Stemphylium vesicarium (Wallr.) Simmons, a lucerne leaf spot pathogen. – Aus. J. of Exp. Agric. 31: 793-795.

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Kohmoto, K. 1992: Determination of host-selective toxins. Modern Methods of Plant Analysis New Series. – Plant Toxin Analysis, vol. 13 (eds. Linskens and Jackson, Springer-Verlag, Berlin): 51-73.

Lacy, M.L. 1982: Purple spot: A new disease of young asparagus spears caused by Stemphylium vesicarium. – Plant Dis. 66: 1198-1200.

Larran, S., Monaco, C. & Alippi, H.E. 2000: Endophytic fungi in beet (Beta vulgaris var. esculenta L.) leaveas. – Adv. Hort. Sci. 14: 193-196.

Llorente, I. & Montesinos, E. 2002: Effect of relative humidity and interrupted wetness periods on brown spot severity of pear caused by Stemphylium vesicarium. – Phytopathology 92: 99-104.

Mehta, Y.R. & Brogin, R.L. 2000: Phytotoxicity of a culture filtrate produced by Stemphylium solani of cotton. – Plant Dis. 84: 838-842.

Montesinos, E., Moragrera, C., Llorente, I. & Vilardell, P. 1995: Susceptibility of selected European pear cultivars to infection by Stemphylium vesicarium and influence of leaf and fruit age. – Plant Dis. 79: 471-473.

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pp. 207 - 211

207

Organic field-testing of compounds to control apple scab (Venturia inaequalis) in combination with alleyway cover crops Hanne Lindhard Pedersen1, Lars P. Christensen2, Marianne Bengtsson4, Klaus Paaske3, John Hockenhull4 1Department of Horticulture 2Department of Food Science and 3Department of Crop Protection, Danish Institute of Agricultural Sciences, Research Centre Aarslev, Kirstinebjergvej 10, Postbox 102, DK-5792 Aarslev, Denmark. [email protected]. 4Section for Plant Pathology, Department of Plant Biology, The Royal Veterinary and Agricultural University (KVL), Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Abstract: Field-testing of new potential fungicides acceptable to organic production preventing apple scab (Venturia inaequalis) infections on leaf and fruits during primary apple scab infection period was carried out in 2003 and 2004. The testing was done on the variety ‘Delorina’ in combination with different cover crop treatments, aimed to establish different nitrogen supplies to support tree growth and yield.

Sulphur, had the best effect in these trials and the use of sulphur resulted in an increase in yield, due to more and bigger fruits. Both in 2003 and 2004 the best disease control was achieved with sulphur and some control was also seen with the alternative compounds in 2003. The use of sulphur in combination with a soil treatment that reduces the level of nitrogen available to the trees increased saleable yields. An apparent correlation was found between severity of scab and the flavanol content of the fruits.

The results of this work show that more work is needed in order to find effective alternatives to sulphur and copper fungicides for control of scab in organic apple orchards. Key words: apple scab, Venturia Inaequalis, cover crops, Phenolics Introduction The aim of the trials was organic in-field testing of new potential fungicides acceptable to organic production preventing apple scab (Venturia inaequalis) infections on leaf and fruits during primary apple scab infection period. Field-testing was carried out with and without combination with cover crops. Materials and methods The trials were carried out in combination with different cover crop treatments in single-tree plots. The formerly resistant variety ‘Delorina’ on rootstock M9, planted 1995 at a planting distance of 3.3 m x 1.6 m, unfertilized and with mechanical weed cleaning in the tree row, were used. The experimental orchard is located at Research Centre Aarslev (100 27´ E, 550 18´N).

In 2003, fungicides were sprayed preventively according to RIMpro warnings in the primary apple scab season, from bud break until the end of June, when ascospore discharge stopped. In 2004, weekly preventive treatments were carried out in the primary apple scab season. Application was done using a nap sac mist blower until incipient run-off.

208

The three alleyway cover crops 1. Grass: A permanent weak grass mixture of Red fescue (Festuca rubtra) and Meadow grass (Poa

pratensis). 2. Clover grass: A permanent clover grass mixture consisting of White clover (Trifolium repens)

and Perennial ryegrass (Lollium perénne). 3. Annual: An annual cover crop consisting of Italian ryegrass (Lolium multiflorum) and Persian

clover (Trifolium resupinatum) sown every year in July and mulched down the following year in April. Mechanical weed cleaning was practiced from April to July.

Results and discussion Control of apple scab Both in 2003 and 2004 the best disease control was achieved with sulphur (Kumulus) (Table 1 and 2) and some control was also seen with the alternative compounds in 2003. The reduction of apple scab infections was greatest on the rosette leaves. In 2004 the alternative compounds were tried alone and in combination with sulphur, but the results were approximately the same as with sulphur alone (Table 2). Phenolics Phenolics were extracted from frozen apples (fruit and peel) of the variety ‘Delorina’ and analysed by analytical HPLC. The major phenolics were identified as chlorogenic acid, catechin, epicatechin, a cyanidin glycoside, rutin and three other flavonol glycosides, respectively. Significant differences in the content of flavonol glycosides were observed between treatments with the highest content being found in the untreated apples and the lowest in apples treated with E15/Kumulus and Plant extract E52/Kumulus, respectively (Table 2). Cover crops The annual cover crop supplied the trees with a high nitrogen level in the soil, but it only resulted in differences in uptake by the trees in 2004 (Table 3 and 4). This gave a slightly higher infection of powdery mildew, a higher infection of apple scab and more green fruits (Table 3). Yield was significantly higher for trees grown in grass alleyways in both years (Table 3 and 4)

02468

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ld k

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3. Plant extract E 524. C-pro.

5. E15

Figure 1. Yield without apple scab for 5 treatments and 3 cover crops in 2003.

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Yield Apple scab control using sulphur increased the yield. Combination of trees grown in alleyway grass using sulphur to control apple scab had the highest yield of fruits not damaged by apple scab (Fig. 1 and 2). Especially for the clover grass alleyway in 2003, the apple scab control using E15, Plant extract E52 seems to have had a positive effect on the yield (Fig 1). In 2004 the use of sulphur increased the yield of not apple scab damaged fruits in the clover grass alleyway and annual cover crop (Fig. 2).

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Figure 2. Yield without apple scab for 6 treatments and 3 covercrops 2004. Table 1. Control of apple scab in the variety ’Delorina´ in 2003 with sulphur and alternative

fungicides: number of treatments, percentage of non infected leaves on annual shoots, rosettes and fruits on 2nd July, two weeks after the end of the primary infection period. Yield and percentage fruits without russeting at harvest.

Alternative fungicides used

Number treatments

2003

Annual shoots:% leaves

without scab

Rosettes: % leaves without

scab

% fruits without

scab

% fruits without

russeting at harvest

Yieldkg/tree

1. Control 0 17,5 c 28,4 d 20,8 c 82,6 a 6,2 b 2. Kumulus S (0,27 %) 8 51,8 a 87,8 a 71,7 a 52,9 b 12,7 a 3. Plant extract E52 (5,0 %) 8 23,1 c 35,9 cd 35,0 bc 60,9 b 8,8 b 4. C-pro (0,3 %). 5 24,2 c 44,0 bc 26,7 c 83,7 a 8,2 b 5. E15 (0,2 %) 7 33,2 b 54,1 b 45,0 b 29,6 c 7,1 b Numbers followed by the same letters are not significantly different for p<0,05.

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Table 2. Control of apple scab in the variety ’Delorina´ in 2004 with sulphur and alternative fungicides: Number of treatments, percentage of non infected fruits on 8th July, two weeks after the end of the primary infection period and yield, fruit quality and content of Flavanols in mg compound per g fruit at harvest.

Alternative fungicides used Number

treatments 2003

% fruits without

scab

% fruits without

russeting at harvest

Yield kg/tree

Fruit size g/fruit

% surface colour

Flavanol glycosides Mg/gram

1. Untreated 0 72,2 b 52,4 a 5,7 b 82 c 59,7 a 0,023 a 2. Kumulus (0,27%) 11 90,0 a 55,1 a 10,3 a 103 a 54,7 bc 0,012 b 3. E15 (0,02 %) 11 77,8 ab 34,7 b 6,7 b 86 bc 51,7 cd 0,016 ab4. E15 (0,02 %)

and Kumulus (0,27 %) 11 90,0 a 41,8 ab 9,6 a 98 a 50,0 d 0,011 b

5. Plant extract E52 (5%) 11 72,2 b 32,2 b 6,0 b 84 bc 57,3 ab 0,016 ab6. Plant extract E52 (5%)

and Kumulus (0,27 %) 11 87,8 a 44,0 ab 9,2 a 95 ab 44,8 d 0,011 b

Numbers followed by the same letters are not significantly different for p<0,05. Table 3. Infections of powdery mildew and apple scab at the end of the primary apple scab season.

Fruit colour and yield at harvest and nitrogen and potassium in leaf samples and available nitrogen in soil for the variety ’Delorina´ in 2003.

Cover crop used

Powdery mildew on

shoots

% fruits without

scab

% surface colour

Yield kg/tree

% Nitrogen in leaves

% potassium in leaves

Nmin in soil 0-50 cm

depth 1. Grass 1,6 ab 42,0 ab 57,6 a 11,6 a 2,01 1,34 26,3 b 2. Clover grass 1,5 b 47,0 a 48,9 b 7,2 b 2,14 1,24 22,9 b 3. Annual 1,7 a 30,5 b 49,8 b 7,0 b 2,22 1,02 51,3 a

Numbers followed by the same letters are not significantly different for p<0,05. Table 4. Infections of powdery mildew and apple scab at the end of the primary apple scab season.

Fruit colour and yield at harvest and nitrogen and potassium in leaf samples and available nitrogen in soil for the variety ’Delorina´ in 2004.

Cover crop used

Powdery mildew on

shoots

% fruits without

scab

% surface colour

Yield kg/tree

% Nitrogen in leaves

% potassium in leaves

Nmin in soil 0-50 cm

depth

1. Grass 1,96 a 78,3 a 53,0 b 10,9 a 2,44 a 1,02 a 39,3 b 2. Clover grass 1,58 b 83,3 a 56,4 a 5,2 c 2,53 a 1,00 a 41,8 ab 3. Annual 1,81 a 83,3 a 52,2 b 7,6 b 2,44 a 0,91 a 70,4 a

Numbers followed by the same letters are not significantly different for p<0,05.

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Conclusion Sulphur, had the best effect in these trials and the use of sulphur resulted in an increase in yield, due to more and bigger fruits.

Both in 2003 and 2004 the best disease control was achieved with sulphur and some control was also seen with the alternative compounds in 2003.

The use of sulphur in combination with a soil treatment that reduces the level of nitrogen available to the trees increased saleable yields.

An apparent correlation was found between severity of scab and the flavanol content of the fruits.

Acknowledgement We kindly acknowledge financial support from Danish Research Centre for Organic Farming (DARCOF).


pp. 213 - 217

213

Evaluating the use of RIMpro and Metos weather stations for control of apple scab (Venturia inaequalis) in Denmark 2002-2005 Hanne Lindhard Pedersen1, Karen Linddal Pedersen2, Klaus Paaske3 1Danish Institute of Agricultural Sciences, Department of Horticulture, Research Centre Aarslev, Kirstinebjergvej 10, Postbox 102, DK-5792 Aarslev, Denmark. [email protected] The Fruit and Vegetable Advisory Service, Rugaardsvej 197, DK-5210 Odense NV, Denmark. E-mail:[email protected] Institute of Agricultural Sciences, Department of Crop Protection, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark. [email protected]. Abstract: The apple scab-warning program RIMpro was tested in an integrated and organic strategy compared to unsprayed and traditional conventional strategy in 2002 to 2004 in the variety Jonagold. The actual discharge of ascospores is compared to the RIMpro estimated value of ejected spores. During the same period a national Internet based scab-warning system build on Metos weather stations and RIMpro software has been developed.

The use of Rimpro scab control strategy with 2 to 4 less sprays during the primary apple scab season controlled apple scab on leaves and fruits at the same level as the traditional strategy. The estimated ascospore discharge seems not to be adjusted to the actual discharge in the beginning of ascospore season under Danish conditions. This is probably because of low temperatures in early spring.

The establishment of the national Internet scab warning system has difficulties collecting the necessary climatic data to run the software program due to break down of the weather stations and data transfer. Key words: Apple scab, Venturia inaequalis, warning, control, ascospore discharge Introduction The aim of the work was to optimize and reduce the use of fungicides to control apple scab in the orchards, by using the apple scab warning system RIMpro. Materials and methods The discharge of ascospores from over-wintered leaves of the variety ‘Jonagold’ infected with apple scab was measured in slide-holder devise, used for collecting ascospores at the Danish Research Centre Aarslev (100 27´ E, 550 18´N). The measured amount was compared to the estimated value of ejected spores from the daily report function in RIMpro. Spores were counted after rain with a maximum of thee times a week during the period of ascospore maturation from 2002 to 2005.

Four control strategies against apple scab were tested in 2002 to 2004 in the variety ‘Jonagold’ on rootstock M9 at planting distance 3,5 x 1,3 m. The trees were planted in 1999 at Research Centre Aarslev. A traditional apple scab warning based on Mills, was compared to a conventional and organic RIMpro strategy. The used fungicides were the allowed in the actual years. Mills and the conventional RIMpro strategies, were treated identical after the primary

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2002Green tip: 28th of March

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Figure 1. Percent ascospore discharge for apple scab (Venturia Inaequalis), present and estimated for 2002 to 2005.

ascospore season. For organic productions only pure sulphur was allowed. Apple scab severity was calculated according to Townsend & Heubergers (1943). Results and discussion Nationwide apple scab warning Metos weather stations were placed in 18 different orchards around Denmark to cover different climatic locations. The stations were sited in the period 2002 to 2003 and include sensors for temperature, relative humidity and leaf humidity. Twice a day a server, was calling all the stations to collect weather data. The data was transformed by RIMpro and transferred automatically into a webpage available to all growers. At first all stations were connected by a cable, to the private computers of the growers however this system was not steady, the growers had to switch a button every time they used RIMpro and they often forgot to do that. In 2003 GSMmodems were added to most weather stations to improve the data collection. The GSM modem worked well the first summer. Ever since, the GSM modem, the battery, the sun cell and the different sensors on the weather stations has gone broke now and then. Caused by all these technical problems it has not yet been possible to get a steady flow of data into the homepage. The homepage has been running the last two years, with the data available at a certain time. The amount of repairs has increased much and in the 2005 season 8 stations have not been running.

Next year the project will only include the weather stations, which are still running, as the project has not enough money for repairs left.

This fall there will be applied for a new project. This project will include The Danish Advisory Service, The Danish Institute of Agricultural Science and The Danish Meteorological Institute (DMI). The hope is that DMI will precipitate and serve the project, with climatically data from their weather stations, which are located around Denmark.

216

Ascospore discharge Most years the start of discharge was around green tip for Jonagold or at the first rain after green tip. However, 0,6 percent of the actual ascospores were caught before green tip at the 3rd of April in 2004.

The estimated ascospore discharge seems not to be adjusted to the actual discharge in the beginning of ascospore season under Danish conditions. In spring 2003 we had a long dry cool period in April, where to estimation did not fit. This was probably due to the relative low temperatures. In 2003 a correction was made in the program and the estimated discharge was better estimated in 2004 and 2005 (Fig. 1). In 2003 to 2005 the actual ascospore discharge ended approximately the 20th of May, two to three weeks before the estimated discharge. Control strategy The use of the Rimpro strategy controlled apple scab on leaves and fruits at the same level as the traditional strategy with 2 to 4 fewer sprays. 2003 was a season with heavy apple scab infections, and the apple scab control in general was not sufficient. The RIMpro strategy had a tendency towards a less sufficient disease control 2003, where also the estimation of ascospore discharge was not adequate. Sulphur reduced apple scab infections, but 2 out of 3 years the disease control in the apple scab susceptible variety Jonagold, was not sufficient. Table 1. Apple scab severity on leaves and fruits on the variety ‘Jonagold’ for 4 strategies and

3 years. 2002 N. of treatments Apple scab severity Strategy

Leaves, 26 June

Leaves, 15 September

Fruits at harvest

1. Unsprayed 0 4,8 a 61,9 a 15,6 a 2. Mills 14 0,0 b 1,3 b 0,0 b 3. RIMpro 10 0,0 b 5,0 b 0,0 b 4. Organic RIMpro 14 0,0 b 4,1 b 0,0 b 2003 N. of treatments Apple scab severity Strategy

Leaves, 25 June

Leaves, 14 August

Leaves, 15 September

Fruits at harvest

1. Unsprayed 0 12,1 a 81,1 a 98,9 a 81,1 a 2. Mills 14 1,1 b 23,2 b 45,2 b 17,7 b 3. RIMpro 10 2,8 b 49,6 ab 67,5 ab 27,9 b 4. Organic RIMpro 14 3,9 b 55,1 ab 68,9 ab 31,9 b 2004 N. of treatments Apple scab severity Strategy

Leaves, 1 June

Leaves, 10 August

Leaves, 8 September

Fruits at harvest

1. Unsprayed 0 0,7 a 42,3 a 63,4 a 19,7 a 2. Mills 15 0,0 b 3,9 b 10,9 b 0,8 b 3. RIMpro 13 0,0 b 8,0 b 11,0 b 2,0 b 4. Organic RIMpro 17 0,3 ab 36,8 a 51,8 a 12,5 ab Means followed by same letter do not significantly differ for P=0,05.

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Conclusion The technical quality of Metos weather stations was not good enough.

RIMpro estimations of ascospores are not adequate, when the temperatures are low during spring, this probably also extended the estimated ascospore season.

The use of RIMpro scab control strategy reduced the need of fungicide sprays against apple scab with 2 to 4 treatments compared to the Mills strategy. Acknowledgements We kindly acknowledge financial support from ‘Plan Danmark’. Plan Danmark is a foun-dation, which contribute money to support fruit growing, agriculture and craft. References Townsend, G.R. & Heuberger, J.W. 1943: Plant Disease Reporter 27 (17): 340-343.


pp. 219 - 221

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Use of bulk ascospore samples for monitoring apple scab fungicide resistance in individual orchards Vincent Philion IRDA, Mont-Saint-Bruno National park research station, 330, chemin des Vingt-Cinq Est Saint-Bruno-de-Montarville, J3V 4P6, Québec Abstract: In regions where apple scab is a problem, growers routinely use fungicides that may be prone to the development of tolerant Venturia inaequalis populations. Reduced fungicide efficacy leads to higher scab levels, additional sprays, and can result in crop failure before the problem is identified. On the other hand, regional resistance surveys can lead to large-scale abandon of products that limit available fungicide management options in orchards where resistance is not a problem. We developed an affordable and reliable method to evaluate the efficacy of fungicides against bulk ascospore samples harvested from leaves of individual orchards. The test is done by evaluating in vitro mycelial growth using a modified line-intercept method after incubating for 48 hours in a liquid suspension of each fungicide (cyprodinil, dodine, flusilazole, kresoxim-methyl, thiophanate-methyl) and compared to a control. We observed population EC50 shifts for certain molecules in a number of commercial orchards as compared to a organic orchard. These results were confirmed with fungicide efficacy trials done using potted trees exposed both to natural ascosporic inoculum in test orchards, and to artificial inoculations using bulk conidia samples in growth chambers. The potted tree results demonstrated practical resistance for both flusilazole and thiophanate-methyl in certain sites, and no resistance to kresoxim-methyl, cyprodinil and dodine. Preliminary results also suggest that flusilazole maintains a higher level of efficacy in curative treatments than for protectant treatments for both tolerant and sensitive spore populations. Ongoing potted tree experiments are aimed at establishing a practical fungicide resistance threshold, which would enable us to advise individual growers based on results from the in vitro tests. Key words: fungicide resistance monitoring, apple scab Introduction In many parts of the world, apple production relies heavily on the use of fungicides effective against apple scab. Since the last 40 years, a number of products that were registered have lost efficacy because of resistance, and products registered only recently such as the strobilurins have already lost field efficacy in certain regions.

Unfortunately, the resistance-monitoring techniques that are currently available are labor intensive which limits their use to large scale regional surveys. Consequently, growers often face important economical losses before resistance is confirmed and the product is abandoned. Conversely, the detection of initial signs of resistance through regional surveys can lead to a large scale abandon of products, even though the product may be fully effective in a large proportion of orchards. Thus, limiting available fungicide rotation options because of the lack of an affordable system for testing individual orchards.

The objective of our project was to develop an affordable in vitro resistance monitoring test and validate results in vivo.

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Materials and methods In vitro tests Ascospores of Venturia inaequalis were harvested from over wintered leaves originating from different orchards using a spore tower modified from Gilpatrick et al. (1972) and designed to collect dry spores in Eppendorf cupules. The samples were then frozen until processed, usually within a few months. The bulk ascospore samples were resuspended in PDB and the concentration adjusted to deposit 400 spores per well in flat bottom ELISA plates. Spores were exposed to a range of concentration of the different fungicides: Equal (65% dodine), Senator (70%Thiophanate-methyl), Nustar (20% flusilazole), Sovran (50% kresoxim-methyl), Vangard (75% cyprodinil) and a PDB control. Mycelial growth was assessed after 48 hours incubation with shaking at 18C. A modified line-intercept method was used to evaluate the number of intersection between mycelial growth and a grid placed in the ocular, as described by Nedoma et al. (2001). The log LD50 of the different fungicides against the V. inaequalis ascospores populations was obtained by a logistic regression of the number of intercepts against the log of fungicide concentration. The average of at least 5 tests was reported. Table 1. Log LD50 for 5 fungicide active ingredients against V. inaequalis populations from 18

differents sites in Quebec.

Origin CyprodinilKresoxim-

Methyl Dodine Thiophanate-

methyl Flusilazole1 -0,2 -3,5 0,2 0,2 -2,12 -0,2 -2,2 -1,8 0,9 -1,63 -0,5 -3,2 -3,2 -2,5 -4,24 -0,6 -2,6 -0,9 0,1 -1,55 -0,6 -3,5 -1,5 0,4 -3,46 -0,2 -3,2 0,9 0,8 -4,07 -1,1 -3,9 -2,2 -0,3 -3,58 -0,8 -3,4 -2,3 1,4 -3,09 -0,3 -3,1 -1,7 -0,5 -3,6

10 -1,6 -4,0 -1,1 -0,6 -4,911 -0,6 -3,6 -0,4 -1,3 -5,112 -0,7 -3,3 -2,1 -0,1 -3,213 -1,4 -4,0 -1,4 -1,6 -3,414 -1,1 -3,4 -1,8 -0,3 -2,715 -0,8 -3,4 -1,7 0,1 -2,916 -0,9 -3,1 -1,5 0,1 -4,117 -0,9 -3,5 -0,7 0,7 -4,618 -1,0 -2,4 -1,4 0,3 -2,2

In vivo validation in orchards Potted McIntosh trees grafted onto M106 rootstock and grown outside were sprayed to runoff on May 13th 2005 with either Equal (400mg/L), Senator (225mg/L), Nustar (36mg/L), Sovran (80mg/L), Vangard (68mg/L), Captan 80WP (725mg/L) or a water control less than 24 hours prior to exposure to a natural primary infection period in 5 different orchards of the “Montérégie Ouest” region of Quebec. At least 6 trees (replicates) were used for each spray/site combination. Trees were removed from the orchards after the infection to prevent

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any exposure to the normal spray schedule in these orchards. The same trees were sprayed again on May 20th and re-exposed for the following infection period. Trees were finally moved away from any orchard and left unsprayed until scab severity assessment was performed on June 20th. Percent leaf area diseased was evaluated for each tree and disease reduction was reported in relation to the water control. Potted tree experiment in growth chambers The same experiment was carried with Potted McIntosh trees grafted onto M106 rootstock were sprayed to runoff with either Equal (400mg/L), Senator (225mg/L), Nustar (36mg/L). Results and discussion In vitro tests We observed population EC50 shifts for certain molecules in a number of commercial orchards as compared to a organic orchard. Criteria for rot risk assessment Twenty years ago, IOBC published a document that can be considered as one of the cornerstones of Integrated Production in Europe.

A PCO patent has been applied for by IRDA on the use of this method in 2003. Acknowledgements We wish to thank Wolfram Köeller for numerous and fruitful suggestions. References Gilpatrick, J.D., Smith, C.A. & Blowers, D.R. 1972: A method of collecting ascospores of

Venturia inaequalis for spore germination studies. – Plant. Dis. Rep. 56(1): 39-42. Nedoma, J., Vrba, J., Hanzl, T. & Nedbalova, L. 2001: Quantification of pelagic filamentous

microorganisms in aquatic environments using the line-intercept method. – FEMS Microbiology Ecology 38(1): 81-85.


pp. 223 - 230

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Temperature and humidity requirements for germination and infection by ascospores of Pleospora allii, the teleomorph of Stemphylium vesicarium Vittorio Rossi, Elisabetta Pattori, Simona Giosuè

Istituto di Entomologia e Patologia vegetale, Università Cattolica S. Cuore, Via E. Parmense 84, I-29100, Piacenza, Italy Abstract: Pleospora allii is the teleomorph of Stemphylium vesicarium, the causal agent of the brown spot disease on pear. The ability of P. allii ascospores to cause infection has not yet been demonstrated, and no information is available on environmental conditions favouring ascospore germination and infection. These ecological aspects were investigated by environment-controlled experiments. Dynamics of ascospore germination were observed between 0.5 and 48 hours of incubation at different temperatures (T 5 to 35°C), in water or in dry conditions, with relative humidity (RH) between 100 and 67%. Maximum germination occurred after 48 hours of incubation in water at 21-23°C; few ascospores germinated below 15°C and at 30-35°C. At 100% RH germination decreased by about one third and no germination was observed below 80%. Ascospores were inoculated on leaves of three pear varieties showing different susceptibility under orchard conditions (‘Abate Fétel’, ‘Conference’, and ‘William’ in decreasing order of susceptibility). Leaves were incubated at different T (5 to 35°C), 100% RH, and observed daily for the appearance of necrotic spots. Ascospores caused higher infection on ‘Abate Fètel’ than on ‘Conference’, while sporadic symptoms were observed on ‘William’. Highest disease incidence occurred at 25°C. Key words: Pyrus communis, environment-controlled conditions, primary inoculum Introduction Pleospora allii is the teleomorph of Stemphyilium vesicarim, the causal agent of brown spot of pear, but its role in the life cycle of the pathogen was not sufficiently known. In recent years, pseudothecia of P. alli have been observed in infected pear leaves on the orchard ground (Llorente & Montesinos, 2004; Maccaferri et al., 2003), maturing and releasing ascospores from February to May (Llorente & Montesinos, 2004). In northern Italy the first disease symptoms usually appear in June, when few or no ascospores are present in the orchard air (Picco et al., 1996; Maccaferri et al., 2003), while conidia are usually abundant (Rossi et al., 2005).

It was also demonstrated that herbaceous plants covering the soil of pear orchards can support saprophytic growth of S. vesicarium strains causing brown spot of pear, the production of pseudothecia during winter, maturation and ejection of ascospores, and abundant production of conidia (Rossi et al., 2004).

The ability of ascospores to cause infection on pear has not yet been demonstrated, and no information is available on environmental conditions favouring ascospore germination and infection. In the present work these ecological aspects of P. allii ascospores were investigated.

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Materials and methods Production of ascospores A strain of S. vesicarium (ISA/94) isolated from pear was used for production of pseudothecia of P. allii. This strain has been used to produce pseudothecia on pear and grass leaves (Rossi et al., 2005). Fungal colonies were grown on V8 agar (100 g of V8 juice, 3 g CaCo3, 20 g of agar per 1 litre of distilled water), pH adjusted to 7, at 20°C, with a daily photoperiod of 12 h of fluorescent light and 12 h of darkness. After 3 weeks, the fungus had formed pseudothecium primordia and colonies were then incubated at 10°C; filter paper was placed on the inner side of the cover of the dishes and moistened with water to maintain high RH (Llorente & Montesinos, 2004). First mature ascospores were observed after about 5 weeks.

Ascospore suspensions were prepared by collecting mature pseudotecia from colonies, crushing them in distilled water, and then filtering with a double layer of mesh cloth. Germination course of ascospores To test the effect of temperature (T) on ascospore germination, spore suspension was placed in test tubes (2 ml per tube) and incubated at 5, 10, 15, 20, 25, 30, and 35°C (3 replicate tubes per T regime). After 0.5, 1, 3, 6, 12, 24, and 48 hours of incubation, a drop (10 µl) of spore suspension was drawn from each tube, deposited on a microscope slide, and observed microscopically; the proportion of germinated ascospores was calculated over a random sample of 50 to 100 spores per drop. Only pigmented ascospores were considered: ascospores were classified as germinated when they had at least one visible germ tube.

To test the effect of relative humidity (RH), two experiments were carried out. In the first, 3 drops (10 µl) of spore suspension were deposited on a microscope slide and dried under an air flow (drops dried in few minutes). Slides were placed on Petri dishes, the base covered with blotting paper soaked with 0.7 ml of water. Dishes were sealed with Parafilm to maintain 100% RH, and incubated for 0.5 to 48 hours at 5, 10 15, 20, 25, 30, or 35°C. Proportion of germinated spores was then determined microscopically as previously described.

In the second experiment, the effect of different RH regimes on ascospore germination was tested. Spores were placed on microscope slides as previously described, and incubated for 24 h at 25°C, with 100, 98, 95.5, 92, 89, 87, 82, 81, 80, 67% RH. To obtain the different RH levels the slides were placed in Petri dishes sealed with Parafilm, soaked with water or different salt solutions, following Dhingra & Sinclair (1985). Maintenance of the defined RH level was recorded by data loggers (Tinytag Plus, Gemini Data Loggers Ltd., Chichester, UK). Microscopic observations were then performed to determine the proportion of germinated ascospores. Infection of pear leaves Ascospore suspensions were used to inoculate leaves of three pear varieties showing different sensibility under orchard conditions: Abate Fétel (high sensitive), Conference (sensitive) and William (resistant).

Leaves were detached from 2-year old potted plants grown in a glasshouse at 20 ± 2°C, washed in running tap water for 30 min, dried under a sterile air flow and placed in sterilised plastic boxes (20x30 cm) over blotting paper wetted with sterile water, the upper surface down. Six 10 µl drops of a conidial suspension were placed on the lower leaf surface (Fig. 6A). Control leaves were inoculated with water. Boxes were enclosed in plastic bags to maintain 100% relative humidity and incubated at different temperature regimes (5, 10, 15, 20, 25, 30, and 35°C), 12 h day-length, for 16 days.

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Leaves were inspected daily to determine the number of inoculated drops causing necrosis of the leaf tissue. Disease incidence was then calculated as a percentage of total inoculations showing necrosis over the total inoculations. Data analyses Percentage data on germination and on disease incidence were transformed using the arcsine function (arcsine % ) and then subjected to a factorial analysis of variance to test significativity of the experimental factors tested.

Percent germinations were then transformed in relative germination by dividing each value (temperature x incubation time x experiment) by the maximum germination observed in each experiment. The same was performed for the incidence data. Relative germinations and incidences were then fitted by a non-linear regression model in the form:

Y = ((α (Teqβ) (1-Teq))γ)/(1+exp(φ-λ t)) [1] where: α, β, γ, φ, and λ are the model parameters; Teq is an equivalent of T, calculated as (T-Tmin)/(Tmax-Tmin), where Tmin and Tmax are minimum and maximum levels of X when Y is equal to zero; t is the incubation length. This model describes germination or incidence progress over time of incubation by a logistic equation; in such an equation the asymptote depends on T, according to a Bete function.

Goodness of fit was evaluated through: the standard error of parameters and distribution of actual data versus the estimated data points; residual mean square (MSRES) and adjusted coefficient of determination (Radj2); number of iterations taken by the Marquardt algorithm to converge on parameter estimates (Quinn & Keough, 2002). All the statistical analyses were performed using SPSS ver. 11.5 (SPSS Inc., Chicago, IL). Results and discussion Germination course of ascospores Ascospore germination was significantly (P<0.0001) influenced by incubation time, temperature, and experiment. These 3 factors accounted for more than 50% of experimental variability, while their interactions, though significant, accounted for less than 30% of variability.

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(%) of P. allii ascospores in water. A, B, and C are three replicate experiments carried out with ascospores of different ages: first ascospores matured on 12 January, experiment A was carried out on 21 February, experiments B and C on 11 April.

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The time the ascospores remained in water accounted for 27% of total variability. In the first experiment, at 25°C, 7% of ascospores germinated in 30 min, 14% in 1 to 1.5 hours, about 45% in 3 to 6 hours, 67% in 12 hours, while all ascospores had produced a germ tube after 24 hours incubation. (Fig. 1A). Temperature accounted for 15% of variability; in all experiments, optimum temperature for ascospores to germinate was 25°C. The experiment accounted for 9% of variability. Germination was higher in the first experiment (13% on average) than in the others (5 and 3%, respectively) (Fig. 1). These differences in germination were likely due to differences in the ascospore age: the first experiment was carried out about 30 days after first ascospores have been matured, while the other 2 experiments 40 days after the first one.

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temperature (T, in °C), and incubation length (in hours), according to equation [1] (A). In B, distribution of model estimates versus observed data.

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(%) of P. allii ascospores over a dry surface, with 100% RH (A), and after an incubation of 48 hours at 25°C, with changing relative humidity (RH, in %) (B); bars in B represent the 95% confidence intervals of average germination.

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Relative germination was well described by equation [1] (Fig. 2A). Estimates of equation parameters and their asymptotic standard errors were: α = 4.36 ± 0.161; β = 1.225 ± 0.0544; γ = 3.012 ± 0.3933; φ = 5.39 - 0.328·T + 0.0085·T2; λ = 0.22 ± 0.028. In this equation, the parameter φ, that expresses the lag phase of the logistic curve, is a function of T. Adjusted R2 was 0.91, MSRES 0.0044, and the residues of estimated versus observed value were regularly distributed (Fig. 2B). Tmin was set at 0°C and Tmax at 40°C.

Germination was lowered when ascospores were incubated over a dry surface, with 100% RH. Maximum germination was 38% in water and 18% in dry conditions, nevertheless, the germination course in dryness showed the same dynamic over time and temperature regimes (Fig. 3A) as in water (Fig. 1).

When ascospores were incubated on a dry surface under different RH regimes the proportion of germinated spores rapidly diminished: at 90-95% RH it was one half the germination at 98-100% RH; at 83% and 86% RH only few ascospores germinated, and no germination occurred below 83% RH (Fig. 3B). Infection of pear leaves Ascospores were able to cause necrosis on detached leaves of the pear varieties considered. The first disease symptoms appeared after 6 days of incubation at 25°C on the 2 sensitive varieties, while in the resistant one symptoms appeared after 14 days (Fig. 4 A). Maximum disease incidence was observed after 12 days of incubation on ‘Abate’ and ‘Conference’, with 67% and 42% of the inoculated tissues producing necrosis, respectively. On ‘William’, disease incidence was 12% after 16 days of incubation (Fig. 4 A).

Pear varieties then reacted to ascospore infection as they do to the disease under orchard conditions (Montesinos et al., 1995); consistently, the leaf area occupied by necrosis around the inoculation sites was higher in ‘Abate Fétel’, than in ‘Conference’, and in ‘William’ (Fig. 5). Host specificity in S. vesicarium is due to the fact that the pathogen produces 2 host-specific SV-toxins (SV-toxin I and II), which selectively induce necrosis only on susceptible cultivars (Singh et al., 1999) by altering the plasma membranes of host cells (Singh et al., 2000).

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Figure 4. Incidence of necrotic spot on leaves of three pear varieties (‘Abate Fétel’,

‘Conference’, and ‘William’, in decreasing order of susceptibility to the brown spot disease in natural epidemics) after inoculation with P. allii ascsopores: incidene is expressed as a percent of the inoculated tissue that showed necrosis; A, dynamics of necrotic spots appearance over incubation time at 25°C, 100% RH; B, effect of temperature (T) on incidence after 16 days of incubation.

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A B C DA B C D Figure 5. Representative examples of necrotic tissue on pear leaves of ‘Abate Fétel’ (B),

‘Conference’ (C), and ‘William’ (D), 16 days after inoculation with P. allii ascsopores. Six drops of ascospore suspension were placed on the lower leaf surface as in A. Differences include the number of inoculation points evolving in necrosis and the extension of the necrotic area. In B, the tissue below the inoculation point was visibly more damaged than the neighbouring necrotic tissues.

Results from this work demonstrated that ascospores of P. allii produced by a S. vesicarium strain causing brown spot of pear are able to cause the disease on pear leaves. Pathogenicity of both conidia and ascospores has been demonstrated also for S. botryosum / P. herbarum on faba bean (Simay, 1992), S. botryosum / P. tarda on asparagus (Leuprecht, 1992), S. vesicarium / P. allii on garlic and onion plants (Basallote-Ureba et al., 1999).

Infection incidence after 16 days of incubation was significantly (P<0.0001) influenced by T, pear variety, and their interaction. T accounted for 27% of experimental variability, and its interaction with host varieties for 14%. No disease was observed for leaves incubated at 5 or 10°C, and sporadic symptoms appeared at 35°C (Fig. 4B). Maximum disease incidence was observed at 25°C for all the pear varieties, but on the susceptible ‘Abate’ infection incidence was high also at 30°C. In the resistant ‘William’ the disease appeared only at 25° and 30°C.

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ascospores on pear leaves of ‘Abate Fétel’ and ‘Conference’, temperature (T, in °C), and incubation length (in days), according to equation [1] (A). In B, distribution of model estimates versus observed data.

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Relative infection in ‘Abate’ and ‘Conference’ was well described by equation [1] (Fig. 6A). Estimates of equation parameters and their asymptotic standard errors were: α = 4.41 ± 0.099; β = 1.162 ± 0.0310; γ = 3.165 ± 0.2504; φ =6.63 ± 0.821; λ = 0.76 ± 0.099. Adjusted R2 was 0.88, MSRES 0.0108, and the residues were regularly distributed (Fig. 6B). This fit was obtained by setting Tmin=10°C and Tmax=38°C.

Comparison between ascospore germination and infection of pear leaves by P. allii showed some differences in ecological requirements. Germination occurred in a wider T interval (0<T<40°C) than infection (10<T<38°C), and optimum T was lower, 21-23°C versus 25°C, respectively.

Conditions for ascospore germination were similar to the lower values of the optimum range for S. vesicarium conidia, that is 20-30°C at 98-100% RH (Montesinos & Vilardell, 1992). On the contrary, optimal conditions for infection by conidia were lower than those found for ascospores: >24 h of continuous wetness are necessary at 22.6°C for 'Passe Crassane’ fruits and 21.1° for ‘Conference’ leaves (Montesinos et al., 1995). In the experiment of Montesinos et al. (1995), fruits and plants were inoculated with conidia at T ranging from 5 to 30°C and wetness durations from 0 to 24 h, and afterwards were maintained for expression of disease symptoms at 20°C and 80% RH. In the experiments carried out in the present work, leaves inoculated with ascospores were maintained at constant T regimes (5 to 35°C), 100% RH, until the appearance of symptoms. Therefore, differences in the environmental conditions during incubation could have influenced the above mentioned discrepancy.

In conclusion, the present work demonstrated that ascospores of P. alli produced by S. vesicarium strains able to cause the brown spot disease on pear are pathogenic on pear leaves. As a consequence, pseudothecia produced by the fungus teleomorph on pear leaf litter or in dead leaves of the herb species grassing the orchard floor are a potential inoculum source for primary infections. Relevance of this inoculum on epidemic development has to be demonstrated, because Pleospora ascospores are usually absent in spore sampling from the orchard air, or they are caught in very low numbers (Picco et al., 1996; Maccaferri et al., 2003).

Acknowledgements This work was funded by the Emilia-Romagna Region and co-ordinated by CRPV. References Basallote-Ureba, M.J., Prados-Ligero, A.M. & Melero-Vara, J.M. 1999: Aetiology of leaf

spot of garlic and onion caused by Stemphylium vesicarium in Spain. – Plant-Pathology 48: 139-145.

Dhingra, O.D. & Sinclair, J.B. 1985: Basic plant pathology methods. – CRC Press Inc., Boca Raton, Florida.

Leuprecht, B. 1992: Gezielte Bekämpfung von Stemphylium botryosum Wallr. an Spargel. Versuche zur Optimierung der Spritztermine. Aufbau eines Warndienstes in Bayern mit Hilfe von agrarmeteorologischen Messstationen. – Gesunde Pflanzen 44: 205-208.

Llorente, I. & Montesinos, E. 2004: Development and field evaluation of a model to estimate the maturity of pseudothecia of Pleospora allii on pear. – Plant Disease 88: 215-219.

Maccaferri, E., Collina, M. & Brunelli, A. 2003: Studies on the epidemiology of Stemphylium vesicarium on pear. – Journal of Plant Pathology 85: 310.

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Montesinos, E. & Vilardell, P. 1992: Evaluation of FAST as a forecasting system for scheduling fungicide sprays for control of Stemphylium vesicarium on pear. – Plant-Disease 76: 1221-1226.

Montesinos, E., Moragrega, C., Llorente, I., Vilardell, P., Bonaterra, A., Ponti, I., Bugiani, R., Cavanni, P. & Brunelli, A. 1995: Development and evaluation of an infection model for Stemphylium vesicarium on pear based on temperature and wetness duration. – Phytopathology 85: 586-592.

Picco, A.M., Betto, A. & Porri, A. 1996: Stemphyllium, Pleospora and Alternaria airspores in a pear tree orchard: a three year quantitative monitoring in Italy. – In: 1st European Symposium on Aerobiology, Santiago de Compostela, Spain: 156-157.

Quinn, G.P. & Keough, M.J. 2002: Experimental design and data analysis for biologists. – Cambridge University Press, Cambridge.

Rossi, V., Pattori, E., Giosuè, S. & Bugiani, R. 2004: Growth and sporulation of Stemphylium vesicarium, the causal agent of brown spot of pear, on herb plants of orchard lawns. – European Journal of Plant Pathology 111: 361-370.

Rossi, V., Bugiani, R., Giosuè, S. & Natali, P. 2005: Patterns of airborne conidia of Stem-phylium vesicarium, the causal agent of brown spot disease of pears, in relation to weather conditions. – Aerobiologia, in press.

Simay, E.I. 1992: Pleospora herbarum and some other fungi on faba bean stalks. – FABIS-Newsletter 31: 39-41.

Singh, P., Bugiani, R., Cavanni, P., Nakajima, H., Kodama, M., Otani, H. & Kohmoto, K. 1999: Purification and biological characterization of host-specific SV-toxins from Stemphylium vesicarium causing brown spot of European pear. – Phytopathology 89: 947-953.

Singh, P., Park, P., Bugiani, R., Cavanni, P., Nakajima, H., Kodama, M., Otani, H. & Kohmoto, K. 2000: Effects of host-selective SV-toxin from Stemphylium vesicarium, the cause of brown spot of European pear plants, on ultrastructure of leaf cells. – Journal of Phytopathology 148: 87-93.


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Equations for the distribution of Venturia inaequalis ascospores versus time during infection periods Vittorio Rossi1, Simona Giosuè1, Riccardo Bugiani2 Istituto di Entomologia e Patologia vegetale, Università Cattolica S. Cuore, Via E. Parmense 84, I-29100 Piacenza, Italy; 2Servizio Fitosanitario, Regione Emilia-Romagna, Via di Saliceto 81, I-40128 Bologna, Italy Abstract: Distribution of Venturia inaequalis ascospores versus time during an infection event was investigated by integrating in a dynamic simulation model the available knowledge on the biology of infection processes and the effect of environmental conditions. Processes of spore immigration on leaf surface, germination, appressorium formation, and successful infection establishment were incorporated into the model by elaborating mathematical equations depending on air temperature and length of the wet period. Survival of spores belonging to different development stages (ungerminated, germinated, with appressorium) was also included in the model as a function of temperature, relative humidity, and duration of wetness interruption. Based on comparison with previously published data the architecture of the model and its algorithms can be considered accurate and robust. Nevertheless, validation of model simulations under orchard conditions will be necessary before its use in management decisions. Key words: apple scab, infection efficiency, simulation model, systems analysis Introduction The apple scab fungus, Venturia inaequalis (Cooke) Winter, overwinters primarily as pseudothecia in affected apple leaves in the leaf litter. Spring rains trigger ascospore ejection from pseudothecia and ascospores cause infection on leaves and other susceptible host tissue (Biggs, 1990). Control of apple scab is achieved by repeated applications of fungicides to prevent ascosporic infections (Creemers & Vanmechelen, 2001; Carisse & Dewdney, 2002). In order to optimize disease control, fungicide applications are applied in response to discrete infection periods. Infection periods were defined by Mills (1944) as the minimum hours of continuous wetness required for leaf infection by ascospores to occur at temperature between 6 and 25°C.

At least two major revisions have been made to the Mills table. MacHardy & Gadoury (1989) introduced the Mills-3 curve: based on the assumption that the delay in ascospore discharge due to rain falling at night had inflated the times reported for ascospore infection by Mills, they suggested that the Mills times be reduced by 3 hours at all temperatures. Stensvand et al. (1997) revised the effect of temperature below 8°C. Minimum infection times do not determine quantitatively the proportion of ascospores establishing a successful infection in each infection period. The original Mills table distinguished ‘light’, ‘moderate’ and ‘heavy’ infections, but the correspondent curves identify the additional times leaves needed to remain wet for a visible increase in scab intensity, and not the time needed to have moderate or severe scab symptoms (MacHardy, 1996).

The use of the ‘minimum infection time’ criterion is widely used in apple scab management, but it is critical when there are intermittent wetting periods. No studies actually provide information on survival of the fungus over all of the various combinations of

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temperatures and wetting and drying intervals (Turechek & Carroll, 2003). As a consequence, different empirical rules have been proposed that generate confusion (Koehler, 2005).

Stensvand et al. (1997) stressed the need of constructing a distribution of infection versus time to better define disease management actions. Therefore equations for the distribution of V. inaequalis ascospores over time during an infection period were developed and integrated in a dynamic simulation model. Materials and methods Simulation model A dynamic simulation model was developed following the systems analysis (Rabbinge et al., 1989) and using “Powersim Studio 2005” (Powersim Software AS, Bergen, Norway), software designed to build advanced dynamic models quickly and efficiently. It allows the creation of a simulation project, to define units of measurement, variables, links, and flows, to set up the simulation, to create data in- and output objects, to view the results of simulation when it runs. Equation development Three literature sources were used to develop equations: Rossi et al. (2003a; 2003b), Turner et al. (1986), Becker & Burr (1994). Information from these works were used to develop new equations to be introduced in the simulation model. Details of this procedure are explained in the results. The SPSS statistical package ver. 11.5 was used for regression analysis (SPSS Inc., Chicago, IL). Results and discussion Simulation model development The simulation model makes the calculation of infection efficiency of ascospores possible for any infection period, with a time step of one hour. The flow diagram of the model is shown in Fig. 1; this diagram comes directly from the Powersim software and then it uses its specific symbols: symbols and variables are explained in Tab. 1 and 2, respectively. Table 1. Symbols used to draw the flow diagram of the model (see Fig. 1).

Symbol Explanation Symbol Explanation

State variable (or stock of material) Copy of an auxillary variable

Stock with undefined content

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Rate variable Flux of material

Auxillary variable Flux of information

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Table 2. Legend of variables used in the model (see Fig. 1). Acronym Explanation Acronym Explanation Auxillary varaibles Rate variables Teve Air emperature (°C) DEP Deposition rate (h-1) Heve Hours of the event (n) GER Germination rate (h-1) LW Leaf wetness (yes / not) APP Rate of appressorium production (h-1) Hdry Hours of wetness interruption (n) INF Infection rate (h-1) RHdry Relative humidity Hdry (%) MOR1 Mortality rate for S1 (h-1) Tdry Temperature during Hdry (°C) MOR2 Mortality rate for S2 (h-1) DS1 First derivative eq [1] MOR3 Mortality rate for S3 (h-1) DS2 First derivative eq [2] State variables DS3 First derivative eq [3] S1 Deposited spores (n·cm-2) DS4 Empirical index (0.01 to 2) S2 Germinated spores (n·cm-2) DM1 First derivative eq [4] S3 Spores with appressorium (n·cm-2) DM2 First derivative eq [5] S4 Infecting spores (n·cm-2) DM3 First derivative eq [6] S1D Spores died in S1 (n·cm-2) Parameters S2D Spores died in S2 (n·cm-2) DAD Deposited ascospore dose (n·cm-2) S3D Spores died in S3 (n·cm-2)

The model includes four state variables; these variables contain stocks of ascospores in four subsequent stages of the infection chain: S1, ungerminated spores on the leaf surface; S2, spores germinated that have not yet produced an appressorium; S3, spores with an appressorium; S4, spores that have successfully completed the infection process.

Spores enter stocks at variable rates, named DEP (deposition), GER (germination), APP (appressorium formation), and INF (infection), respectively. Rates depends on duration of the wet period and on temperature over such a period (Heve and Teve, respectively).

Total spores available during the infection process are defined by the parameter DAD, that is the deposited ascospore dose, the total number of spores that actually land on susceptible apple tissue during the infection event (MacHardy & Jeger, 1982).

When there is an interruption of the wet period (LW, leaf wetness, equal to zero) the infection process stops and spores being in S1, S2 or S3 can survive or die. Dead spores go in three state variables named S1D, S2D, and S3D, for spores in S1, S2, or S3, respectively, at the moment of wetness interruption. Spores enter the stocks of dead spores at three mortality rates (MOR1 to MOR3), which depend on duration of wetness interruption, temperature, and relative humidity during the dry period (Hdry, Tdry, and RHdry, respectively).

Some variables acting in the model come from outside (symbol ) while other are calculated within the model by specific equations. The former variables are the meteorological data of air temperature, relative humidity and wetness duration, recorded at hourly intervals. The latter variables are auxillary variables that make the calculation of rates possible by using meteorological data.

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Figure 1. Flow diagram of the model simulating the dynamic of infection by Venturia

inaequalis ascospores, drawn using the Powersim software (see Tab.1 and 2 for symbols and acronym explanations).

Three equations were developed to calculate the proportion of spores being in stages S1 to S3 over time of the infection event (Heve) as a function of air temperature (Teve):

[1]: DS1 = 1/(1+exp(2.999-(0.067·Teve+0.0000142)·Heve)) [2]: for Teve ≤ 20°C DS2 = 1/(1+exp((5.23-0.1226·Teve+0.0014·Teve2)-(0.093+0.0112·Teve+ -0.000122 Teve2)·Heve)) for Teve > 20°C DS2 = 1/(1+exp((-2.97+0.4297·Teve-0.0061·Teve2)-(0.416-0.0031·Teve+ -0.000245·Teve2)·Heve)) [3]: for Teve ≤ 20°C DS2 =1/(1+exp((6.33-0.0647·Teve-0.000317·Teve2)-(0.111+0.0124·Teve+ -0.000181·Teve2)·Heve)) for Teve > 20°C DS2 =1/(1+exp((-2.13+0.5302·Teve-0.00913·Teve2)-(0.405+0.00079·Teve+ -0.000347·Teve2)·Heve))

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Equation [1] was developed by Rossi et al. (2003b) to estimate the dynamic of airborne ascospores after a rainfall triggering ejection from pseudotecia, as a function of air temperature. In this work, equation [1] was used to estimate ascospore deposition (Fig. 2A); this simplification seems to be reasonable for the purpose of this work, because deposition depends mainly on density of the ascospores into the orchard air, when rainfall, wind, and tree canopy remain quite constant over the period when ascospores are airborne.

Equations [2] and [3] were developed starting from equations of Turner et al. (1986), that determine dynamics of 3 categories of ascospores (ungerminated, germinated, with appressorium) over time of wetness as a function of temperature in the interval 5 to 20°C. Turner’s equations were used to calculate proportions of ascospores that have germinated or have formed the appressorium on each Heve, with Teve ≤ 20°C. For higher temperatures, these proportions were estimated using data from Boric (1985). For each temperature regime (5 to 30°C), a new data set was formed, as follows: (i) it was considered that during an infection event some cohorts of ascospores immigrate on leaves, a cohort being formed by ascospores immigrating on each Heve, the number of cohorts being equal to the time that ascospores remain airborne; (ii) dynamics of germination and appressorium formation were calculated by averaging over Heve the proportions of ascospores that have germinated or formed the appressorium, respectively, in the different cohorts. Data were then fitted using a logistic model in the form Y=1/(1+exp(a-b·X)) (Fig. 2B and 2C); in such a model the parameters a and b were estimated by means of parabolas depending on Teve, the former parameter accounting for length of the lag-phase, the latter one being the rate parameter.

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Figure 2. Proportions of spores that deposit on leaves (A), germinate (B), and produce an

appressorium (C) over time of the infection event (Heve), at different temperature regimes (Teve).

The proportion of spores with an appressorium that successfully complete the infection process (spores in S4), i.e. produce a sub-cuticolar stroma, secondary hyphae, and finally disease symptoms, was determined by the empirical index DS4. This index ranges between 0.1 and 2, and was calculated by comparing the minimum time for infection to occur calculated by the model and that reported by MacHardy & Gadoury (1989).

Other equations were developed to determine mortality of ascospores in S1, S2, or S3 when there is an interruption of the wet period, as a function of interruption length (Hdry), temperature (Tdry) and relative humidity (RHdry) during such an interruption:

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[4]: DM1 = 0.263·(1-0.97315Hdry) [5]: DM2 = (-1.538+0.253·Tdry-0.00694·Tdry2)·(1-0.977Hdry)·(0.0108·RHdry-0.08) [6]: DM3 = (0.0028·Hdry)·(-1.27+0.326·Tdry-0.0102·Tdry2)

These equations were developed starting from the work of Becker & Burr (1994) for the

survival of conidia in different development stages (ungerminated, germinated, with appressorium) as a function of temperature and relative humidity during wetness interruption. These data concern conidia and not ascospores because no sufficient information is available for the survival of ascospores during discontinuous wetness (MacHardy, 1996).

To develop equations [4] to [6], the original regression equations of Becker & Burr (1994), that fit experimental data for each combination of temperature x relative humidity separately, were solved and the calculated proportions of viable spores were combined in a new data set, as mortality over time of interrupted wetness. These data were then fitted by the right regression model. For S1, the only significant independent variable was Hdry, that influenced mortality following a non-linear asymptotic model: Y=a·(1-bX) (Fig. 3A). For S2, Hdry, Tdry, and RHdry all influenced mortality, according to an asymptotic model, where the asymptote a depends on Tdry, multiplied by a correction factor that increases linearly with RHdry (Fig. 3B). For S3, Hdry and Tdry influence mortality in a linear combination, irrespective of RHdry (Fig. 3C).

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Model runs The model was used to simulate the development of apple scab infection under different environmental scenarios. For all the simulation runs DAD was set at 1 ascospore per cm2 leaf,

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so that the simulation results can be regarded as a proportion of ascospores in the different stages of the infection process. Duration of simulation was set at Heve = 60 h, with 1 h being the hour of rainfall triggering ascospore discharge. Meteorological conditions were changed with the simulation run.

In a first step, the model was operated with continuous leaf wetness, so that progress of infection depends only on Teve: Fig. 4 shows dynamics over Heve of spores being in S1, S2, and S3 when Teve is 5 to 30°C, step 5°C; Fig. 5 shows spores in S4, i.e. spores that have successfully completed the infection process.

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(ungerminated), S2 (germinated) and S3 (with appressorium) calculated by the model for 6 simulation runs with no interruption of leaf wetness and changing air temperature: A, 5°C; B, 10°C; C, 15°C; D, 20°C; E, 25°C; F, 30°C.

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Model outputs are in total agreement with the data on V. inaequalis ascospore development used to build the model, so that it can be concluded that both model architecture and algorithms correctly simulate the biology of the fungus.

Comparison of the model outputs with data not used in model building is difficult, because precise information on the effect of environmental conditions on infection severity is not available. The infection curves of Mills (1944) identify the approximate hours of leaf wetness required for ‘light’, ‘moderate’, and ‘heavy’ infections in an orchard with abundant inoculum, but these curves must be intended to identify the additional times leaves needed to remain wet for a visible increase in scab intensity (Mac Hardy, 1996), and not a robust measure of infection efficiency of ascospores under changing environmental conditions. Nevertheless, two data sets were used for a preliminary validation of model outputs.

Stensvand et al (1997) defined minimum time required after arrival of inoculum for successful infection and establishment in leaf tissue. These hour numbers were compared with that produced by the model, the latter being calculated by determining the Heve when S4 is ≥0.015, for Teve between 2 and 30°C, step 2°C (Fig. 6A).

Schwabe (1980) determined wetting and temperature requirements for leaf infection using artificial inoculation with ascospores. Relative scab infection was calculated from the Schwabe’s data for two leaf wetting periods, 18 and 24 h, and 6 mean temperatures during leaf wetting, 6 to 30°C. These data were compared with that produced by the model, as stocks of spores in S4, at Heve equal to 18 and 24 h, for the above mentioned Teve values (Fig. 6B and 6C).

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Figure 6. Comparison between model outputs (white dots) and data not used in model building (black dots): minimum time for infection (A) (Stensvand et al., 1997); relative infection after 18 h (B) and 24 h (C) of wetness (Schwabe, 1980).

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Considering that model outputs and literature data express scab development in different ways, there is a substantial agreement between the different series of data (Fig. 6), so it can be concluded that the model produces consistent simulations of ascospore dynamics during the infection event.

In a second step, the model was operated with intermittent wetting, so that progress of the infection depends on temperature and characteristics of wetness interruption, including duration of the dry period, temperature and relative humidity during such a period.

Fig. 7 shows dynamics over Heve of spores in S1 to S3, with a 8 h dry period with different conditions of temperature and relative humidity during such a period.

Fig. 8 shows these dynamics for an initial wetting of 10 h, with intermittent dry periods between 2 and 32 h, as in the experiments of Schwabe (1980).

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(INF), rates of mortality for spores in S1 (MOR1), S2 (MOR2), and S3 (MOR3), and stocks of spores being in S1 (ungerminated), S2 (germinated), and S3 (with appressorium) calculated by the model for 3 simulation runs with 8 h of wetness interruption (Hdry) and the following conditions for Teve, Tdry and RHdry, respectively: A, 15°C, 17°C, 70%; B, 20°C, 24°C, 85%; C, 25°C, 28°C, 90%.

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Figure 8. Stocks of spores in S1 (ungerminated), S2 (germinated), S3 (with appressorium),

and S4 (infecting) calculated by the model for 6 simulation runs with 10 h of initial wetting, and 0 (A), 2 (B), 4 (C), 8 (D), 16 (E), and 32 (F) h of wetness interruption. Teve and Tdry 18.8°C, RHdry 90%.

Simulations of Fig. 8 were compared with the data of Schwabe (1980): he measured the effect of different discontinuous wetting on scab development in apple trees after inoculation with ascospores. Relative infection was calculated by dividing the percent infection observed on trees exposed to 2, 4, 8, 16, and 32 h of dry to that obtained with continuous wetting. Schwabe performed 4 experiments with 16 h of wetting split in two periods of 1+15 to 4+12. To make simulations uniform to these experiments, the initial wetting was set at 10 h, so that all the spores entered S1 before the initial of drying. Averages of Schwabe’s experiments were then compared with model outputs, the latter calculated as the ratio between S4 with

intermittent wetting and that with continuous wetting. Model outputs were in general agree-ment with Schwabe’s data, that have not been used in the model building: model simulations were within the confidence limits of the experimental data, with the only exception for the simulation with 8 h of dry. .

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Fig. 9. Comparison between model output (white dots) and data not used in model building (black dots): relative infection with 2 to 32 h of dryness (1 is infection with no wetting interruption). Whiskers are the 95% confidence intervals for the means

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No more comparison was possible, because few studies have been conducted with the aim of solving the problem of evaluating the effect of intermittent wetting periods, but none of them provided answers for all of the various combinations of temperatures and wetting and drying intervals (Turechek & Carroll, 2003). As a consequence, different empirical rules have been proposed that generate some confusion, with minimum hours of dryness ranging from few as 4 hours to as many as 32 hours (Koehler, 2005).

Becker & Burr (1994) proposed the following rule based on their experimental results: if the interval of drying is less than 48 h in length, the initial and subsequent intervals of wetting should be summed to calculate Mills infection periods. MacHardy (1996) suggested a more conservative rule, by reducing the dry period to less than 24 h, regardless of weather conditions (sunshine, temperature, and relative humidity) during the intervening dry period. This rule was less conservative than the limit of 10 h suggested in Canada (Braun & Pelletier, 2003; Solymár, 2005), or the widely used rule of summing wetting periods separated by less than 6 h (Teviotdale & Gubler, 2002) or 8 h (Penrose, 2004) of sunny weather, this periods being extended by a safety margin of 3 to 4 h if drying is slow, and humidity remains high.

In conclusion, this work is a first attempt to construct distribution of V. inaequalis ascosporic infection versus time during an infection event, by integrating in a dynamic simulation model available knowledge on the biology of infection process and on the effect of environmental conditions. Based on comparison with previously published data the architecture of the model and its algorithms can be considered accurate and robust. Nevertheless, there are some critical aspects to be considered prior to using this model for apple scab management.

A first criticism concerns the inoculum dose available for any infection event, i.e. the dimension of DAD. This parameter could be estimated by integrating the measurement of PAD, i.e. the potential ascospore dose (Gadoury & MacHardy, 1986), with some available tools: a model estimating the proportion of the seasonal ascospores becoming airborne over time of the primary season (Rossi et al., 2000), rules for ascospore discharge (Rossi et al., 2001), a model estimating the rate of deposition of the airborne ascospores (Rossi et al., 2003a). A second critical aspect is that data used for developing equations for spore mortality concern conidia and not ascospores. The use of these equations is possible only by assuming that ascospores react to dryness as the conidia do, but this assumption needs to be verified. A third aspect concerns the need to test the model under orchard conditions, where different environmental and host conditions both change with complex interactions.

Acknowledgements This work was funded by the Emilia-Romagna Region and co-ordinated by CRPV. References Becker, C.M. & Burr, T.J. 1994: Discontinuous wetting and survival of conidia of Venturia

inaequalis on apple leaves. – Phytopathology 84: 372-378. Biggs, A.R. 1990: Apple scab. – In: Compendium of apple and pear diseases, eds Jones &

Aldwinckle, APS Press, St. Paul Minnesota: 6-9. Boric, B. 1985: Uticaj temperature na klijavost spora Venturia inaequalis (Cooke) Winter I

uticaj starosti na nijhovu vitalnost. – Zastita Bilja 36: 295-302. Braun, P.G. & Pelletier, J.R. 2003: Producing apples in eastern and central Canada. – Atlantic

Food and Horticulture Research Centre (AFHRC), http://res2.agr.ca/kentville/pubs/ pub1899/n1899_e.htm

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Carisse, O. & Dewdney, M., 2002: A review of non-fungicidal approaches for the control of apple scab. – Phytoprotection 83(1): 1-29.

Creemers, P. & Vanmechelen, A. 2001: Strategies and product selection in apple scab control. – Parasitica 57(1/3): 13-31.

Gadoury, D.M. & MacHardy, W.E. 1986: Forecasting ascospore dose of Venturia inaequalis in commercial apple orchards. – Phytopathology 76: 112-118.

Koehler, G.W. 2005: New England Apple Pest Management Guide. – www.umass.edu/ fruitadvisor/NEAPMG/, pp. 7-17.

MacHardy, W.E. & Gadoury, D.M. 1989: A revision of Mill’s criteria for predicting apple scab infection periods. – Phytopathology 79: 304-310.

MacHardy, W.E. 1996: Apple Scab: Biology, Epidemiology, and Management. – APS Press, St. Paul, Minnesota.

MacHardy, W.E. & Jeger, M. 1982: Integrating control measures for the management of primary apple scab, Venturia inaequalis (Cke.) Wint. – Prot. Ecol. 5: 103-125.

Mills, W.D. 1944: Efficient use of sulfur dusts and sprays during rain to control apple scab. – Cornell Extension Bulletin 630: 1-4.

Penrose, L. 2004: Apple and pear scab. – www.agric.nsw.gov.au/reader/pome-pests-diseases/ h4ab4.htm.

Rabbinge, R., Ward, S.A. & van Laar, H.H. 1989: Simulation and system management in crop protection. – Pudoc, Wageningen.

Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 2000: A new model estimating the seasonal pattern of airborne ascospores of Venturia inaequalis (Cook) Wint. in relation to weather conditions. – J. Plant Pathol. 82: 111-118.

Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 2001: Environmental factors influencing the dispersal of Venturia inaequalis ascospores in the orchard air. – J. Phytopath. 149: 11-19.

Rossi, V., Giosuè, S. & Bugiani, R. 2003 a: A model simulating deposition of Venturia inaequalis ascospores on apple trees. – EPPO Bull. 33: 407-414.

Rossi, V., Giosuè, S. & Bugiani, R. 2003 b: Influence of air temperature on the release of ascospores of Venturia inaequalis. – J. Phytopathol. 151: 50-58.

Schwabe, W.F.S. 1980: Wetting and temperature requirements for apple leaf infection by Venturia inaequalis in South Africa. – Phytophylactica 12: 69-80.

Solymár, B. 2005: Apple Scab. Ontario Ministry of Agriculture, Food and Rural Affairs. – www.omafra.gov.on.ca/english/crops/facts/apscab.htm.

Stensvand, A., Gadoury, D.M., Amundsen, T., Semb, L. & Seem, R.C. 1997: Ascospore release and infection of apple leaves by conidia and ascospores of Venturia inaequalis at low temperatures. – Phytopathology 87: 1046-1053.

Teviotdale, B.L. & Gubler, W.D. 2002: IPM Pest Management Guidelines: Apple. – www. ipm.ucdavis.edu/PMG/ crops-agriculture.htm.

Turechek, B. & Carroll, J. 2003: Probability of scab infections resulting from intermittent wetting periods. – Scaffold Fruit Journal, Update on Pest Management and Crop Development 12(7). www.nysaes.cornell.edu/ent/scaffolds/.

Turner, M.L., MacHardy, W.E. & Gadoury, D.M. 1986: Germination and appressorium formation by Venturia inaequalis during infection of apple seedling leaves. – Plant Dis. 70: 658-661.


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Climatic conditions prior to green tip of apple affect ascospore maturation in Venturia inaequalis Arne Stensvand1, Håvard Eikemo1, David M. Gadoury2, Robert C. Seem2 1Department of Plant Pathology, Norwegian Crop Research Institute, Plant Protection Centre, 1432 Ås, Norway; 2Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA Abstract: Apple leaves infected with Venturia inaequalis (overwintered on the ground) were collected in the following countries: Australia, Belgium, Brazil, Chile, Denmark, France, Germany, Italy, New Zealand, Norway and Sweden (1 to 3 years of sampling in each country, 21 site/year combinations). Samples were collected at green tip, air dried at room temperature and sent via air mail to Norway, where they were kept frozen (-18 ºC) until tested. Leaf disks cut from the samples were incubated moist, but without surface wetness at 20 ºC to allow continuous ascospore maturation. Each sample was immersed in water twice weekly over four weeks, until the supply of ascospores was exhausted. Ascospores ejected into water were collected and counted. Based upon number of days with rain prior to bud break for the two most extreme sites/years (lowest and highest number of days with rain), we adjusted the degree day accumulation for the biofix of an existing degree-day model of ascospore maturation. To improve performance at drier sites, 11.5 degree days were added to the model biofix for each additional day with ≥ 0.2 mm rain. At wetter sites 11.5 degree days were subtracted for each additional wet day. Regression analysis confirmed a significant correlation between the number of pre-bud break rain events and the subsequent pattern of ascospore release. There was no difference if using 0.2, 1.0 mm or 2.0 mm per day as limits for adjusting the degree day accumulation. These preliminary changes represent simple rainfall-based rules to adjust the ascospore maturity model to improve accuracy based upon weather conditions during the month before bud break of apple. Key words: aerobiology, apple scab, epidemiology, spore discharge, spore maturation Introduction Apple scab is caused by the ascomycete Venturia inaequalis. Ascospores of the fungus released during periods of rain in spring and early summer are the primary source of inoculum. Several temperature-based models have been developed to estimate ascospore maturity of V. inaequalis (Gadoury & MacHardy, 1982; James & Sutton, 1982b; Lagarde, 1988; Massie & Szkolnik, 1974). Protracted periods of dry weather can delay ascospore maturation in V. inaequalis (James & Sutton, 1982a, 1982b; Keitt & Jones, 1926; O'Leary & Sutton, 1986; Schwabe et al., 1989; Wilson, 1928). In North Carolina, it was found that the pseudothecial development rate was retarded in the period between 1 February and bud break if the water saturation deficit became greater than 85%, and there was no pseudothecial development in dry leaves. If rainfall was 0.25 mm or less and hours of 100% RH was 12 or less per day there was no change in pseudothecial maturity (James & Sutton, 1982b). The New Hampshire model which is based upon accumulated degree days after bud break of apple (Gadoury & MacHardy, 1982), was recently revised and now uses the impact of extended dry periods after bud break to refine estimates of ascospore maturity (Stensvand et al., 2005). By halting degree-day (base = 0 ºC) accumulation if 7 consecutive days without rain occurred, accuracy of the New Hampshire model was greatly improved.

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However, there are anecdotal reports throughout Europe regarding what seems to be a difference in the duration of ascospore release between the predominantly wet regions of north-western Europe and the drier regions south of the Alps. These reports further indicate that delayed maturation and release in dry regions is not always associated with dryness after bud break, but is sometimes associated with dry conditions before bud break, as was presumed to occur in North Carolina (James & Sutton, 1982a). Our objective was to experimentally determine the impact late-winter dryness (prior to initial spore maturity) has on the subsequent pattern of ascospore release.

Materials and methods

Apple leaves infected with V. inaequalis (overwintered on the ground) were collected in the following countries: Australia, Belgium, Brazil, Chile, Denmark, France, Germany, Italy, New Zealand, Norway and Sweden (1 to 3 years of sampling in each country, 21 site/year combinations altogether). Samples were collected at green tip, air dried at room temperature and sent via air mail to Norway, where they were kept frozen (-18 ºC) until tested. Leaf disks cut from the samples were incubated moist (100% RH), but without surface wetness at 20 ºC to allow continuous ascospore maturation. Each sample (containing 3 replicates) was immersed in water for two hours twice weekly over four weeks (for 5-600 degree days, base = 0 ºC), until the supply of ascospores was exhausted. Ascospores ejected into water were collected and counted in a light microscope.

Based upon experience from North Carolina of when wetness and temperature started to affect ascospore maturation in early spring, which was approximately one month prior to bud break (James & Sutton, 1982a), we concentrated on this period (last 30 days prior to bud break). Three parameters were calculated based on data from the driest and wettest location (the locations with the lowest and highest number of days with rain) for the last 30 days prior to bud break; range = difference in number of rainy days above 0.2 mm between the two locations, mean rain = mean number of rainy days with ≥ 0.2 mm for the two locations, difference in DD = difference in degree day accumulation at 50% ascospore accumulation in the laboratory for the two locations. The effect of number of days with rain during the month prior to bud break was estimated and applied as a correction factor to the assumed degree-day accumulation at the time of bud break for all site/year combinations.The procedure was repeated, including only events with ≥ 1 mm or ≥ 2 mm precipitation. Furthermore, we also tested the effect of number of rain days above 0.2 mm in combination with temperature. The effect of temperature was determined as for rainy days, by using the warmest and coldest locations to calculate the temperature range, mean temperature, and difference in DD. The accumulated percentages of spores released were transformed into probits of the percentage, and linear regressions were run against the adjusted degree days.

The shift in start of the season’s ascospore release based on pre-bud break weather conditions was evaluated using historical spore trapping data from Norway. Ten site/year combinations categorized as ”wet years”, i.e. less than 200 degree days between corrected and uncorrected degree day accumulation at 95% estimated maturity of ascospores (Stensvand et al., 2005) were included in linear regression analysis on probit transformed data.

Results and discussion

When plotting all the data for accumulated ascospore release, we found a variation in the percentage of spores released at the first time of testing for each sample (equal to time of bud break) from 0 to more than 30%. The more frequent rains there had been prior to bud break, the higher the percentage of spores were released early in the simulated season. There was an

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obvious shift in the release pattern during the simulated season according to moisture before bud break, but pre-bud break conditions did not seem to affect the steepness (rate of release) of the cumulative curves for each location and year.

Of all 21 site/year combinations tested, the sample from Brazil in 2003 had been exposed to the least number of rainy days for the last month prior to bud break, and the sample from Chile in 2002 had been exposed to the highest number; (i) range = 19 (the difference between Brazil 2003 and Chile 2002 in number of rainy days), (ii) mean rain = 14.5 (mean number of rainy days of ≥ 0.2 mm), (iii) = difference in DD = 218 (difference in degree day accumulation at 50 % spore maturation). The effect of each day with ≥ 0.2 mm rain prior to bud break on the shift in degree day accumulation was then: difference in DD/range = 218/19 = 11.5. Thus one day difference from the mean number of rainy days (which was 14.5) shifted the degree day accumulation with 11.5. The degree days for each location/year were then adjusted for days with rain according to the following equation: Actual degree day + (number of days with rain last 30 days before bud break – (mean rain) x 11.5. The resulting degree day accumulations from the international collection were then transformed into probits of the percentage, and the effect of the different adjustments was compared to the original degree days by linear regression. The regression for probit transformed numbers of events with ≥ 0.2 mm is shown in Fig. 1. Using 1 or 2 mm instead of 0.2 mm did not improve the R2 significantly.

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in 12 countries (21 site/year combinations) after simulated seasons in the laboratory. Degree day (DD) accumulation of ascospore release for each location was adjusted according to days with rain ≥ 0.2 mm the last 30 days prior to bud break. Data were probit transformed. Y = 0.008X – 1.13, R² = 84.7.

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Figure 2. Historical data of ascospore trapping from Norway (10 site/year combinations)

adjusted for number of days with rain ≥ 0.2 mm for the last 30 days prior to bud break. Data were probit transformed. Y = 0.807X + 3.079, R²= 84.7.

For the historical data from Norway, adjusting the degree days according to days with rain (events with ≥ 0.2 mm, ≥ 1 mm or ≥ 2mm rain per day) one month prior to bud break increased the R2 from 0.61 (no adjustments) to 0.72 (adjusted for rain). Results from using rain events ≥ 0.2 mm are shown in Fig. 2. There was no significant improvement if using higher rain thresholds (1 or 2 mm), but if including temperature, this improved the R² to 0.86.

Our results clearly demonstrated that the weather conditions prior to bud break affects the onset of ascospore release for V. inaequalis, but there was no change in the release pattern itself due to pre-bud break conditions. We suggest (preliminary) an adjustment of the biofix for mature ascospores based on the following: If there are 15 days (14.5 days according to our results) with ≥ 0.2 mm rain during the last 30 days prior to bud break, the first ascospores are mature and ready to be released at bud break. For each additional day with or without rain, the degree day accumulation should be adjusted with 10 degree days (11.5 degree days according to our results). Thus, if there are 5 days with rain (≥ 0.2 mm) during the last month prior to bud break, the degree day accumulation should be adjusted with 10 x 10 = 100, i.e. the first ascospores are ready to be released 100 degree days after bud break. If there are 25 days with rain prior to bud break, the first ascospores are mature approximately 100 degree days prior to bud break.

Our results indicate that extraordinarily wet or dry weather during the one-month-period before bud break can affect the maturity of the population at the time of bud break. The net effect on the model is to shift the curve depicting the distribution of spore maturity to the left or right. Excessively wet weather shifts it to the left, shortens the lag phase, and increases the percentage of the population that is mature at the time of bud break. Excessively dry weather has the opposite effect. Internal development of the ascocarp at 30 days before bud break is generally typified by presence of pseudoparaphyses, and is just prior to the time when asci are beginning to form in the centrum (James & Sutton, 1982a; Gadoury & MacHardy, 1982). James & Sutton (1982a) began accumulating environmental data to estimate ascocarp maturity on 1 February in North Carolina. We found little evidence that the environment prior to appearance of mature asci had an effect on the subsequent rate of maturation. Instead, we focused on the development of a correction factor that would apply specifically to the brief

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period immediately preceding ascus formation (i.e., one month before bud break). Although data accumulation beginning at an earlier date or stage of development, and incorporating the one month period immediately before bud break, as was done by James & Sutton (1982a) and Rossi et al. (1999), might also improve accuracy of the degree day model (Gadoury & MacHardy, 1982), our results indicate that focusing on the period immediately before bud break is likely to yield the same benefit. References Gadoury, D.M. & MacHardy, W.E. 1982: A model to estimate the maturity of ascospores of

Venturia inaequalis. – Phytopathology 72: 901-904. James, J.R. & Sutton, T.B. 1982 a: Environmental factors influencing pseudothecial develop-

ment and ascospore maturation of Venturia inaequalis. – Phytopathology 72: 1073-1080. James, J.R. & Sutton, T.B. 1982 b: A model for predicting ascospore maturation of Venturia

inaequalis. – Phytopathology 72: 1081-1085. Keitt, G.W. & Jones, L.K. 1926: Studies of the epidemiology and control of apple scab. –

Wisconsin Agricultural Experiment Station Research Bulletin No. 73: 104 pp. Lagarde, M.P. 1988: Etudes sur la maturation des ascospores de Venturia inaequalis (Cke.)

Wint. en vue de l’elaboration d’un modele. – Second International Conference on Plant Diseases 8-10 November 1988, Palais des Congres, Bordeaux, Lac. Annales II/III (4): 1093-1098.

Massie, L.B. & Szkolnik, M. 1974: Prediction of ascospore maturity of Venturia inaequalis utilizing cumulative degree-days. – Phytopathology 64: 140.

O’Leary, A.L. & Sutton, T.B. 1986: The influence of temperature and moisture on the quantitative production of pseudothecia of Venturia inaequalis. – Phytopathology 76: 199-204.

Rossi, V., Ponti, I., Marinelli, M., Giosuè, S. & Bugiani, R. 1999: Field evaluation of some models estimating the seasonal pattern of airborne ascospores of Venturia inaequalis. – Journal of Phytopathology 147: 567-575.

Schwabe, W.F.S., Jones, A.L. & van Blerk, E. 1989: Relation of degree-day accumulations to maturation of ascospores of Venturia inaequalis in South Africa. – Phytophylactica 21: 13-16.

Stensvand, A., Eikemo, H., Gadoury, D.M. & Seem, R.C. 2005: Use of a rainfall threshold to adjust a degree-day model of ascospore maturity of Venturia inaequalis. – Plant Disease 89: 198-202.

Wilson, E.E. 1928: Studies of the development of the ascigerous stage of Venturia inaequalis. – Phytopathology 18: 375-420.


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Application of the BSPCast model to control Stemphylium vesicarium in a district of the Emilia-Romagna region Clelia Tosi1, Massimo Liboni2, Riccardo Bugiani1 1Servizio Fitosanitario (Plant protection Service), Via Saliceto 81, I-40128, Bologna, Italy; 2OVR-Ortofrutticola Valle del Reno, Ferrara, Italy Abstract: Severe brown spot outbreaks occurred in 1970s and 1980s in the Emilia-Romagna region (northern Italy); from that moment several researches started to study epidemiology of Stemphylium vesicarium. At the end of 1990s, the Ferrara district organized, together with the Plant Protection Service, a team to coordinate and provide farmers (about 180000 ha) with all the technical information useful to rationalize the disease control. Information from simulation model was used to compile weekly bulletins for farmers. In those years a meteorological network was developing on the district area, and the data produced by the meteorological stations were used as inputs for model running. At the moment, the BSP-cast model, after a wide validation in the Ferrara district, works daily on all the meteorological stations and the results are spread to technicians and to farmers through bulletins and meetings. In 2002, a new service was activated, in collaboration with the Plant Protection Service service, to spread outputs of the BSP-cast model using SMS. Messages are produced three times a week, starting from the end of flowering to mid august, signalling the risk level for brown spot outbreaks. Since 2004, the service was improved by the daily quantification of spore presence in the orchard air using spore traps. Key words: pear, brown spot, Stemphylium vesicarium, forecasting model, warning service Introduction Brown spot of european pear has been first reported in Emilia-Romagna (Italy) in the mid ’70. From that moment on many efforts were made to develop strategies to control effectively the disease (Ponti et al., 1984; Brunelli et al., 1984; Ponti et al., 1993; Ponti et al., 1996). The severe epidemics stimulated the research and experimentation either towards the biology of the fungus and rationalization of the chemical applications that, in years climatically favourable for the disease, reach more than 20 sprays.

At the end of the ’80, a joint collaboration between the University of Girona (Spain), the Plant Protection Service of Emilia-Romagna, and the University of Bologna (Italy) led to the development of a forecasting model simulating the risk of infection on the bases of climatic conditions during the pear growing season (Montesinos et al., 1995; Llorente et al, 2000; Llorente at al., 2002; Bugiani & Gherardi, 1998; Bugiani et al., 2000). Demonstrative field trials From 1998 to 2000 demonstrative field trials were carried out.

The field trials aimed to compare the disease control strategy using BSPCast with a risk threshold of 0.6 and a calendar strategy. In all the years, the BSPCast strategy as well the calendar strategy made use of thiram. In 1998, the schedule strategy made use of thiram, kresoxym-methyl and the mixture fludioxonil+cyprodinil at 7 days interval. At harvest, the percentage of fruits affected was estimated.

On the whole, the BSPCast has been able to reduce 36%, 26% and 56% of the chemicals applied in 1998, 1999 and 2000 respectively (Table 1).

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Table1. Demonstrative field trials from 1998 to 2000 showing the percentage of spray reduction due to BSPCast application.

Year Strategy % of fruits affected at

harvest

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Disease pressure

Schedule using thiram 10 – Schedule using kresoxym- methyl 10 – Schedule using fludioxonil+cyprodinil 10 – 1998

BSPCast (0.6) using thiram 8 36

Medium

Schedule using thiram 0.3 – 1999 BSPCast (0.6) using thiram 0 26 High

Schedule using thiram 1 – 2000 BSPCast (0.6) using thiram 0 56 Low

The presence of Abbée Fétél, the most widely grown pear variety in the pear growing area of Emilia-Romagna and also the most susceptible to S.vesicarium infections has stimulated in the following years the birth of a regional warning service which provides farmers with information about the risk of infection elaborated by the BSPCast model.

Figure1. Information flow from weather data collected and elaborated by BSPCast, through technical meetings, up to disease strategy recommendations diffused to farmers.

The Brown Spot Warning service The warning service in Ferrara province uses the BSPCast model over a network of meteorological stations (Fig.1) covering 180.000 ha. The information is elaborated by

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specialised technicians who collect meteorological data from a network of weather stations located in pear growing areas in Ferrara province and validate by means of unsprayed pear plots located in the pear growing area. The information once validated, is diffused by means of : • Weekly technical bulletin on provincial magazines. • Ferrara province website • Weekly technical meeting with field technicians. • Weekly farmers meeting • Local Television programme • SMS service for farmers and field technicians and advisors (Fig.2).

Figure 2. Network of meteorological station located in pear growing areas in Ferrara

province. Table 2. Numbers of sms service subscribers over the years.

Year S.m.s. delivered N° subscribers 2002 8,500 171 2003 11,000 213 2004 15,000 290 2005 19,000 389

The information about the risk of brown spot infection (low, medium, high) elaborated

by BSPCast is nowaday updated and available three times a week from petal fall to harvest for the most susceptible pear cultivars. In the last four years, with aim to ever more differentiate the BSPCast information for the different pear growing areas of the province, a new service based on sms was set up. The BSPcast information is sent three times a week to mobile mobile phone for those farmers and technicians subscribing the service.

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Besides, since 2004, the concentration of S.vesicarium conidia carried out by spore-traps located in untreated pear plots, have been also provided by the warning service.

The number of subscribers for pear brown spot recommandation is ever more increasing (Table 1). So far at least 1.120 pear growing hectars and 280 farms follow the sms recommen-dations sent by the warning service.

References Brunelli, A., Di Marco, G., Contarelli, G. & Ponti, I. 1984: Prove di lotta contro la maculatura

bruna delle pere. – ATTI Giornate Fitopatologiche, I: 203-212. Bugiani, R. & Gherardi, I. 1998: Nuovi indirizzi per la razionalizzazione della difesa del pero

dalla maculatura bruna. – Informatore-Fitopatologico 48(6): 65-70. Bugiani, R., Gherardi, I., Brunelli, A. & Ponti, I. 2000: Maculatura bruna del pero: nuove

conoscenze epidemiologiche e strategie di difesa. – Rivista di Frutticoltura e di Orto-floricoltura 62(9): 36-40.

Llorente, I., Vilardell, P., Bugiani, R., Gherardi, I. & Montesinos, E. 2000: Evaluation of BSPcast disease warning system in reduced use of fungicide use programs for manage-ment of brown spots of pear. – Plant Disease. 84: 631-637.

Llorente, I., Moragrega, C., Vilardell, P., Montesinos, E., Bugiani, R., Govoni, P. & Gherardi, I. 2002: Field evaluation of a brown spot disease predictor as a system for scheduling fungicide sprays for control of Stemphylium vesicarium on pear. – Acta-Horticulturae (596): 539-542.

Montesinos, E., Moragrega, C., Llorente, I., Vilardell, P., Bonaterra, A., Ponti, I., Bugiani, R., Cavanni, P. & Brunelli, A. 1995: Development and evaluation of an infection model for Stemphylium vesicarium on pear based on temperature and wetness duration. – Phyto-pathology 85: 586-592.

Ponti, I., Cavanni, P. & Brunelli, A. 1982: Maculatura bruna delle pere: eziologia e difesa. – Informatore Fitopatologico. 3: 35-40.

Ponti, I., Brunelli, A., Tosi, C., Basaglia, M., Bevilacqua, T., Emiliani, G., Cont, C. & Viccinelli, R. 1993: Verifica dell’attività di diversi preparati contro la maculatura bruna del pero. – Informatore Fitopatologico 5: 45-52.

Ponti, I., Brunelli, A., Tosi, C., Cavallini, G. & Mazzini, F. 1996: Aggiornamenti sull’attività dei fungicidi contro la maculatura bruna del pero. – ATTI Giornate Fitopatologiche 2: 165-177.


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Resistance management in Vf resistant organic apple orchards Marc Trapman Bio Fruit Advies, Dorpsstraat 32, 4111 KT Zoelmond, [email protected] Abstract: Modern Vf scab resistant apple varieties open the way for organic growers to lower chemical input, higher yields, better skin quality, improved biological control against mites and insect pests and better consumer acceptance of their management practices. Many examples in the past years have shown however that the Vf resistance can be easily overcome by local scab populations in North-Western Europe. Discussions during the meetings of the IOBC pome fruit work group in 2000 in Fontevraud (France) and in 2003 in Lindau (Germany) lead to a set of management practices necessary to prevent gene-flux, and selection towards Vf virulence in the local apple scab populations. Eleven orchards of the Vf resistant apple variety Santana that were planted between 1998 and 2000 were monitored for apple scab lesions on fruits from 2002 to 2005. The results were evaluated in relation to the resistance management practices. We concluded that fungicide treatments against the major primary scab infections are the key factor in the resistance management on Vf resistant apple varieties. From a practical viewpoint, these early season fungicide applications are also necessary for the control of powdery mildew, as the main Vf- resistant apple cultivars appear to be relatively susceptible to powdery mildew. Growers that are not willing treat their orchards with fungicides al all should not plant Vf resistant varieties otherwise, they may well contribute to the rapid demise of Vf in their region. Introduction The potential of Vf resistant apple varieties Organic production of common commercial apple varieties like Elstar, Jonagold and Golden Delicious in North-Western Europe require 20 to 30 applications of sulfur, lime sulfur and or copper salts per year for the control of apple scab. Even with this high input, successful control of the disease is not guaranteed.

Modern Vf scab resistant apple varieties like Topaz, Red Topaz and Santana have a great potential for organic fruit growers. These varieties combine Vf resistance against apple scab with high yields, good storability and a consumer quality that is equal to better than common commercial varieties.

Moreover, the advantages go beyond that. A total of 20 to 30 fungicide treatments per year make consumers doubtful about what is meant by ‘organic’ production.

Furthermore, the management of apple scab with moderately effective fungicides put a enormous pressure on the growers during the entire growing season. This stress is mostly alleviated by growing Vf varieties. Additionally, the phytotoxicity of the inorganic fungicides reduce the overall production and skin quality of the fruits.

And last but not least, the toxic effects of sulphur and lime sulphur on beneficial insects and mites reduce the possibilities for natural control of spider mites and insect pests in organic orchards. Growing Vf resistant apple varieties reduces chemical input, increases yields, improves quality, and increases biological pest control and overall results in a more sound organic production system. The need for ‘resistance management’ During the past 20 years, many cases of apple scab lesions have been reported on Vf resistant varieties in Europe. In the Netherlands, as early as in 1979 symptoms of apple scab have been

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found on the varieties Priam, Prima, Priscilla and Sir Pize in the former experimental orchard De Schuilenburg. (Blommers et al., 1983) In the former national research station at Wilhelminadorp the first scab symptoms on Vf resistant varieties where noted in 1984 and a very severe outbreak occurred in 1997 (Kemp & Schouten, 1998). Ever since we had repeatedly cases in which scab symptoms where found in orchards planted with Vf resistant varieties in the Netherlands as well as in other European countries.

As Vf scab resistance seems to be overcome so quickly by the scab fungus, organic growers and advisors doubt whether Vf resistant varieties can hold their promises, and if it isn’t better to save the necessary investments in marketing an new variety and stick to the common commercial varieties.

During the meetings of the IOBC working group Diseases in Orchards in 2000 in Fontevraud (France) and in 2003 in Lindau (Germany) we discussed the problem and concluded that genetic information to overcome Vf resistance is probably percent in local apple scab populations in North Western Europe. Thus we agreed that specific management practices are necessary to prevent selection towards Vf virulence in the local apple scab populations.

Based on present knowledge and discussions, the following measures are advised for planning and management of Vf resistant orchards in The Netherlands: Table 1. Measures for resistance management in Vf resistant apple orchards.

Measures Impact on local apple scab population 1 Do not plant Vf- varieties together

with susceptible apple varieties ● Limits the scab population in the orchard ● Limits the genetic variability in the scab

population 2 Keep distance between Vf- orchards

and orchards with susceptible varieties

● Prevents Gen-flux towards the resistant orchard

3 Treat with fungicides on major primary infections

● Fungicides further limit the scab population that can infect leaf tissue

● Limits the absolute scab population in the orchard

4 Apply sanitary measures during winter.

● Limit over wintering scab population

These recommendations have a sound scientific background, but are they practical? And do these measurements deliver measurable results in retarding or preventing the local scab population to overcome of the Vf resistance ?

This contribution has to be seen as a feedback from the field to proceed the discussion on the necessity and practical possibilities for resistance management in Vf scab resistant orchards. Materials and methods From 1998 onward large blocks of the Vf resistant variety Santana where planted by organic growers. The need and principles of resistance management were communicated to the

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growers by advisers and researchers. Orchard layout and management practices outlined in Table 1 were adopted at various levels. Table 2 summarizes the relevant data and management praxis of the 11 orchards the orchards that where followed up. The last Orchard is not an orchard, but a collection of 6 different Vf resistant cultivars in a private garden.

1. In one orchard, 10% pollinator trees of the low scab susceptible varieties Alkmene and Discovery were planted in-between the Santana trees. In another orchard, a Malus floribunda selection was planted as a pollinator. In all other orchards Vf resistant varieties were used for pollination.

2. The distance of the Santana orchards to the scab susceptible orchards was variable. Grubbing out an old plantation and replanting it with Vf resistant varieties often does not always allow a reasonable distance between the Vf orchard and existing orchard blocks with susceptible varieties.

3. In orchard 1 to 8 (Table 2) fungicide treatments were made in relation to the major primary apple scab infections during spring. Infections where calculated by the apple scab management software RIMpro. Additional treatments with a low dosage of sulphur (2-3 kg /ha) where made to control mildew as Santana is very susceptible to apple mildew. The total number of fungicide applications in orchard 1 to 8 ranged from 3 to 13 treatments per year. In orchard 9, 10 and 11 no fungicide applications were made.

4. The application of urea is not allowed in organic farming, and materials with comparable effects on leaf decay and prevention of perithecium formation that could be used in organic farming are currently not available. Sanitary measures are limited to shredding and mulching leaves in autumn, and turning the soil with mechanical weed cleaning machines. The majority of the growers apply these sanitary measures. Due to higher soil microbial activity because the use of organic fertilisers, the leaf decomposition in organic orchards is noticeably better than in integrated orchards, even in absence of any specific sanitary measures. Contrary to conventional blocks, we can observe at but break that most of the leaves are decomposed. Table 2. Management details in the orchard blocks that were monitored.

Resistance management 1 2 3 4

Orchard

Plan

ting

(yea

r)

Surf

ace

(ha.

) Grass or mechanical weed cleaning

Susceptible varieties between ?

Distance to susceptible varieties (m)

Treatments on mayor primary infections

Sanitary measures

1 Dronten 1998 1.0 Mechanical Yes *) 0 Yes Yes 2 Lisserbroek 1998 1.5 Mechanical No 50 Yes Yes 3 Zeeland 1999 1.5 Mechanical Yes **) 25 Yes Yes 4 Meijel 1999 1.0 Mechanical No 50 Yes No 5 Tuil 2000 0.6 Grass mulch No 0 Yes No 6 Geldermalsen 1998 0.5 Mechanical No 0 Yes No 7 Randwijk 1999 0.8 Mechanical No 50 Yes Yes 8 Rees 2000 0.5 Mechanical No 50 Yes Yes 9 Lobith 1998 1.5 Long grass No 500 No No 10 Nispen 2001 0.5 Long grass No >1000 No No 11 Burgh-H. 2000 Few

trees Long grass No 25 No No

*) 10% pollinator trees of low scab susceptible varieties Alkmene and Discovery **) 10% pollinator trees of Malus Evereste

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The Santana orchards are now 4 to 8 years old. During the last 4 years the occurrence of apple scab was monitored by checking at least 500 randomly chosen fruits shortly before harvest. In cases where no scab was found in the sample, but fruits with scab symptoms were found outside the sample the incidence was recorded as <0.1 % scabbed fruits. Results Table 3. Percentage fruits with apple scab at harvest.

Orchard 2002 2003 2004 2005 1 Dronten 0 0 0 0 2 Lisserbroek 0.8 0 < 0.1 < 0.1 3 Zeeland 0 0 0 0 4 Meijel < 0.1 0 0 0 5 Tuil < 0.1 < 0.1 < 0.1 0 6 Geldermalsen 0 0 0 0 7 Randwijk < 0.1 0 < 0.1 0 8 Rees 0 0 9 Lobith 0.5 3 83.0 98.4 10 Nispen > 1% *) 54.5 100 11 Burgh-Haamsteede ~ 5 *) 65.3

*) Growers estimation

In orchard 3, apple scab symptoms were found on leaves in the planting year (1999) but never since. In most of the orchards, some fruits with apple scab symptoms were found in the last four years. In orchards 1 to 8, the scab incidence was kept at a low level. In orchards 9, 10 and 11 however scab incidence increased dramatically. In 2004 and 2005 we observed in orchards 9 and 10 that a large part of the leaves had dropped already in June because of severe apple scab infections (Table 3). Discussion and conclusions The most obvious difference between orchards with effective resistance management and orchards without is that orchard 9,10 and 11 did not receive fungicide applications on key moments during primary season. This seems to be the most important factor to prevent the scab epidemic to build up in the Vf resistant orchard.

From these data no conclusions are possible on the contribution of sanitary measurements to the problem as also other orchards lacked these treatments. As outlined before, leaf degradation in organic orchards is very rapid, and at but break very little leaves were still present, even without specific sanitary measures.

The relatively large distance to susceptible orchards in the case of orchards 9 and 10 did not prevent the build-up of a Vf virulent scab population.

Our conclusion is that fungicide treatments aimed against the major primary scab infections are the key factor in the resistance management on Vf resistant apple varieties. This conclusion is supported by the observation that in north western Europe both on isolated Vf resistant trees planted in private gardens, and Vf resistant orchards blocks on research institutes or commercial fruit farms that do not receive any fungicide treatments apple scab mostly develops within a few years.

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From a practical viewpoint these early season fungicide applications are also necessary for the control of powdery mildew, as the main Vf- resistant apple cultivars appear to be relatively susceptible to powdery mildew.

Leaving Vf resistant apple cultivars completely unsprayed as still is done on many research stations in Europe, as well as in private gardens is unwise, and poses a great danger on the commercial Vf resistant orchards in the region. For private gardens apple varieties with field resistance to apple scab should be advised, not Vf resistant varieties. Commercial fruit growers that are not willing treat their orchards with fungicides al all should not plant Vf resistant varieties otherwise, they may well contribute to the rapid demise of Vf . References Blommers, L., Freriks, J. & Trapman, M. 1983: Perceel IX/X Schuilenburg. Verslag van

onderzoek 1979-82 S240. – Internal report, March 1983. Kemp, H. & Schouten, H.J. 1998: “Gebrauchswert von Schorfresistenz (Vf) und resistenten

Appfelsorten“. – Dokumentation der Fachtagung 9-10 März 1998 der Universität Hohenheim.


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Infection risk and biological parameters: automating fungal spore count and leaf growth measurements Stijn Van Laer, Peter Jaeken, Piet Creemers RSF-Royal Research Station of Gorsem, De Brede Akker 13, B-3800 Sint-Truiden, Belgium, E-mail: [email protected], [email protected] Abstract: Beside climate parameters also biological factors are important for estimating fungal disease infection risk. The airborne fungal spore dose and the presence of susceptible plant parts determine the severity of infection. In 2004, the monitoring of these biological factors was optimized and automated for apple scab. Counting of ascospores collected with a Burkard spore trap was automated using a microscope with a motorized object table and a digital camera, and by means of image analysis based on the specific morphology of the scab ascospores. Automated counting started in the scab season of 2005. The detection limit based on the analysis of the spore count of 2004 was ± 25 spores/cm2 slide surface (± 20 spores/m3 air). Spores laying next to each other or touching another object on the slide are disregarded by the analysis. The efficacy observed had a high variation (efficacy range 5-50%) partly due to this reason. The efficacy obtained also depended on the background noise present, which in itself depends on climate conditions. Manual counting of these frames with the highest number of ascospores detected overcomes this problem. Until now plant growth monitoring consisted of determining growth of individual bloom clusters and following the phenological evolution. In 2004 vegetation area index (VAI) measurements were incorporated. By up scaling sampling, from a limited numbers of bloom clusters to a whole tree, the effect of biological variation during growth on the measurement is reduced. The utility value of VAI measurement for apple scab warnings are discussed and illustrated with the measurements done in 2005. Key words: apple scab, ascospore, image analysis, spore counting, vegetation area index Introduction At present infection risk for fungal diseases is for most diseases determined making use of only climatic data. Apart from the climatic factors also epidemiology and the sensitivity of the plant (variety tolerance, infectable plant tissue, phenological stage) play an important role. Infections which are classified as light infections can lead to very heavy infections when these periods are accompanied with the occurrence of high concentration of fungal spores and/or large infectable tissue masses present on the plant. A good example is apple scab caused by Venturia inequalis (Creemers, 2002; Machardy et al., 2001). In the past, only the beginning and the end of the ascospore flights, markers for the start and end of the primary infection period of scab, were determined. Now also the “Airborne Ascospore Dose” and the phenological evolution of the apple tree are integrated to determine the infection risk during a climatological infection period. A very strong correlation was found between both biotic parameters and scab infestation.

The determination of biotic parameters is enormous labor consuming and therefore not included in the prediction of infection risk for many diseases. Automating the monitoring of biotic parameters and implementing image analysis in the setup can be the solution. In the past several attempts were made for the automation of the fungal spore count, however non are implemented at present (Bernier & Landry, 2000; Benyon et al., 1999). The daily spore

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count must be made available to the agronomic advisers as soon as possible, however image acquisition and analysis requires a lot of calculating power, which equaled a lot of time in the past. The methods developed in the past took this in consideration, but by doing so decreased the accuracy of the measurements. The recent development of high resolution digital cameras for microscopy and the ever increasing calculating power of computers obligatory for image analysis makes it possible to refine the image analysis without increasing the measuring time (Perner et al., 2005).

LAI (Leaf Area Index) is defined as the ratio of the total projected leaf area over the corresponding ground area. VAI (Vegetation Area Index) also includes the non-photosynthetic active vegetation elements, namely stems, twigs, branches and dead leaves. Both parameters are commonly used in a wide range of applications as e.g. forest ecology, climatic change and canopy development. Recently a protocol was developed for measuring VAI/LAI in plantation systems in a non-destructive way (Jaeken et al., 2004). Although the method was developed for describing crop characteristics important for spray applications, it can be adapted for determining the formation of newly formed green tissue during the primary apple scab season. Materials and methods Automated fungal spore counting Scab ascospores were collected using a Burkard spore trap placed into a shallow pit covered with death apple leaves from previous growth season. The microscope slides with vaseline coming from the spore trap were fixated before counting (fixative: 5g gelatine, 50g glycerine, 0.5g phenol crystal, 50ml water).

Image analysis was conducted using a microscope with a motorized object table (Axioskop with an MCP4 controlled table; Carl Zeiss AG, Germany), a high resolution digital camera (Axiocam MRc5; Carl Zeiss AG) and image processing software that also controls the mobile object table (KS300, Carl Zeiss AG). Specimens were imaged at magnification 100x. Vegetation Area Index measurements The method used was developed recently by Jaeken et al. (2004). VAI values for the apple orchard were measured using a digital camera (Nikon Coolpix 900, Nikon Co., Tokyo, Japan) with a screwed on fish eye lens converter (FC-E8, Nikon Co.). The orchard was photographed twenty times in a zig-zag line between two tree rows to average out the variability attributed to the shot position. The camera itself was positioned 50 cm above ground level on top of water leveled tripod. The polar pictures were processed using standard image software (Corel Photo Paint) for masking the background and grey scale conversion. Winphot (Winphot 5.0, ter Steege, Tropenbos, Netherlands) was used to extract the VAI-values. Results and Discussion Automated scab ascospore counting A first attempt made in our laboratory to automate the counting of apple scab ascospore was performed in 1999. The results obtained were disappointing. Analysis had to be done at magnification 400x. As a result the amount of pictures to be analyzed was very high. Automatic counting of a slide coming from the spore trap took one day. The accuracy was moderate as only spores completely in focus could be detected and the results obtained depended greatly on the light conditions of the microscope. The amount of falls positives detected was also too high. The recent introduction of high resolution digital camera’s in microscopy and the increasing calculating power of computers made it possible to revise this method (Table 1). The method developed to detect the apple scab ascospores comprises

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thresholding-based image segmentation. This method has some limitation as objects overlapping or touching another object can not be distinguished. However the calculation time needed is less than case-based object recognition and user friendly software is available to perform thresholding-based image segmentation. Furthermore the number of spores that remain undetected due to this limitation is at an acceptable level (<10%). The revision of the original method from 1999 comprised several adjustments. The calibration performed at the beginning of the measurement was made independent of the lighting conditions. The selection criteria of the original method were made less strict in order to detect also ascospores out of focus. However this enhanced detection of false positives. Countermeasures taken to exclude false positives consisted out of an extra image analysis of the middle point of the region selected. This extra step focuses on the septum present in the middle of apple scab ascospores. The detection of ascospores at the end of the procedure is based on a comparison between the densitometric characteristics of the whole spore against the densitometric characteristics of the middle of the spore. Table 1. Image analysis steps performed for selecting and identifying/counting apple scab

ascospores, including the specific parameters of the different steps and input image used.

Image analysis steps Parameters Input image

Output image

greyscale conversion 256 grey values original image

1

contrast enhancement input grey values = 0-51 output grey values = 0-255

1 2

adaptive grey value segmentation matrix size of lowpass filter = 29 Discrimination level = -5

2 3

fill holes in all region 3 4 binary morphology opening octagon opening 4 5 scrap regions scrap hole size limits = 0-400

pixels 5 6

True colour threshold segmentation hue thresholds = 199-32 lightness thresholds = 106-153 saturation thresholds = 6-70

original image

7

First measurement using image 6 as a mask over image 7 Data to be measured: densitometric mean (A), standard deviation of densitometric mean (B),

minimum grey value (C), maximum grey value (D) (region select conditions: area 450-850 pixels; feret ratio 0.36-0.62; perimeter ratio(perim. of convex shell/perim.) >0.98; feret maximum 34.1-48.0 pixels; densitometric mean 108.5-158.0;

standard deviation of densitometric mean 15.1-27.5; minimum grey value of a region < (0.8841*densitometric mean)-28 )

marking middle point of the regions 6 8 binary morphology dilation octagon dilation 9 10

Second measurement using image 10 as a mask over image 7 Data to be measured: densitometric mean (E), standard deviation of densitometric mean (F)

(region select conditions: area >1 pixel) Ascospore recognition making use of densitometric measurements

Conditions to be fulfilled: D-C > 65

A+(2*B)-E-(2*F) < 8.5 D-C > (-3.6607*(A+(2*B)-E-(2*F))

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Not only image analysis was optimized. Focusing of the microscope on 10 focus planes (+ extra focus planes if the optimal focus plane was at the boundary plane evaluated) every 9 images taken and calculating the focus value on basis of all pixels and all grey values in the picture increased the robustness of method under evaluation. Despite this it is still impossible to focus all the objects visible on one image, due to the irregular surface of the slide. The ejection of ascospores occurs after/during rainfall and during dew events. Counting ascospores is therefore only necessary when these events occur. The macro developed contains the possibility of selecting the time windows to be counted, avoiding unnecessary counting. Because a high resolution digital camera is used, the analysis can be done at 100x magnification. This reduces the number of pictures to be taken and analyzed. Part of the time gained is however lost later during analysis due to the lager file size of the pictures.

The evaluation of the automated spore counting method was done making use of the spore captures performed during the primary scab season of 2004. The reference was the manual count performed in 2004. This analyses pointed out a major disadvantage of the automated counting, namely the dependency of the method on the quality of the slide and the background noise present (debris, pollen, other fungal spores), which itself depends on the climate conditions at that time point. Furthermore a lot of spores stick together when ascospore discharge is very high. The efficacy obtained ranged between 5-50% due to these reasons. In order to solve this problem, manual counting of the 5 frames on which the highest number of ascospores was detected with image analysis is included at the end of the procedure. By doing so, the difference in efficacy is accounted for. The detection limit based on the analysis of the spore count of 2004 was ± 25 spores /cm2 slide surface (± 20 spores/m3 air). Pear scab ascospores, spores with a similar morphology as apple scab ascospores, remained undetected, confirming the high accuracy of the method. The automated counting method was proven to be valuable and automatic spore counting started in 2005. At the start of primary scab season in 2005 one last problem arose linked to the difference in color of fresh and old ascospores. Ascospores darken while aging. As the evaluation of the method was done on the ascospore captures from 2004, only old spores were looked upon. The densitometric selection criteria had to be adjusted so that also fresh new spores were included. VAI measurement as a marker for leaf growth detection During the primary scab season the amount of newly formed green tissue is an important factor for determining actual scab infection risk and the dilution of fungicides present on the leaves. Until now plant growth monitoring in relation to infection risk determination consisted of measuring the growth of individual bloom clusters and following the phenological evolution. The introduction of VAI equals an up scaling of sampling as whole trees are looked upon. In correlation with up scaling, biological variation decreases as the number of bloom clusters monitored increases. However no differentiation is made between different bud types. Sometimes this can be important as a delay in flower development can occur ranging from 5 to 10 days. The utility value of VAI measurements are discussed and illustrated with the measurements done in 2005 (Figure 1).

Changes in phenology and leaf growth only came to expression in the VAI measurements after the trees in the orchard reached green cluster (BBCH 55). Before that time point, visual observations were still necessary. A better detection limit is needed in order to detect leaf growth in the earliest stages of flower development. The limiting factors here are the resolution of the pictures taken and the software which only allows pictures of a certain size. The importance of the early phenological stages may not be underestimated. Although the absolute increase in newly formed green tissue is low, the relative increase is already high. Fungicide applications during these pre green cluster stages will have a short protection time window due to the fungicide dilution effect. From green stage on, VAI

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measurements detected all growth events during the growth season. Most important growth event of the flower bud formation is the transition between green cluster and green bud (BBCH 56) as this moment coincides with the largest relative and absolute increase of newly formed green tissue. The two measurements performed during the transition were able to discriminate the growth accurately as a gradual increase is visible when looking at the VAI values. The change between green bud and balloon stage (BBCH 57) caused by cell expansion and not cell division was also clearly visible as an increase in VAI. VAI normally does not discriminate between leaves and non photosynthetic active parts of the plants like petals, however because apple blossoms are white, they are disregarded in the determination of VAI which is performed on the grey scale hemispheric image. Therefore changes observed during the stages where petals are present can be addressed to the start of shoot growth that occurs at the time of bloom (BBCH 61-65). The start of shoot growth is accompanied with a large increase of newly formed green tissue. Also this event could be observed clearly in VAI index. Although VAI does not give an exact value of the amount of green tissue formed, changes in VAI during the primary scab infection season coincided nicely with the time points were new green tissue was formed. The relation between VAI and the actual leaf surface is complex due to the specific morphology of a developing bloom cluster. Measurements of the mean leaf surface present at different phenological stages done in the past make correlation possible. The only thing missing is a good calibration method which is important for estimating the maximum VAI of an orchard reached for the different growth stages. This problem and also the problem concerning the detection limit of the VAI measurement will be addressed in the near future. VAI measurement can be a valuable tool for estimating the occurrence of newly green tissue when in the future the outcome can be expressed as relative and absolute increase of newly green tissue formed during the last days.

0

0.05

0.1

0.15

0.2

0.25

0.3

21/03

/2005

28/03

/2005

4/04/2

005

11/04

/2005

18/04

/2005

25/04

/2005

2/05/2

005

Phenological stage & Date

VAI

52 54 55 56 56 57 57 59 59 61 65

Figure 1. VAI measurements during the primary scab infection period and the phonological

stage at time of measuring (+/- standard error, n=20).

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Acknowledgements Research subsidized by the Ministry of the Flemish Community and the IWT (Institute for the Promotion of Innovation by Science and Technology in Flanders). References Benyon, F., Jones, A., Tovey, E. & Stone, G. 1999: Differentiation of allergenic fungal spores

by image analysis, with application to aerobiological counts. – Aerobiologia 15: 211-223. Bernier, T. & Landry, J. 2000: Algorithmic recognition of biological objects. – Can. Agri.

Eng. 42: 101-109. Creemers, P. 2002: Warning systems as basis for ecological agriculture and horticulture. –

Proceedings of V Entfrute meeting, Fraiburgo, Brazil, 35-52. Jaeken, P., Broers, N., De Moor, A. & Vercammen, J. 2004: Vegetation area index

determination in fruit crops and its link to spray application. – Aspects of Applied Biology 71: 311-319.

Machardy, W.E., Gadoury, D.M. & Gessler, C. 2001: Parasitic and biological fitness of Venturia inaequalis: Relationship to disease management strategies. – Plant Disease 85 (10): 1036-1049.

Perner, P., Jänichen, S. & Perner, H. 2005: Case-based object recognition for airborne fungi recognition. – Artificial Intelligence in Medicine, in press.


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Alternaria alternata, causal agent of dead (dormant) flower bud disease of pear M. Wenneker1, L.T. Tjou-Tam-Sin2, A.S. van Bruggen2, P. Vink3 1Applied Plant Research, Research Unit Fruit, P.O. Box 200, 6670 AE Zetten, The Netherlands ([email protected]); 2Plant Protection Service, P.O. Box 9102, 6700 HC Wageningen, The Netherlands; 3Applied Plant Research, Research Unit Flower Bulbs, P.O. Box 85, 2160 AB Lisse, The Netherlands Abstract: Dead (dormant) flower buds of pear are an important phenomenon in pear production in the Netherlands. Vigourous or unbalanced tree growth and Pseudomonas syringae pv. syringae are mentioned as likely causes of dead flower buds. Several tree growth control treatments including ethephon, Regalis (Prohexadione-Ca) and root pruning were evaluated. Regalis increased disease incidence. The plant stimulant (foliar fertilizer) Resistim (potassium phosphonate) reduced disease incidence. Pseudomonas syringae pv. syringae was occasionally isolated from diseased flower buds. However, Alternaria alternata was nearly always isolated from diseased buds. Pathogenicity of isolated A. alternaria was proven on detached dormant flower buds. By identifying the causal agent of dead flower buds disease, an effective control strategy can be developed. Key words: Ethephon, growth control, Pseudomonas syringae, Regalis, Resistim Introduction Dead flower buds are a common phenomenon in pear culture in the Netherlands, Belgium and Mediterranean countries (Deckers & Schoofs, 2001; Montesinos & Vilardell, 2001). However, also disease incidences are reported from South America; e.g. Uruguay and Brasil (E. Leoni, pers. comm.). The disease is characterized by partial or complete necrosis of flower buds during dormany or budbreak. Depending on disease severity, symptoms vary from reduced number of flowers per bud to buds completely killed.

The disease is present in most years but does not cause problems, due to the abundance of flower buds in normal years. However, in years with low bud numbers per tree, the disease causes significant (financial) losses, which was the case in 2001 in the Netherlands. Disease incidences may be as high as 80% – 90%.

First reports about the disease in the Netherlands date from the 1960's. The problem is mostly found in the main pear cultivar ' Conference', but also cultivars such as 'Doyenne du Comice', 'Verdi' and 'Gieser Wildeman' are also affected. Adequate control strategies are not available.

Until now, the bacterium Pseudomonas syringae pv. syringae (P.s.s.) was commonly regarded as the causal agent of dead flower buds in pear. However, the relation between P.s.s. and dead flower buds in orchards has never been proven in the Netherlands. It was concluded that population levels of P.s.s. were not significantly correlated to the amount of disease, in an extensive study over ten years in Spain (Montesinos & Vilardell, 1996). Other possible causes mentioned are unbalanced (vigourous) tree growth, abiotic stresses, incompatibility between scion and cultivar, and other plant pathogens and pests. However, dead flower buds caused by the pear bud weevil (Anthonomus pyri) are easily distinguished from dead flower bud disease.

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The objectives of the project are (i) to evaluate the effect of tree growth regulation, (ii) to test the effect of plant stimulant (foliar fertilizer) application, (iii) to monitor disease development, (iv) to isolate possible pathogens and (v) to develop possible control strategies. Material and methods Tree growth regulation and plant stimulant application Trials were performed in a pear orchard located at the experimental station at Randwijk, the Netherlands. The orchard was of spindle shaped pear trees (cultivar Conference) on Quince MC rootstock. Trees were planted in 1999 in a single row planting system (3.5 m x 1.5 m). Spray applications were carried out with a handheld spray gun (manufacturer EMPAS) with a 1.2 mm ceramic hollow cone nozzle at 1.1 – 1.2 Mpa and a spraying volume of 1000 l ha-1. The experiment was done in a randomised block design with five replicates. Each replicate consisted of 5 trees. Observations were made on the middle 3 trees.

The experiments consisted of the following treatments: Growth regulation:

1) Non-treated control. 2) Luxan ethephon (a.i. ethephon 48%): four applications with seven to ten days

interval. First application starting ten to fourteen days after bloom. First application: 250 ml ha-1; second application 150 ml ha-1, third and fourth application 100 ml ha-1.

3) Regalis (a.i. prohexadione-Ca, 10%): three applications with tree weeks interval. First application starting at three to five leaf stage. Rate: 1 kg ha-1.

4) Root pruning: two-sided, at 35 cm from the trunk. Pruning was carried out in two periods. First period east side of the trees at the end of May. Second period west side of the trees at the begin of June.

Plant stimulant (foliar fertilizer): 5) Resistim (potassium phosphonate: potassium 139 g l-1; phosphor 75 g l-l): seven

applications (weekly). First application begin of May. Rate: two litres ha-1. The experiments (treatments) started in 2002, and were continued in 2003 and 2004 on

the same trees to study over-year effects of the treatments. Disease development and pathogen assessment During bud development samples were taken regularly, and buds were microscopically examined for the presence of symptoms (i.e. lesions or necrotic flower primordia). Samples of dormant flower buds were collected and transported to the laboratory, in autumn and winter of 2002 – 2003. Bulk samples of 20 buds were analyzed for the presence of Pseudomonas syringae pv. syringae, according to the standard procedures of the Plant Protection Service. Other buds were cut into two pieces and assessed for the presence of diseased flower primordia or necrotic lesions. Also, individual buds were plated onto Potato Dextrose Agar (PDA) and analyzed for the presence of (pathogenic) fungi.

In 2004 in 8 commercial pear orchards random samples of 100 flower buds per orchard were taken. In the laboratory 50 buds were individually assessed for the presence of symptoms, and 50 buds were individually assessed for infection with Alternaria. The buds used for determination of infections were surface sterilized and cut into two pieces. The flower primordia of each bud were plated onto PDA. Disease assessment Disease incidence was assessed at the beginning of bloom (April), in the subsequent year after the treatments were carried out. All flower buds per tree were counted and the disease incidence per tree was calculated from the overall count (as a percentage dead flower buds).

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Mean disease incidence of all trees for each replicate was used for statistical analysis. Effect of treatments was determined with ANOVA at a 0.05 probability level. Results Pear tree growth regulation and plant stimulant In 2003 – 2005 the dead flower bud incidence varied between the subsequent years. In 2003 there was a high disease incidence, as in 2005 disease incidence was relatively low. In 2003 no statistical differences of the treatments were found compared to the untreated control. In 2004 Regalis and root pruning increased the occurrence of dead flowers buds, as Resistim decreased the number. In 2005 the same trends were observed, however the effects were not statistically significant (Figure 1).

abb

ab

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101520253035404550

2003 2004 2005

% d

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control ethephon Regalis root pruning Resistim

Figure 1. Effect of treatments on dead flower bud incidence in 2003 – 2005.

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1015202530354045505560

control ethephon Regalis root pruning Resistim

% d

ead

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> 1 year twigs one year twigs terminal buds (1-year)

Figure 2. Effect of treatments on dead flower bud incidence in 2004.

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In 2004 significant effects of the treatments were found. However, the effect was dependend of the position of the flower bud in the tree. Figure 2 shows that lowest disease incidences are found on older twigs, relatively most dead buds are found on the terminal buds on first year twigs. With exception of the resistim treatment. Resistim resulted in low disease incidences, especially on one year twigs and terminal buds. Regalis application increased dead flower bud incidence, especially on first year twigs (Figure 2). Disease development and pathogen assessment Microscopic examination revealed that first symptoms of the disease developed in autumn and early winter. The expression of symptoms started with the senescence of the top flower of the flower cluster. The disease progressed during winter and spring. Eventually, resulting in the death of most flowers and decay of buds at flowering.

In 2002 – 2004 the bacterium Pseudomonas syringae pv. syringae was only isolated from non-sterile bulk samples in relatively low numbers. Indicating a low presence of P.s.s. in the orchards. The bacterium was only sporadically isolated from dead flowers in dormant buds. However, the fungus Alternaria alternata was (nearly in all samples) found in diseased flower buds and also often in symptomless flower buds. Alternaria assessment in commercial orchards In the commercial orchards the internal visual symptoms (i.e. necrotic spots and dead flower primordia) ranged from 2% – 50%. The infection rate ranged from 10% - 85%. There was a very good correlation between the occurrence of visible symptoms and infection with Alternaria alternata (Figure 3).

R2 = 0.902

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Figure 3. Relation between visual symptoms and infection of A. alternaria. Discussion and conclusions Effect of growth regulation and Resistim One of the objectives of the experiments was to evaluate the effect of tree growth regulation and the plant stimulant Resistim on dead flower bud incidence. However, no positive effect of tree growth regulation was observed in the trials. It gave proof to the contrary; treatments like root pruning had a tendency of increased disease incidence. Regalis (a.i. prohexadione-Ca) increased disease incidence, and also lowered the number of flower buds per tree.

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Only Resistim had a positive effect in controlling dead flower buds. Especially in reducing disease incidence in buds on young twigs and terminal buds. Possibly, Resistim acts as a systemic fungicide and is transported to growing points (sinks). From these results it can be concluded that tree growth control is not an option for reducing dead flower buds incidence on pear.

It should be taken into account that ethephon and Resistim are phosphonate derivates. Phosphonate derivates as potassium phosphonate, aluminium tris-o-ethylphosphonate (fosetyl-Al), and 2-chloroethylphosphonate (ethephon) appeared to be effective at high doses to reduce infections of P. syringae pv. syringae on pear under controlled conditions (Mora-grega et al., 1998). Fosetyl-Al (Aliette) is used to control diseases caused by fungi (Coffey & Joseph, 1985; El-Hamalawi et al., 1995). Potassium phosphate is known to induce systemic resistence in barley to powdery mildew (Mitchell & Walters, 2004). Also prohexadione-Ca is known to have antibacterial properties (Aldwinckle et al., 2002; Maxson & Jones, 2002).

In this study it was observed that first symptoms of the disease process are already visible in October, i.e. long before bloom. This implies that a possible pathogen has penetra-ted the flower bud during summer. This makes it more easy to isolated the causal agent. Because from (rotting) dead flower buds as observed at bloom buds all sorts of secundary pathogens can be isolated, including Pseudomonas-species.

Until now, it was commonly accepted that the bacterium Pseudomonas syringae pv. syringae was the causal agent of dead flower buds of pear. This was partly due to the fact that Pseudomonas syringae is proven to be the causal agent of blossom blast. However, the symptoms of blossom blast are characterized by blast of blossom and leaves which occur in periods of cool wet weather during bloom and post-bloom stages (Jones & Aldwinckle, 1990). However, these symptoms differ from the symptoms of dead flower bud disease; which are characterized by partial or complete necrosis of flower buds during dormany or budbreak. Mainly due to ice-nucleation activity (INA) of Pseudomonas syringae and often large resident populations of this bacterium in orchards a relation with dead flower buds is assumed, and Koch's postulates have been completed (Montesinos & Vilardell, 1991).

In contrast, extensive research in Spain (Montesinos & Vilardell, 2001) did not reveal a significant relation between dead flower bud incidence and Pseudomonas levels. In addition, antibacterial treatments control (copper and kasugamycin) did not prevent the occurrence of dead flower buds. In our study P.s.s. was only isolated sporadically from bulk samples and individually diseased flower buds. Indicating that the bacterium plays a minor role in disease development in winter and early spring in the Netherlands. However, the fungus A. alternata was (nearly in all samples) found in diseased flower buds and also in symtomless flower buds.

Apparantely, A. alternata is capable of penetrating and infecting pear flower buds. A. alternata is known to cause late blight in pistachio (Pryor & Michailides, 2002; Evans et al., 1999) and several diseases in fruit crops such as moldy-core in apple (Reuveni et al., 2002) and brown rot in citrus (Timmer et al., 1998). Therefore, it is assumed that A. alternata is the causal agent of dead flower buds of pear in the Netherlands.

The correlation between the occurence of visual symptoms and the pressure infection with Alternaria alternata supports this hypothesis. In laboratory tests the pathogenicity of A. alternata was proven on flower buds of detached pear twigs (Wenneker et al., in prep.). The survey in commercial orchard revealed that the infection rate of dormant flower buds with A. alternata can be over 80%. Indicating the potential (financial) risk for individual growers in years with favourable conditions for disease expression. Future research should focus on the difference in infection rates between orchards; i.e. effect of inoculation pressure and/or spraying scheme.

It is possible that standard fungicides control most (antagonistic) fungi, with the exception of Alternaria alternata, and thereby creating conditions for massive growth of

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Alternaria on pear buds. By identifying the causal agent of dead flower bud disease of pear, an effective control strategy can be developed, e.g. fungicide schemes with Alternaria specific fungicides as Rovral (a.i. Iprodione). In field trials it was already proven that fungicide treatments can reduce dead flower bud incidence significantly (Wenneker et al., in prep.). Acknowledgements This research is funded by the Dutch Product Board for Horticulture. The authors wish to thank Ron Anbergen, Patrick Hendrickx and Kaisa Kiesi for their excellent co-operation in this research. References Aldwinckle, H.S., Reddy, M.V.B. & Norelli, J.L. 2002: Evaluation of control of fire blight

infection of apple blossoms and shoots with SAR inducers, biological agents, a growth regulator, copper compounds, and other materials. – Acta Horticulturae 590: 325-334.

Coffey, M.D. & Joseph, M.C. 1985: Effects of phosphorous acid and Fosetyl-Al on the lifecycle of Phytophtora cinnamoni and P. citricola. – Phytopathology, 75: 1042-1046.

Deckers, T. & Schoofs, H., 2001: Bacterial Problems in Belgian pear growing. – The compact fruit tree 34 (4): 121-124.

El-Hamalawi, Z.A., Menge, J.A. & Adams, C.J. 1995: Methods of fosetyl-Al application and phosphonate levels in avocade tissue needed to control stem canker caused by Phytophtora citricola. – Plant Disease 79: 770-778.

Evans, N., Michailides, T.J., Morgan, D. & Felts, D. 1999: Studies on sources of inoculum of Alternaria Late Blight of Pistachio. – KAC Plant Protection Quarterly 9 (2): 4-6.

Jones, A.L. & Aldwinckle, H.S. 1990: Compendium of apple and pear diseases. – American Phytopathological Society, St. Paul, Minnesota, USA.

Mitchell, A.F. & Walters, D.R. 2004: Potassium phosphonate induces systemic protection in barley to powdery mildew infection. – Pest management science 60: 126-134.

Maxson, K.L. & Jones, A.L. 2002: Management of fire blight with gibberellin inhibitors and SAR inducers. – Acta Horticulturae 590: 217-233.

Montesinos, E. & Vilardell, P. 1991: Relationship among population levels of Pseudomonas syringae, mount of ice nuclei, and incidence of blast on dormant flower buds in commercial pear orchards in Cataluna, Spain. – Phytopathology 81 (1): 113-119.

Montesinos, E. & Vilardell, P. 2001: Effect of bactericides, phoshonates and nutrient amendments on blast of dormant flower buds of pear: a field evaluation for disease control. – European Journal of Plant Pathology 107: 787-794.

Moragrega, C., Manceau, C. & Montesinos, E. 1998: Evaluation of drench treatments with phosphonate derivates against Pseudomonas syringae on pear under controlled conditions. – European Journal of Plant Pathology 104: 88-94.

Pryor, B.M. & Michailides, T.J. 2002: Morphological, pathogenic, and molecular characterization of Alternaria isolates with Alternaria Late Blight of Pistachio. – Phytopathology 92: 406-416.

Reuveni, M., Sheglov, D., Sheglov, N., Ben-Arie, R. & Prusky, D. 2002: Sensitivity of Red Delicious apple fruit at various phenologic stages to infection by Alternaria alternata and moldy-core control. – European Journal of Plant Pathology 108 (5): 421-427.

Timmer, L.W., Solel, Z., Gottwald, T.R., Ibanez, A.M. & Zitko, S.E. 1998: Environmental factors affecting production, release, and field populations of conidia of Alternaria alternata, the cause of brown spot of citrus. – Phytopathology 88 (11): 1218-1223.


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Testing alternative chemicals against apple scab and powdery mildew Xiangming Xu, Joyce Robinson, Angela Berrie East Malling Research, New Road, East Malling, Kent ME19 6BJ Abstract: Experiments were conducted in glasshouse compartments or polytunnels to investigate the efficacy of several organic-compatible chemicals in controlling apple powdery mildew and apple scab when applied as a protectant, curative and antisporulant fungicides. Several products resulted in statistically significantly reduction of mildew or scab severity; however, the reduction in disease severity achieved by these products, compared to the untreated or conventional fungicides, was very small and still unacceptable in commercial organic production. Only two traditional products, copper and sulphur, controlled scab and mildew effectively. We conclude that in the UK where environmental conditions are very conducive to scab and mildew epidemics, the only feasible solution to control scab and mildew in organic production is to grow cultivars which are resistant to the diseases, especially scab. Key words: copper, apple, scab, mildew, sulphur, organic Introduction One of the main reasons for the poor performance of current organic apple production methods is inadequate pest and disease control. Apples are subject to attack by a wide range of highly damaging pests and diseases. The diseases scab and mildew are particularly debilitating. They severely reduce tree growth, yield and quality. In conventional production, they can be managed satisfactorily by fungicides coupled with disease warnings generated by Adem™ (Berrie & Xu, 2003). The range of plant protection products available for disease control in organic production in the UK is very limited (copper carbonate and oxychloride, potassium soap, sulphur). There is an urgent need to discover novel organic-compatible products that can effectively be used to manage apple scab and mildew.

There is a considerable range of plant protection products based on clay, mineral, compost and algal extracts that are claimed to have fungicidal properties against diseases on several crops (Fallik et al., 1997a; 1997b; Pasini et al., 1997; Petsikos-Panayotarou et al., 2002; Scheuerell & Mahaffee, 2002; Gamagae et al., 2003; Mann et al., 2004). Recent work in Australia has also suggested that raising the pH of the leaf and fruit surface and using calcium hydroxide controlled scab. Sodium bicarbonate may be a safer alternative. Most reports on controlling diseases by natural products often do not provide sufficient details, which does not instil confidence in some claims resulting from these studies. It is essential that we should investigate the effectiveness of various alternative products in controlling apple scab and powdery mildew under UK conditions. Materials and methods This study was conducted in three phases. A wider range of products were included in the first phase, but less effective/useful ones were discarded in subsequent experiments to enable a more detailed study of the most promising ones. Table 3.1 gives the alternative chemicals tested in this research. In the first phase, preliminary experiments were conducted in glass-

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house compartments or polytunnels to investigate the efficacy of selected organic-compatible chemicals in controlling apple powdery mildew and apple scab when applied as a protectant, curative and antisporulant fungicide using MM106 rootstock plants. In the second phase, experiments were conducted on small potted trees in a sand-bed to determine the activity and relative persistence of the alternative products selected from the first phase experiments. Finally, a few products were selected and taken further to orchard trials.

New products were constantly added to the screening experiments, particularly in the first phase experiments, during the entire five-year period. Hence, not all products were evaluated in a single experiment. Therefore, the efficacy of the products was only compared with the untreated controls within the same experiment. First phase on rootstock plants Plant materials and inoculation. Rootstock MM106 plants were used for testing; they were potted up in batches and grown in a ‘mildew-free’ and ‘scab-free’ glasshouse compartment at about 20°C (18-23°C) and 70% relative humidity (rh) with a 16 h light/8 h dark daily regime. Leaf positions were identified by tagging the youngest fully unrolled leaf at the time of inoculation or spraying. Plants were randomly placed in the compartment/polytunnel before spray application.

For powdery mildew, on each plant to be inoculated, the shoot tip was labelled; the four youngest leaves on each labelled shoot tip were inoculated by shaking conidia from the mildew-infected leaves onto their surface. For apple scab, conidia were washed off previously infected and stored leaves with distilled water; a spore suspension was prepared with its spore concentration adjusted to 2.5x105 conidia per ml. Inoculation was conducted in glasshouse compartments (c. 25 m2) with three misting nozzles to maintain surface wetness (high humidity). Misting nozzles were switched on immediately after inoculation and turned off 24 hours later (i.e. giving 24 h duration of wetness). The tagged shoot tip was sprayed with the spore suspension using a fine hand-held aerosol sprayer. Each shoot tip received approximately 0.3 ml spore suspension, i.e. approximately 75000 conidia, which thoroughly wetted the shoot tip.

Treatments. We conducted three separate experiments in this phase. In the first experiment, ten chemicals were included for testing: Ca(OH)2, Milk, Herb silica, Liquid silica, Ulmasud B, Mycosin, Neudo vital, Sulphur, Wetcol 3 and Equisetum. They were applied either at 100% of the labelled recommended full rate and at the rate used in previous research (Table 3.1). In addition to these chemicals, two control treatments were also included: water-treatment and untreated. The second experiment was conducted to investigate the efficacy of compost teas in controlling apple powdery mildew and apple scab when applied as a protectant fungicide. We have tested compost tea in a mixture with a surfactant (Agral) as well as on its own. In total, there were seven treatments: fungal compost tea with or without Agral, bacterial compost tea with or without Agral, Systhane, Agral and untreated. In the third experiment, the efficacy of Serenade against scab was evaluated. In all three experiments, a complete randomised design was used and the experiments were repeated. In each repeat, there were at least three plants each with up to 11 shoots for each treatment.

In the first experiment, each chemical was tested as a protectant, curative and anti-sporulant treatment against powdery mildew, and as a protectant and curative treatment against scab. For the protectant test, chemicals were first applied with a hand-held sprayer to the tagged shoots until runoff. Then these treated shoots were inoculated with scab conidia 3 or 8 days after the spray, or with mildew conidia 4 or 8 days after spray. For testing curative effects, selected shoots were first inoculated with mildew or conidia and then treated with chemicals 2 or 4 days later. For anti-sporulant testing, chemicals were similarly applied to sporulating mildew colonies on rootstock plants; these colonies resulted from inoculation

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done 14 days before the application of chemicals. In the second experiment, only the protectant effects were evaluated: products were applied 1 day after inoculation. For the Serenade test against scab, plants were either treated with Serenade 2 days before inoculation or inoculated 2 days before being treated with Serenade.

Table 1. Products used in the screening studies and their rate of use.

Product Active ingredient Concentration Rate/L Phase of the study

Mycosin Aluminium sulphate + Horsetail extract

1% 10 ml/L 1

Ulmasud B Activated alumina meal 2% 20 ml/L 1 Herb silica Not available 5 L/ha 2.5 ml/L 1 Ca(OH)2 Ca(OH)2 4 kg/100L 40 g/L 1 Headland Sulphur Sulphur 560 ml/100L 5.6 ml/L 1-3 Neudo vital Plant extract 5 ml /500 ml 10 ml/L 1 Lime Sulphur Calcium polysulfide 15 g/L 2 Equisetum Horsetail extract 2% 20 ml/L 1 Wetcol 3 Copper sulphate/lime 50 L/1000L 50 ml/L 1-3 Milk Not available 50% 500 ml/L 1 Milsana Extract of Reynoutria 1.2% 12 ml/L 1-3 Serenade Bacillus subtilis 6 kg/ha 6 g/L 1-3 Liquid silica Not available 5L/ha 2.5 ml/L 1-3 Compost Tea – fungal Not available 100 ml/L 1, 3 Compost Tea – bacteria Not available 100 ml/L 1, 3 MaxiCrop Seaweed extract 100 ml/L 3 Polyversum Fungi-like organism

Pythium oligandrum 0.5 g/L 2

Farmphos Potassium phosphite 2.5 ml/L 2

Disease assessment. Scab was assessed on each of all the fully unrolled leaves above the tag (including the tagged leaf) on each inoculated shoot tip, up to a maximum of five leaves for rootstock plants. The upper surfaces of each leaf were examined for scab colonies 12-14 days after inoculation and the approximate percentage of leaf area with scab colonies was recorded for each leaf. The number of mildew colonies were counted on each leaf of the treated shoots from the leaf ‘0’ (youngest fully extended leaf) to the tagged leaf about 10-14 days after inoculation for the protective/persistence and curative tests. For antisporulant tests, 10-14 days after treatment, roughly 3 cm2 pieces of sticky tape were cut and put on the top of the treated lesions; they were then peeled off and stuck onto glass slides. The imprint of mildew colonies left on the sticky tape was examined under a microscope. The percentage of healthy (non-damaged) mildew conidia was estimated by examining 50 conidia per slide.

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Second phase on potted trees Experiments in this phase were conducted over two years (2001 and 2002). In the sand-bed tests, no artificial inoculation was used and hence disease development relied on natural inoculum and infections.

In 2001, several products were selected on the basis of glasshouse results for testing to evaluate their efficacy against powdery mildew and scab on potted trees of 5-year-old cv. Queen Cox. Products tested were Headland sulphur, Lime sulphur, Wetcol 3, Milsana and Liquid silica. In addition, Systhane and an untreated control were also included. They were sprayed every 10 days from June 8th until 30th July 2001, a total of 6 times. Polyversum, a root drench treatment, was also applied from 28th June, a total of four times. There were 10 trees per treatment.

Eight growing shoots were labelled per tree and the fruits removed to encourage them to keep growing. On each tree, the tip of each labelled shoot was sprayed with an appropriate product. On each tree scab and mildew were assessed on five shoots, randomly chosen from the eight treated shoots, on 22nd June, 6th July and 10th August. On the first two occasions leaves ‘–1’ to ‘–4’ were recorded for number of lesions. For the last record leaves ‘–1’ to ‘–8’ were recorded, again, for number of lesions for the two diseases. Leaf ‘0’ is the youngest fully unrolled leaf and ‘–1’ is the leaf immediately below (i.e. older) the leaf ‘0’. A random block design was used with two blocks. Within each block, there were five trees for each treatment.

In 2002, tests were conducted on potted trees of 5-year-old cv. Queen Cox and 3-year-old cv. Gala to evaluate the following products: Wetcol (pre-bud burst application), Wetcol, Distillery by-product, FarmFos42, Headland Sulphur, Lime Sulphur, Liquid Silica, Milsana, Polyversum and Serenade. As in 2001, Systhane and an untreated control were also included. They were sprayed every 10 days from bud-burst until 30th July 2002, a total of 12 times, except the bud-burst application of Wetcol 3. There were eight trees (five Cox and three Gala) per treatment.

Unlike in 2001, the whole tree was sprayed thoroughly with an appropriate product. On each tree, five shoots were randomly chosen for assessment of scab and mildew in mid-May. In August, diseases on both leaves and fruits were assessed. Leaves ‘–1’ to ‘–4’ were recorded for number of lesions of scab and mildew whereas all fruit on each tree were assessed for fruit scab. Orchard test Orchard evaluation was carried out in 2003 and 2004 in an organic orchard (VF211) at East Malling Research. In 2003, seven products were evaluated: early copper (Wetcol) at pre-bud-burst, routine copper at low rate, Milsana, Serenade, Liquid Silica, Sulphur and compost tea. Unfortunately, the compost tea making facility was not delivered from The Netherlands until mid-July and hence compost tea was not included as a treatment. In addition, an untreated control was also included. Tests were conducted on two cultivars: Red Pippin and Saturn. The orchard was divided into three blocks and in each block there were three trees of each cultivar for each treatment. From April, each product (apart from the early copper treatment) was applied routinely (every 10-14 days weather permitting). However, in early summer, because of bad weather (either too windy or wet), maintaining the interval of spray was not possible.

Scab and mildew were assessed three times: June, July and August. For each assessment, one tree from each cultivar in each block, located in the middle of the three trees, was assessed for each treatment. For assessing mildew, five shoots were randomly selected and presence of mildew was recorded on the top five fully unrolled leaves. For scab assessment, it was the same as mildew for the first two assessments whereas only fruit scab was assessed in the last assessment. All the fruits on the tree were assessed for the presence of scab lesions and number of scab lesions was counted on those infected fruits.

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In 2004, nine treatments were evaluated in the same orchard as in 2003: fungal compost tea, bacterial compost tea, Maxicrop, three pair-wise combinations of Liquid Silica, Maxicrop and Milsana, Sulphur and untreated. These were selected on the basis of results obtained in the previous years. Again, each treatment was evaluated on both Red Pippin and Saturn. The orchard was divided into two blocks and in each block each treatment had four trees for each of the two varieties. From April, each product was applied routinely (every 10-14 days weather permitting). However, by early June, mildew was very severe and incidence of fruit scab on Red Pippin was very high, the experiment was hence terminated then.

Scab and mildew were assessed in early June. Two trees from each block of each cultivar, located in the middle of the four trees, were assessed for each treatment. For mildew assessment, five shoots were randomly selected and presence of mildew was recorded on the top five fully unrolled leaves. Scab assessment on leaves was the same as mildew. For the assessment of fruit scab, all the fruits on the tree were assessed for the presence of scab lesions and number of scab lesions was counted on those infected fruits. Statistical analysis Disease incidence data (p), i.e. proportion of leaves or fruit infected, were logit-transformed

( ⎟⎟⎠

⎞⎜⎜⎝

⎛− pp

1ln ) before analysis of variance. Disease density data, i.e. number of colonies per

leaf/fruit, were logarithm-transformed before analysis of variance. Analysis of variance was used to assess the overall significance of treatment effects. The significance of each pair-wise treatment difference was evaluated using the least significant different (LSD) test on the transformed scale. Results Rootstock (glasshouse test) Table 2 presents the summary of the protectant tests in the first experiment. Overall there were significant differences between treatments in both scab and mildew severity. Several products had significantly reduced the number of mildew lesions, compared to the untreated; these included Ca(OH)2, Sulphur, Wetcol 3. In contrast, none of treatments had significant control effects on scab; indeed several products apparently significantly increased the scab severity.

Table 3 presents the summary of the curative tests in the first experiment. Overall there were significant differences between treatments in both scab and mildew severity. However, none of the products showed any significant curative control effects against either scab or mildew. Similarly, none of the treatments had significant anti-sporulant effects against mildew, except Ulmasud B (Table 3).

Table 4 gives the summary of the second experiment using compost tea products. None of the alternative products had any harmful effects on scab and mildew development. Indeed, the combination of Agral and bacterial compost tea resulted in even greater incidence than the untreated (P < 0.01). Interestingly, Systhane did not control scab effectively either. Further tests indicted that the isolates used were not sensitive to Systhane (results not shown). Serenade did not reduce scab significantly compared with the untreated when applied either as a curative or protectant product (the third experiment). Sand-bed tests 2001 Test Apple Scab: Analysis of variance showed that there were no significant block effects for both years, and so data were pooled over the two blocks. Results from 2001 tests on potted trees of cv. Queen Cox are given in Table 5. For the first two assessments, scab lesions were only

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observed on a few leaves. There were also no significant differences between treatments. For the last assessments, only 202 out of 2270 leaves had developed scab lesions. However, there were significant differences between treatments (Table 5). All the treatments significantly reduced the number of lesions, compared with the untreated. Of the products tested, the two sulphur products and Wetcol 3 were the best; their performance is statistically similar to Systhane. Table 2. Average number of mildew lesions or average percentage of scabbed leaf area on

each treated/inoculated leaf in the testing for protective action of the chemicals on MM106 rootstock. There were no significant differences between treatments with the same letter after their means (LSD tests).

Powdery mildew Scab (%) 1-4 days 5-8 days 3 days 8 days

Ca(OH)2 1.3 ab 2.2 ab 9.8 d 0.5 a Equisetum 12.1 b 5.0 bc 8.2 cd 5.6 c Herb silica 12.2 b 5.4 bc 9.5 d 2.9 abc Liquid silica 4.1 ab 8.0 c 8.8 cd 2.0 abc Milk 7.5 ab 2.2 ab 8.2 cd 4.5 bc Mycosin 11.0 ab 4.2 bc 4.4 bc 2.4 abc Neudo Vital 13.0 b 8.8 c 3.9 abc 3.0 abc Sulphur 0.1 a 0.1 a 0.7 ab 2.3 abc Ulmasud B 6.6 ab 4.7 abc 4.4 cd 1.7 abc Untreated 8.8 ab 8.6 c 3.3 ab 1.8 ab Water 8.8 ab 6.1 bc 6.2 cd 0.8 ab Wetcol 3 3.6 ab 4.9 ab 0.5 a 1.9 ab

Table 3. Average number of mildew lesions or average percentage of scabbed leaf area on

each treated/inoculated leaf in the testing for curative action of the chemicals on MM106 rootstock as well as antisproulant effects (expressed as average percentage of healthy conidia on colonies sampled 10 days after chemical application). There were no significant differences between treatments with the same letter after their means (LSD tests).

Powdery mildew Scab (%) 2 days 4 days Antisporulant 2 days 4 days

Ca(OH)2 0.9 ab 5.5 abc 76.9 abc 0.00 a 0.15 Equisetum 10.7 c 15.9 c 87.8 bc 0.04 a 0.69 Herb silica 7.3 bc 11.5 bc 77.1 ab 0.02 a 0.01 Liquid silica 1.8 ab 6.9 abc 91.3 c 0.01 a 0.24 Milk 4.4 abc 8.6 abc 87.3 bc 0.02 a 0.36 Mycosin 2.3 ab 3.0 abc 77.3 abc 0.46 b 0.17 Neudo Vital 4.7 abc 11.9 abc 75.3 ab 0.05 a 0.06 Sulphur 0.0 a 0.0 a 82.4 abc 0.01 a 0.15 Ulmasud B 1.4 ab 11.2 bc 67.7 a 0.20 a 0.05 Untreated 3.2 ab 9.8 abc 89.1 bc 0.01 a 0.20 Water 2.3 ab 6.1 abc 90.2 bc 0.30 a 0.55 Wetcol 3 0.4 a 2.0 ab 75.8 ab 0.01 a 0.22

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Table 4. Average number of mildew lesions and percentage of scabbed leaf area on each treated/inoculated leaf in the testing for protective action of the chemicals on MM106 rootstock. There were no significant differences between treatments with the same letter after their means (LSD tests).

Chemicals Mildew Scab Systhane 1.6 c 28.2 a Untreated 23.5 a 30.0 a Agral 35.8 a 22.6 a Bacteria tea 36.8 a 27.8 a Fungal tea 27.7 a 25.5 a Bacterial tea + Agral 52.0 b 27.2 a Fungal tea + Agral 25.5 a 25.3 a

Table 5. Average number of scab lesions per leaf on potted 5-y-old trees of cv. Queen Cox in

sandbed. Products were applied every ten days. There were no significant differences between treatments with the same letter after their means (LSD tests).

Scab Powdery mildew Products 1st & 2nd

assessment 3rd

assessment 1st

assessment 2nd

assessment 3rd

assessment Headland Sulphur

0.196ab 0.28a 0.70b 0.54b

Lime Sulphur 0a 0.32ab 0.08a 0.12a Liquid Silica 0.283bc 1.15d 1.40cd 1.13d Milsana 0.680d 1.02d 1.63e 1.64d Polyversum 0.473cd * 1.33de 2.45f Systhane 0.177b 0.35b 0.65a 0.34ab Untreated 0.801e 1.43e 2.41f 2.01e Wetcol 3

There were no

significant differences between all

the treatments

0.033a 0.73c 1.06c 1.01c

Apple powdery mildew: On the first assessment, mildew lesions were observed on 497 out of 1070 leaves. On those leaves with lesions, number of lesions ranged from 0 to 15. There were significant differences between treatments; treatment means are given in Table 3.5. All the treatments significantly reduced the number of mildew lesions, compared with the untreated. Of the products tested, the two sulphur products were the best and their performance is statistically similar to Systhane, with average 0.3 lesions per leaf compared to 1.4 lesions per leaf for the untreated control. Similar results were also obtained for the other two assessments (Table 3.5). In general, Lime Sulphur was the best and its efficacy is comparable to Systhane. All treatments except Polyversum had some efficacy against powdery mildew 2002 Test Apple Scab: Table 6 shows the average number of scab lesions and incidence of scabbed leaves for each treatment when assessed in mid-May. ANOVA showed that there were no significant treatment effects. Gala had significantly more scab than Cox; there were no scabbed leaves on the bud-burst Wetcol treatment on Gala. However, the overall scab incidence was very low in May.

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Table 6. Average number of scab lesions per leaf and incidence of scabbed leaves for each treatment whether alternative products were applied in sand-bed at a 10-day interval in 2002. Scab was assessed in mid-May. There were no significant differences between all the treatments.

Cox Gala

Treatment No of lesions/leaf

% leaves infected

No of lesions/leaf

% leaves infected

Pre-bud burst Wetcol 0 0 0 0 Distillery Waste 0 0 0.12 7% FarmFos42 0 0 0.1 5% Headland Sulphur 0 0 0.1 3% Lime Sulphur 0.03 2% 0.03 2% Liquid Silica 0 0 0.1 5% Milsana 0 0 0.33 10% Polyversum 0 0 0.5 5% Serenade 0 0 0.05 2% Systhane 0 0 0.02 2% Untreated 0.03 3% 0.23 2% Routine Wetcol 3 0 0 0.18 7%

In the August assessment, virtually all fruits on the trees were scabbed. Thus it was not possible to assess incidence of fruit infection. Furthermore, most infected fruit had multiple lesions, many of which had merged. Therefore, it was not possible to count the number of lesions. For marketable yield, incidence and severity measures for scab are equivalent, i.e. a fruit with a single scab lesion is equally unmarketable as one with many lesions. Despite this fact, scab severity on fruits of trees treated with pre-bud break Wetcol did appear to be less than on other treatments. Because of difficulties in assessing diseases, we have taken digital pictures of each treatment to illustrate the severity of fruit scab (Figure 1). It is also interesting to note the poor skin finish associated with some treatments - sulphur, lime sulphur and Wetcol (routine).

Apple powdery mildew: Table 7 shows the average number of mildew lesions per leaf for each treatment. ANOVA showed that there were significant treatment effects. Overall, all the treatments significantly reduced the number of mildew lesions, compared with the untreated. Of the products tested, the lime sulphur was the best and its performance was not statistically different from Systhane, with average 0.2 lesions per leaf compared to 1.2 lesions per leaf for the untreated control. Broadly speaking, the products can be divided into four groups with decreasing efficiencies: (1) Systhane, Sulphur, (2) Distillery waste, Liquid Silica, Serenade, Milsana, Wetcol 3, (3) FarmFos42, Polyversum, and (4) Bud burst Wetcol.

In the August, assessment of mildew was made virtually impossible by the high incidence of scab on leaves and by the general poor status of leaves. Therefore, no assessment was made. Orchard test 2003 Powdery mildew: In the June assessment, the percentage of mildewed leaves was very high, reaching 73% for the untreated. Only routine low copper had significantly less mildew (61%), whereas Liquid Silica and Milsana appeared to increase mildew (83%) on Red Pippin. Results

279

on Saturn were generally similar apart from the fact that mildew level was generally lower than on Red Pippin (Table 3.8). Table 7. Average number of mildew lesions per leaf and incidence (percentage) of leaves

infected on potted 5-y-old tress of cv. Queen Cox and cv. Gala in a sand-bed in 2002. Products were applied every ten days.

Cox Gala Treatment Lesions Incidence Lesions Incidence Pre-bud burst Wetcol 0.6 30% 1 40% Distillery 0.19 16% 0.4 22% FarmFos42 0.28 22% 0.9 47% Headland Sulphur 0.15 13% 0.23 18% Lime Sulphur 0.11 9% 0.23 6.7% Liquid Silica 0.13 11% 0.82 28% Milsana 0.06 6% 0.52 27% Polyversum 0.35 21% 0.88 35% Serenade 0.06 6% 0.77 37% Systhane 0.06 4% 0 0% Untreated 0.32 24% 1.9 60% Wetcol 3 0.18 14% 0.62 32%

Table 8. Summary of mildew (% of leaves infected in June and July) epidemics on Red

Pippin and Saturn, and fruit scab epidemics on Red Pippin trees subjected to various treatments in 2003.

Mildew Fruit scab June July Red Pippin Saturn Red Pippin Saturn

Incidence (%)

Lesions per infected fruit

Early Copper 76 56 97 59 16 1.6 Low Copper 61 56 92 47 5 3.3 Milsana 80 63 99 76 19 2.8 Serenade 73 60 97 73 22 3.2 Silica 83 63 100 63 13 3.6 Sulphur 72 59 91 42 2 2.5 Untreated 73 63 93 55 18 3.6

For the July assessment, almost all the leaves on Red Pippin were infected by mildew

(Table 8). In contrast, mildew remained at the similar level to that in June on Saturn. In addition, both sulphur and low rate copper had significantly less mildew than untreated, whereas Milsana, Silica and Serenade resulted in significantly more mildew than untreated. Because of the hot weather in the summer, all the extension shoots stopped growth by the time of third assessment in August, hence the mildew was virtually unchanged from July.

Scab: There were no scab lesions observed on Saturn. There were very few leaves infected by scab particularly for the June assessment. Hence data on leaf scab were not presented. About 18% of fruit was infected with scab for the untreated. Of all the treatments,

280

only the treatment (low rate copper and sulphur) had significantly reduced the scab incidence (5% and 2%, respectively) (Table 3.8). For early copper treatment, even though the incidence was similar to the untreated the severity of the scab was significantly less than the untreated: 1.6 lesions per infected fruit compared to 3.6 lesions per infected fruit.

Figure 1. Photos of harvested fruit from potted 5-year-old Cox trees subjected to various treatments.

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2004 Powdery mildew. The percentage of mildewed leaves was very high; nearly all leaves were infected (Table 9). Of the treatments, five had significantly (P < 0.05) reduced the incidence of mildew; sulphur clearly was the most effective one (Table 9). However, even for these five treatments, the incidence of mildew was still too high. For example, about 60% of leaves were infected on Red Pippin treated with sulphur, compared to 99% of the untreated. Both types of compost teas did not reduce mildew significantly. Results on Saturn were generally similar apart from the fact that mildew level was generally lower than on Red Pippin (Table 9).

Scab. There were no scab lesions observed on Saturn. There were very few leaves infected by scab particularly for the June assessment (Table 9). Sulphur and low rate copper had resulted in lowest incidence of fruit scab. All other treatments had very high incidence (> 35%, Table 9), similar to the untreated. Table 9. Percentage of leaves infected by powdery mildew and scab on VF211 field in 2004

(based on 100 leaves – 5 leaves per shoot – 5 shoots per tree) and fruit scab (based on all fruits from four trees per treatment, Red Pippin only). There were no significant differences between treatments with the same letter after their means (LSD tests); the LSD test for mildew is pooled over both Red pippin and Saturn.

Red pippin Saturn Treatment Fruit scab Mildew Scab Mildew Scab Compost Tea (bacteria) 47 de 90 ab 1 70 0 Compost tea (fungal) 54 e 94 a 3 70 0 Liquid silica + Maxicrop 50 d 82 abc 2 71 0

Liquid silica + Milsana 40 abc 88 bc 1 63 0 Low rate copper 10 ab 74 bcd 0 60 0 Maxicrop 45 e 97 ab 7 63 0 Maxicrop + Milsana 37 bcd 73 c 2 51 0 Sulphur 8 a 60 cd 3 19 0 Untreated 60 e 99 e 4 74 0

Discussion and conclusions Overall, it was very disappointing that almost all the alternative products tested during the five year period were not very effective in controlling apple mildew and scab, especially against scab. Of all the products tested, only sulphur and frequent application of low rate copper were effective against powdery mildew and scab, respectively. Interestingly, it also appears that sulphur was also effective against apple scab.

Several published research studies indicated that compost tea products controlled diseases in various crops (Mcquilken et al., 1994; Litterick et al., 2004; Scheuerell & Mahaffee, 2004). However, it is also known that the efficacy of such control depends critically on many factors such as aeration, pH value, compost type, microbial population etc. (Scheuerell & Mahaffee, 2002). Most importantly, our understanding of the exact disease suppressive mechanisms is very limited. This limited knowledge has severally hampered our ability to exploit compost tea for disease management more consistently. Further research is needed to understand the control mechanisms before effective as well as consistent disease control can be achieved with compost tea. It was claimed that application of compost tea with

282

an adjuvant would improve the coverage of microbial population of the host surface. However, in this study inclusion of adjuvant did not improve disease control. From the current data, we conclude that compost tea as we used in this study might not contain appropriate microbial populations at sufficient high concentrations. However, unless we know the identity of these populations, disease control based on compost tea is more of an art than science.

Of the products tested, Milsana is known to induce resistance to powdery mildew on cucumber (Wurms et al., 1999; Fofana et al., 2002; Petsikos-Panayotarou et al., 2002). Here we have also shown that it has some effects in controlling apple powdery mildew; this partial effectiveness of Milsana against powdery mildew was also observed on rose (Pasini et al., 1997). However, on its own it is unlikely to manage powdery mildew effectively in the UK.

Many research studies have indicated various bicarbonate salts can be used to suppress disease development, such as pepper powdery mildew, postharvest pepper rot (Fallik et al., 1997a; 1997b), anthracnose rot in papaya (Gamagae et al., 2003), citrus foliar diseases (Mcgovern et al., 2003), botrytis (Palmer et al., 1997) and cucumber powdery mildew (Reuveni et al., 1996). We did not include any bicarbonate salts in the present study because these were being tested in other trials against apple mildew. Results from those trials were also very disappointing. Interestingly, in a field plot at East Malling application of potassium bicarbonate (without a surfactant) also failed to control powdery mildew satisfactorily on strawberry. In addition, a few studies also indicated that phosphate salts can also control some diseases (Reuveni et al., 1996); but it did not have any effects on apple powdery mildew as shown in this study. Ca(OH)2 did control mildew satisfactorily in the glasshouse trials; however, the treated leaves became white, which does not convey the ‘green’ essence of organic production well; for this reason, we did not test this product any further.

In the UK, environmental conditions are very conducive to scab and mildew develop-ment and furthermore in our testing experiments disease pressure (inoculum) was very high. In general it is expected that these ‘natural’ products are not as effective as synthetic fungicides in controlling diseases. Therefore, the efficacy of these products might be even worse in areas where disease risks are high, as in the UK. We conclude that for a successful organic apple production in the UK conditions it is essential to plant cultivars that are resistant to scab and preferably to powdery mildew as well. Acknowledgements This research is funded by the British Department of Environment, Food and Rural Affairs (Defra) and several other commercial bodies via the LINK scheme. References Berrie, A.M. & Xu, X.-M. 2003: Managing apple scab and powdery mildew using Adem™. –

International Journal of Pest Management 49: 243-250. Fallik, E., Grinberg, S. & Ziv, O. 1997: Potassium bicarbonate reduces postharvest decay

development on bell pepper fruits. – Journal of Horticultural Science 72: 35-41. Fallik, E., Ziv, O., Grinberg, S., Alkalai, S. & Klein, J.D. 1997: Bicarbonate solutions control

powdery mildew (Leveillula taurica) on sweet red pepper and reduce the development of postharvest fruit rotting. – Phytoparasitica 25: 41-43.

Fofana, B., McNally, D.J., Labbe, C., Boulanger, R., Benhamou, N., Seguin, A. & Belanger, R.R. 2002: Milsana-induced resistance in powdery mildew-infected cucumber plants correlates with the induction of chalcone synthase and chalcone isomerase. – Physio-logical and Molecular Plant Pathology 61: 121-132.

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Gamagae, S.U., Sivakumar, D., Wijeratnam, R.S.W. & Wijesundera, R.L.C. 2003: Use of sodium bicarbonate and Candida oleophila to control anthracnose in papaya during storage. – Crop Protection 22: 775-779.

Litterick, A.M., Harrier, L., Wallace, P., Watson, C.A. & Wood, M. 2004: The role of uncomposted materials, composts, manures, and compost extracts in reducing pest and disease incidence and severity in sustainable temperate agricultural and horticultural crop production - A review. – Critical Reviews in Plant Sciences 23: 453-479.

Mann, R.L., Kettlewell, P.S. & Jenkinson, R. 2004: Effect of foliar-applied potassium chloride on septoria leaf blotch of winter wheat. – Plant Pathology 53: 653-659.

McGovern, R.J., Winston, A.E., Rouse, R.E. & Welch, A.W. 2003: Reduction of defoliation in citrus caused by Mycosphaerella citri with a novel biocompatible fungicide. – Plant Disease 87: 134-138.

McQuilken, M.P., Whipps, J.M. & Lynch, J.M. 1994: Effects of water extracts of a composted manure-straw mixture on the plant pathogen Botrytis cinerea. – World J. Microbiol. Biotechnol. 10: 20-26.

Palmer, C.L., Horst, R.K. & Langhans, R.W. 1997: Use of bicarbonates to inhibit in vitro colony growth of Botrytis cinerea. – Plant Disease 81: 1432-1438.

Pasini, C., Daquila, F., Curir, P. & Gullino, M.L. 1997: Effectiveness of antifungal com-pounds against rose powdery mildew (Sphaerotheca pannosa var. rosae) in glasshouses. – Crop Protection 16: 251-256.

Petsikos-Panayotarou, N., Schmitt, A., Markellou, E., Kalamarakis, A.E., Tzempelikou, K., Siranidou, E. & Konstantinidou-Doltsinis, S. 2002: Management of cucumber powdery mildew by new formulations of Reynoutria sachalinensis (F. Schmidt) Nakai extract. – Journal of Plant Diseases and Protection 109: 478-490.

Reuveni, M., Agapov, V. & Reuveni, R. 1996: Controlling powdery mildew caused by Sphaerotheca fuliginea in cucumber by foliar sprays of phosphate and potassium salts. – Crop Protection 15: 49-53.

Scheuerell, S. & Mahaffee, W. 2002: Compost tea: Principles and prospects for plant disease control. – Compost Science & Utilization 10: 313-338.

Scheuerell, S.J. & Mahaffee, W.F. 2004: Compost tea as a container medium drench for suppressing seedling damping-off caused by Pythium ultimum. – Phytopathology 94: 1156-1163.

Wurms, K., Labbe, C., Benhamou, N. & Belanger, R.R. 1999: Effects of Milsana and benzo-thiadiazole on the ultrastructure of powdery mildew haustoria on cucumber. – Phytopathology 89: 728-736.

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