International Workshop on Arthropod Pest Problems in Pome Fruit Production

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IOBC / WPRS

Working Group “Integrated Plant Protection in Orchards”

Sub Group “Pome Fruit Arthropods”

International Workshop on Arthropod Pest Problems in Pome Fruit Production

Proceedings of the meeting

Lleida (Spain) 4 – 6 September, 2006

Edited by:

Jesús Avilla, Jerry Cross and Claudio Ioriatti

IOBC wprs Bulletin Bulletin OILB srop Vol. 30 (4) 2007

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)

The Publication Commission of the IOBC/WPRS:

Horst Bathon Luc Tirry Federal Biological Research Center University of Gent for Agriculture and Forestry (BBA) Laboratory of Agrozoology Institute for Biological Control Department of Crop Protection Heinrichstr. 243 Coupure Links 653 D-64287 Darmstadt (Germany) B-9000 Gent (Belgium) Tel +49 6151 407-225, Fax +49 6151 407-290 Tel +32-9-2646152, Fax +32-9-2646239 e-mail: [email protected] 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 92-9067-199-6 Web: http://www.iobc-wprs.org

Local organisers

of the

International Workshop on Arthropod Pest Problems in Pome Fruit Production

UNIVERSITAT DE LLEIDA (UdL) University of Lleida

INSTITUT DE RECERCA I TECNOLOGIA AGROALIMENTÀRIES (IRTA) Institut for Food and Agricultural Research and Technology

FUNDACIÓ 700 ANIVERSARI UdL

Sponsor

MINISTERIO DE EDUCACIÓN Y CIENCIA (ESPAÑA) Ministryf Education and Science (Spain)

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007

Preface

The present IOBC/wprs Bulletin issue contains the contributions to the meeting “Arthropod Pest Problems in Pome Fruit Production”, organized by the Working Group “Integrated Protection of Fruit Crops”, Subgroup “Pome Fruit Arthropods”. More than 65 scientists from 12 European countries and the USA attended and delivered 51 oral and poster presentations. The volume is divided into four parts, which correspond to the organization of the meeting itself: Biological Control and Arthropod Biology, Behaviour and Behavioural Control, Insecticide Resistance and Other Topics. Each main part was ended by a general discussion on the future, constraints and research needs. Taking into account the number of presentations, codling moth (Cydia pomonella) remains the most important pest in pome fruit production. Many presentations focused on the use of mating disruption for its control, stressing the need to better understand how it works, in order to be able to improve its efficacy, and on the extension of its resistance to pesticides. How to measure resistance was also discussed, as many different methods have been developed and are currently used. A standardization of the methodology was deemed necessary in order to better compare results from different countries. Conservation biological control was another important topic. Pome fruit orchards are quite stable environments in time and space, and are surrounded by natural vegetation that can be used as ecological infrastructures. The importance of augmenting and conserving important natural enemies affected by pesticide applications, such as earwigs, was pointed out. The possible use of semiochemicals to control pests other than lepidopterous ones, such as aphids and midges, was also presented and discussed. The manageable size of the audience facilitated personal interactions, lively discussions at the sessions and, mainly, during coffee breaks in front of the posters. A final techno- recreational journey allowed the participants to visit a couple of orchards, just at harvest time, and an olive mill and museum. The scientific committee wishes to thank all contributors to this lively and worthwhile conference.

Lleida, East Malling, San Michele all’Adige, 2006, December

Jesús Avilla, Jerry Cross, Claudio Ioriatti

ii Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007

List of participants

AGNELLO, ARTHUR 38010-S. Michele a/A (Italy) Cornell University Phone: +390461615143 NYS Agricultural Experiment Station - Fax: +390461650872 Entomology e-mail: [email protected] 630 W. North St. NY 14456-Geneva (USA) ARTIGUES MARTIN, MIQUEL Phone: 3157872341 IRTA. Centre UdL-IRTA Fax: 3157872326 Avda Rovira Roure 191 e-mail: [email protected] 25198-Lleida (Spain) Phone: +34973702629 ALAPHILIPPE, AUDE Fax: +34973238301 Istituto Agrario S.Michele a/A e-mail: [email protected] Centre SafeCrop - Via E. Mach 1 AVILLA, JESUS 38010-S. Michele a/A (Italy) University of Lleida Phone: +390461615148 Centre UdL-IRTA Fax: +340461615148 Avda Rovira Roure 191 e-mail: [email protected] 25198-Lleida (Spain) Phone: +34973702581 ALBÀ TERRATS, JOSEP Mª Fax: +34973238301 SUMI AGRO e-mail: [email protected] 25005-Lleida (Spain) Phone: +34973237907 BEERS, ELIZABETH Fax: +34973237907 Washington State University Tree Fruit Research and Extension Center - ALINS, GEORGINA 1100 North Western Avenue IRTA WA 98801-Wenatchee (USA) Avda Rovira Roure 191 Phone: +15096638181 ext234 25198-Lleida (Spain) Fax: +15096628714 Phone: +34973702579 e-mail: [email protected] Fax: +34973238301 e-mail: [email protected] BOSCH, DOLORS IRTA. Centre UdL-IRTA AMARAWARDANA, LAKMALI Avda Rovira Roure 191 Natural Resources Institute 25198-Lleida (Spain) University of Greenwich - Chemical Ecology Phone: +34973702645 Group Fax: +34973238301 Central Avenue, Chatham Maritime e-mail: [email protected] ME4 4TB-Kent (UK) Phone: +441634883207 BRIAND, FRANÇOISE Fax: +441732883379 Agroscope Changings - Wädenswil e-mail: [email protected] Entomology Case postale 254 ANFORA, GIANFRANCO 1260-Nyon 1 (Switzerland) Istituto Agrario S.Michele a/A Phone: +41223634381 Safecrop Center - Plant Protection Department Fax: +41223634394 Via E. Mach 1 e-mail: [email protected]

BUTTURINI, ALDA DUNLEY, JOHN Servizio Fitosanitario Regione Emilia Romagna Washington State University Via di Saliceto 81 Tree Fruit Research and Extension Center - 40129-Bologna (Italy) 1100 North Western Avenue Phone: +39514159283 WA 98801-Wenatchee (USA) Fax: +39514159277 Phone: +15096638181 ext 236 e-mail: [email protected] Fax: +15096628714 e-mail: [email protected] CASADO, DANIEL University of Lleida EPSTEIN, DAVID Centre UdL-IRTA Michigan State University Avda Rovira Roure 191 Center for Integrated Plant Systems - 25198-Lleida (Spain) Entomology Phone: +34973702646 B18 NFSTC Fax: +34973238301 MI 48824-East Lansing (USA) e-mail: [email protected] Phone: +15174324766 Fax: +15173534995 CROSS, JERRY e-mail: [email protected] East Malling Research Entomology ESCUDERO COLOMAR, ADRIANA New Road, East Malling Fundació Mas Badia 25198ME19 6BJ-Kent (UK) Irta- Estació Experimental Agrícola - Protecció Phone: +441732523748 de conreus Fax: +441732849067 La Tallada d’Empordà e-mail: [email protected] 17134-La Tallada d’Empordà (Spain) Phone: +34972780275 DE CRISTOFARO, ANTONIO Fax: +34972780517 Università degli studi del molise - campobasso e-mail: [email protected] Departamento di science animali, vegetali e dell'ambiente FEMENIA FERRER, BEATRIU Via de Sanctis Universidad Politécnica de Madrid I-86100-Campobasso (Italy) Centro de Ecología Química Agrícola (CEQA) Phone: +39874404686 Camino de Vera s/n Fax: +39874404855 46022-Valencia (Spain) e-mail: [email protected] Phone: +34963879058 Fax: +34963879059 DERRIDJ, SYLVIE e-mail: [email protected]

INRA FITZEGERALD, JEAN Versailles - Insect Physiology East Malling Research Route de St. Cyr New Road, East Malling 78046-Versailles Cedex (France) ME19 6BJ-Kent (UK) Phone: +33130833164 Phone: +441732523758 Fax: +33130833119 Fax: +441732849067 e-mail: [email protected] e-mail: [email protected]

DOMINGO PIÑOL, Mª TERESA FOUNTAIN, MICHELLE ADV-AITONA East Malling Research 27 de gener, 67 Entomology Aitona (Spain) New Road, East Malling Phone: +34606594627 ME19 6BJ-Kent (UK) Fax: Phone: +441732523749 e-mail: [email protected] Fax: +441732849067 e-mail: [email protected] v

GEMENO, CESAR JAUSET, ANA MARIA University of Lleida University of Lleida Centre UdL-IRTA Centre UdL-IRTA Avda Rovira Roure 191 Avda Rovira Roure 191 2519825198-Lleida (Spain) 25198-Lleida (Spain) Phone: +34973702531 Phone: +34973702829 Fax: +34973238301 Fax: +34973238301 e-mail: [email protected] e-mail: [email protected]

GOBIN, BRUNO JAY, CHANTELLE PCFruit East Malling Research Gorsem - Zoology New Road, East Malling De Brede Akker 13 ME19 6BJ-Kent (UK) B-3800-Gorsem (Belgium) Phone: +441732843833 Phone: +3211586968 Fax: +441732849067 Fax: +3211674318 e-mail: [email protected] e-mail: [email protected]

GUT, LARRY KRAWCZYK, GREG Pennsylvania State University Michigan State University Fruit Research and Extension Center - Center for Integrated Plant Systems - Entomology Entomology 290 University Dr, PO Bx 330 205 CIPS PA 17307-Biglerville (USA) MI 48824-East Lansing (USA) Phone: +17176776116 Phone: +15173538648 Fax: +17176774112 Fax: +15173535598 e-mail: [email protected] e-mail: [email protected]

HARVEY REISSIG, W. LETHMAYER, CHRISTA Cornell University AGES, Institute for Plant Health NYS Agricultural Experiment Station - Spargelfeldstraβe 191 Entomology A-1226-Vienna (Austria) 630 W. North St. Phone: +435055533311 NY 14456-Geneva (USA) Fax: +435055533303 Phone: 3157872336 e-mail: [email protected] Fax: 3157872326 e-mail: [email protected] LIGHT, DOUGLAS USDA HELSEN, HERMAN Western Regional Research CenterAgricultural Applied Plant Research - Fruit Research Service Wageningen Universtity and Research Centre - 800 Buchanan Street P.O. box 200 25198CA 94710-Albany (USA) 6670 AE-Zetten (Netherlands) Phone: 5105595831 Phone: +31488473754 Fax: 5105595777 Fax: +31488473717 e-mail: [email protected] e-mail: [email protected] LINGREN, BILL IORIATTI, CLAUDIO Trecé Inc. Istituto Agrario S.Michele a/A P.O. Box 129 Research Center - Plant Protection Department 74330-Adair (Oklahoma) (USA) Via E. Mach 1 Phone: +19187853061 38010-S. Michele a/A (Italy) Fax: +19187853063 Phone: +390461615222 e-mail: [email protected] Fax: +390461650872 e-mail: [email protected] vi

MARTÍ, SANTIAGO PASQUALINI, EDISON Suterra University of Bologna Suterra España - Product Development Agricultural Faculty - DiSTA Cervantes 22A Viale G. Fanin 42 25243-El Palau d’Anglesola (Lleida) (Spain) 40127-Bologna (Italy) Phone: +34973712810 Phone: +390512096297 Fax: +34973712910 Fax: +390512096281 e-mail: [email protected] e-mail: [email protected]

MAYER, CHRISTOPH JULIAN PEDERSEN, KAREN Federal Biological Research Centre for Fruit and Vegetable Advisory Centre Agriculture and Forestry Rugaardsvej 197 Institute for Plant Protection in Fruit Crops - 5210-Odense ND (DK) Entomology / Chemical Ecology Phone: +4563167585 Schwabenheimer Str. 101 e-mail: [email protected] 69221-Dossenheim (Germany) Phone: +4962218680510 QUENIN, HERVÉ e-mail: [email protected] Cotesia "Le Bourg" MELANDRI, MASSIMILIANO 64360 Lucq de Bearn Agronomica R&S Terremerse 25198- (France) Agronomica Research and Development - Phone: +33559343285 Via Sant’ Alberto 325 Fax: +33559343283 48100-Ravenna (Italy) e-mail: [email protected] Phone: +390544483621 Fax: +390544483611 REYES, MARITZA e-mail: [email protected] INRA Centre Avignon - MIARNAU, XAVIER Site Agropac, Domaine St Paul University of Lleida 84914-Avignon (France) Centre UdL-IRTA Phone: +33432722615 Avda Rovira Roure 191 Fax: +33432722602 25198-Lleida (Spain) e-mail: [email protected] Phone: +34973702645 Fax: +34973238301 RIEDL, HELMUT e-mail: [email protected] Oregon State University Mid-Columbia Agricultural Research and MIÑARRO, MARCOS Extension Center - Entomology SERIDA 3005 Experiment Station Drive Apdo 13 OR 97031-Hood River (USA) 33300-Villaviciosa (Spain) Phone: +15413862030 ext 14 Phone: +34985890066 Fax: +15413861905 Fax: +34985891854 e-mail: [email protected] e-mail: [email protected] RODRÍGUEZ, MARCELA NAGY, CSABA University of Lleida East Malling Research - Entomology Centre UdL-IRTA New Road, East Malling Avda Rovira Roure 191 ME19 6BJ-Kent (UK) 25198-Lleida (Spain) Phone: +441732523748 Phone: +34973702546 Fax: +441732849067 Fax: +34973238301 e-mail: [email protected] e-mail: [email protected]

vii

SARASÚA, MARIA JOSE TASIN, MARCO University of Lleida Istituto Agrario S.Michele a/A Centre UdL-IRTA Research CenterSafe Crop Center Avda Rovira Roure 191 Via E. Mach 1 25198-Lleida (Spain) 2519838010-S. Michele a/A (Italy) Phone: +34973702582 Phone: +390461615143 Fax: +34973238301 Fax: +390461650872 e-mail: [email protected] e-mail: [email protected]

SAUPHANOR, BENTOÎT THOMSON, DONALD INRA, Centre Avignon DJS Consulting Site Agropac, Domaine St Paul 3015 S.W. 109 St 84914-Avignon (France) -Seattle (Wa) (USA) Phone: +33432722607 Phone: +12064445770 Fax: +33432722602 e-mail: [email protected] e-mail: [email protected] TORNÉUS, CHRISTER SCHMIDT, SILVIA Swedish Board of Agriculture Istituto Agrario S.Michele a/A Plant protection Centre - Research Center, Safecrop Centre - Plant Box 12 Protection Department SE-230 53-Alnarp (Sweden) Via E. Mach 1 Phone: +4640460418 38010-S. Michele a/A (Italy) Fax: +4640460782 Phone: +390461615143 e-mail: [email protected] Fax: +390461650872 e-mail: [email protected] VALERO, JOSEPH BIOTEPP Company SHEARER, PETER Mont-St-Hilaire - Rutgers University 909 Laudance Rutgers Agricultural Research & Extension -Quebec (Canada) Center - Entomology Phone: 4186580232 121 Northville Road Fax: 4186585846 New Jersey 08302-5919-Bridgeton (USA) e-mail: [email protected] Phone: +18564553100 ext 4110 Fax: +18564553133 VARELA, NÉLIA e-mail: [email protected] University of Lleida Centre UdL-IRTA SIGSGAARD, LENE Avda Rovira Roure 191 Royal Vet & Agricultural University 25198-Lleida (Spain) Ecology Phone: +34973702687 Thorvaldsensvej 40 Fax: +34973238301 DK 1871-Frederiksberg C (Denmark) e-mail: [email protected] Phone: +4535282674 Fax: +4535282670 VILAJELIU, MARIÀ e-mail: [email protected] Fundació Mas Badia

IRTA- Estació Experimental Agrícola - STELINSKY, LUKASZ Protecció de conreus Michigan State University La Tallada d’Empordà Center for Integrated Plant Systems Entomology 17134-La Tallada d’Empordà (Spain) 205 CIPS Phone: +34972780275 MI 48824-East Lansing (USA) Fax: +34972780517 Phone: +15174329514 e-mail: [email protected] Fax: +15173535598 e-mail: [email protected] viii

VITAGLIANO, SILVIA Universita' degli studi del molise-campobaso Dipartimento di scienze animali, vegetali e dell'ambiente Via de Sanctis I-86100-Campobaso (Italy) Phone: +39874404686 Fax: +39874404855 e-mail: [email protected]

WALGENBACH, JAMES North Carolina State University Mountain Horticultural Crops Research & Extension Center - Entomology 455 Research Drive NC 28732-Fletcher (USA) Phone: 8288643562 Fax: 8286848715 e-mail: [email protected]

WALSTON, ALLISON Oregon State University Mid-Columbia Agricultural Research and Extension Center - Entomology 3005 Experiment Station Drive OR 97031-Hood River (USA) Phone: +15413862030 ext 25 Fax: +15413861905 e-mail: [email protected]

WELTER, STEPHEN University of California Environmental Science, Policy and Management 201 Wellman Hall, UC Berkeley CA 94720-Berkeley (USA) Phone: +15106422355 Fax: +15106420477 e-mail: [email protected]

ZARAGOZA, ABEL Suterra Suterra EspañaSales & Marketing Tenor Masini 93 3-6 2519808028-Barcelona (Spain) Phone: +37934906128 Fax: +34934906128 e-mail: [email protected]

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007

Contents

Preface...... i List of participants...... iii

Biological Control and Arthropod Biology

Biological control in pear orchards under seasonal pest management programs with and without organophosphate insecticides Helmut Riedl, Allison T. Walston, Dong-Soon Kim, Deborah J. Brooks ...... 1-8 Beneficial effects of hedgerow plants for insect predators in adjacent orchards – the value of pollen and nectar to Anthocoris nemorum (L.) Lene Sigsgaard, Johannes Kollmann...... 9-13 Adapting orchard management to re-establish earwigs in Belgian pome fruit orchards (abstract only) B. Gobin, A. Marien, S. Davies, H. Leirs ...... 15 Abundance and seasonal distribution of natural enemies in treated vs untreated pear orchards in Lleida (NE Spain) Ana María Jauset, Miquel Artigues, María José Sarasúa ...... 17-21 Pressure of predation in treated vs untreated pear orchards in Gimenells (Lleida, Spain) during spring and summer 2004 (abstract only) Miquel A. Díaz, Miquel Artigues, María José Sarasúa ...... 23 Impact of alternative insect management programs on the predatory arthropod complex of green apple aphids James F. Walgenbach, Raul T. Villanueva ...... 25-30 Presence of the common earwig Forficula auricularia L. in apple orchards and its impact on the woolly apple aphid Eriosoma lanigerum (Haussmann) Herman Helsen, Marc Trapman, Matty Polfliet, Jan Simonse ...... 31-35 Biology and management of woolly apple aphid, Eriosoma lanigerum (Hausmann), in Washington state E. H. Beers, S. D. Cockfield, G. Fazio ...... 37-42 Effects of exclusion or supplementary honey feeding of the common black ant, Lasius niger (L.), on aphid populations and natural enemies on apple Csaba Nagy, Viktor Markó, Jerry Cross...... 43-50 The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in UK pome and stone fruit orchards Michelle T. Fountain, Jerry V. Cross...... 51-60 Ecology and management of the obliquebanded leafroller, Choristoneura rosaceana, in New York apple orchards H. Reissig, M. Sarvary, J. Nyrop...... 61-66 Temperature effect on egg-laying timing in Cydia pomonella (L.) (abstract only) Daniel Casado, Peter Witzgall, César Gemeno, Jesús Avilla, Magí Riba...... 67 Biological interactions between the apple leaf curling midge, Dasineura mali (Kieffer), and its inquiline, Macrolabis mali Anfora Gianfranco Anfora, Nunzio Isidoro, Claudio Ioriatti ...... 69-73

Effect of different pest control strategies on phytophagous and predatory mites in apple orchards of Girona (NE of Spain) (abstract only) Adriana Escudero, Mariano Vilajeliu, Josep Lluis Batllori, Francisco Ferragut ...... 75

Behaviour and Behavioural Control

Effect of flat anti-hail nets on Cydia pomonella (L.) reproductive behaviour Marco Tasin, Camilla Ryne, Vittorio Veronelli, Anna-Carin Bäckman, Claudio Ioriatti...... 79-83 Competitive attraction as a primary mechanism of moth mating disruption in tree fruit crops Larry J. Gut, James R. Miller, Lukasz L. Stelinski, David L. Epstein ...... 85-93 New insights into codling moth, Cydia pomonella (L.), distribution and implications for mating disruption David L. Epstein, Larry J. Gut, James R. Miller, Lukasz L. Stelinski...... 95-100 Mating disruption of codling moth, Cydia pomonella (L.), using Puffer® CM, on apple orchards Santiago Marti, Abel Zaragoza, Tom Larsen...... 101-105 Field assays of new biodegradable controlled-release pheromone dispensers for mating disruption of Cydia pomonella (L.) Beatriu Femenia-Ferrer, Dolors Bosch, Pilar Moya, Jesús Avilla, Jaime Primo ...... 107-114 Towards high performance mating disruption of codling moth, Cydia pomonella (L.) Lukasz L. Stelinski, Larry J. Gut, Peter McGhee, James R. Miller ...... 115-122 Pheromone mating disruption of Cydia pomonella (L.) in California pears: Balancing dispenser emission rates and program performance (abstract only) S. Welter, F. Cave...... 123 Olfactory sensitivy of different populations of Cydia pomonella (L.) to sex pheromone E8E10-12:OH and kairomone ethyl (2E, 4Z)-2,4-decadienoate (abstract only) A. De Cristofaro, G. Anfora, G.S. Germinara, C. Ioriatti, S. Vitagliano, M. Guida, G. Rotundo ...... 125 Use of ethyl (E, Z)-2,4-decadienoate for the control of Cydia pomonella (L.) on apple orchards Silvia Schmidt, Cristina Tomasi, Massimiliano Melandri, Gianfranco Pradolesi, Edison Pasqualini, Claudio Ioriatti ...... 127-132 Experimental use of the micro-encapsulated pear ester kairomone for control of codling moth, Cydia pomonella (L.), in walnuts Douglas Light ...... 133-140 Behavioural responses of Cydia pomonella (L.) neonate larvae to a microencapsulated formulation of ethyl (2E, 4Z)-2,4-decadienoate (abstract only) S. Vitagliano, G.S. Germinara, B. Lingren, C. Ioriatti, E. Pasqualini, G. Rotundo, A. De Cristofaro...... 141 Effect of Madex® (granulovirus) on Cydia pomonella (L.) egg laying on two apple varieties – Relationships with plant surface metabolites Nadia Lombarkia, Claudio Ioriatti, Edouard Bourguet, Sylvie Derridj...... 143-148

Effect of a potential biocontrol agent of apple diseases on the egg laying of Cydia pomonella (L.) A. Alaphilippe, Y. Elad, S. Derridj, C. Gessler ...... 149-152 Field assessment of behaviorially-based management tactics for Conotrachelus nenuphar (Herbst), and Rhagoletis pomonella (Walsh) in the northeastern US Arthur Agnello, Jaime Piñero, Ronald Prokopy ...... 153-156 Four years of mediterranean fruit fly (Ceratitis capitata Wied.) control in fruit orchards of Girona (NE of Spain) by using the mass trapping method (abstract only) Josep Lluis Batllori, Adriana Escudero, Mariano Vilajeliu ...... 157 Exploiting the sex pheromone of the apple leaf midge, Dasineura mali, for pest monitoring and control Jerry Cross, David Hall, Peter Shaw...... 159-167 Investigations on the sex pheromone of pear leaf midge, Dasineura pyri (Bouché), and other gall midge pests of fruit crops Lakmali Amarawardana, David Hall, Jerry Cross...... 169-173 The effect of aphid sex pheromone and plant volatiles on the behaviour of Dysaphis plantaginea and its parasitoid Aphidius matricariae (abstract only) J. Fitzgerald, C. Jay, C. James, L. Wadhams, S. Dewhirst, C. Woodcock, G. Poppy, A. Stewart-Jones...... 175 Different host plant odours influence migration behaviour of Cacopsylla melanoneura (Förster), an insect vector of the apple proliferation phytoplasma Christoph J. Mayer, Jürgen Gross...... 177-184

Insecticide Resistance

Reliability of resistance monitoring on diapausing larvae of codling moth, Cydia pomonella (L.) M. Reyes, J. Olivares, C. Ioriatti, E. Pasqualini, P.J. Charmillot, P. Franck, B. Sauphanor...... 187-194 A new bioassay to test insecticide resistance of Cydia pomonella (L.) first instar larvae: results from some field populations of Lleida (Spain) Dolors Bosch, Marcela Rodríguez, Jesús Avilla...... 195-199 Susceptibility of codling moth, Cydia pomonella (L.) to tebufenozide in Trentino apple growing area Claudio Ioriatti, Cristina Tomasi, Pierre Joseph Charmillot, Denis Pasquier, Benoît Sauphanor, Maritza Reyes...... 201-204 Evolution of codling moth, Cydia pomonella (L.), resistance in Swiss orchards, tested by topical application of insecticides (abstract only) P.J. Charmillot, F. Briand, C. Salamin, D. Pasquier ...... 205 Resistance to insecticides and baseline sensitivity in codling moth populations from Pennsylvania, USA orchards Greg Krawczyk, Larry A. Hull ...... 207-213 Baseline toxicity of new insecticides for Grapholita molesta management Peter W. Shearer, James F. Walgenbach, Greg Krawczyk...... 215-219 Evaluation of pear psylla, Cacopsylla pyri (L.), susceptibility to cypermethrin in pear orchards of Lleida, Spain (abstract only) Xavier Miarnau, Miquel Artigues, María José Sarasúa ...... 221

xii

Other topics

Is there a link between fruit pest phenology and climate change in Belgium? A comparative overview with the codling moth, Cydia pomonella (L.), as a case study (abstract only) B. Gobin, E. Bangels ...... 225 Implementation of non-organophosphate pest management programs on pears in northern Oregon, USA Allison Walston, Deborah Brooks, Steve Castagnoli, Helmut Ried...... 227-234 Population dynamics of Cydia pomonella (L.) in an area wide management program (abstract only) Manel Ribes-Dasi, Esther Tort, María José Sarasúa, Ramon Albajes, Darío Fernández, Jesús Avilla...... 235 Effect of centrifugal tree training on pests and pathogens in apple orchards Sylvaine Simon, Carlos Miranda, Laurent Brun, Hubert Defrance, Pierre-Eric Lauri, Benoît Sauphanor...... 237-245 Abundance, spatial distribution and sampling of leafminers in cider apple orchards: a 3-year survey from Asturias (NW Spain) Marcos Miñarro, Gabriela Fernández-Mata, Iván Fernández, Tania Iglesias, Josep-Anton Jacas...... 247-254 Population injury levels of the apple rust mite Aculus schlechtendali (Nal.) on Golden Delicious and Red Delicious apple fruits Gino Angeli, Claudio Rizzi, Alberto Dorigoni, Claudio Ioriatti ...... 255-260 Can apple aphids be vectors of “Candidatus Phytoplasma mali”? Christian Cainelli, Flavia Forno, Luisa Mattedi, Maria Stella Grando...... 261-266 Transmission of “Candidatus Phytoplasma mali” by psyllid vectors in Trentino Luisa Mattedi, Flavia Forno, Christian Cainelli, Stella Grando, Wolfgang Jarausch...... 267-272 A secondary sexual character for sex determination of Cydia pomonella (L.) (Lepidoptera: Tortricidae) adults, trapped with kairomone lures Darío Fernández, Dolors Bosch, Liliana Cichón, Jesús Avilla...... 273-278 Electrophysiological and behavioural responses of Dysaphis plantaginea and the parasitoid Aphidius matricariae to host plant volatiles (abstract only) Chantelle Jay, Jean Fitzgerald, Celia James, Tom Pope, Lester Wadhams, Sarah Dewhirst, Christine Woodcock, Guy Poppy, Alex Stewart-Jones ...... 279

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 1-8

Biological control in pear orchards under seasonal pest management programs with and without organophosphate insecticides

Helmut Riedl1, Allison T. Walston1, Dong-Soon Kim2, Deborah J. Brooks3 1 Mid-Columbia Agricultural Research & Extension Center, Oregon State University, 3005 Experiment Station Drive, Hood River, OR 97031, USA. [email protected]; [email protected] 2 Department of Plant Resources Science, Cheju National University, Jeju, 690-756, South Korea [email protected] 3 Washington State University-Prosser, 24106 N. Bunn Road, Prosser, WA 99350, USA. [email protected]

Abstract: Although there is considerable potential for biological control in pears, there has only been limited success in taking advantage of it in the past because of the intensive use of broad-spectrum insecticides, in particular organophosphates (OPs) for control of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), and other pests. In recent years, resistance development and regulatory actions because of worker safety and environmental concerns have led to a reduction in OP insecticide use in pome fruits. New control tactics such as mating disruption, microbials and new insecticide chemistries such as insect growth regulators (IGRs) and neonicotinyls have now become available to take the place of OPs and other broad-spectrum insecticides. Although several of these new control tactics, such as mating disruption and microbials, have little impact on natural enemies, little is known about the selectivity of the newer insecticide chemistries which have begun to replace OP insecticides. Between 2001 and 2005, we had an opportunity to assess biological control in pear orchards under pest management programs which either included or excluded OP insecticides. This study was conducted as part of a federally funded collaborative project to promote the use of mating disruption and other selective control tactics on tree fruits in the western United States, to enhance opportunities for biological control and to demonstrate the feasibility and effectiveness of pest management programs on pears which excluded the use of OP insecticides. These field studies showed consistently higher numbers of natural enemies in pear orchards where OP insecticides were not used. Laboratory studies complemented the field studies and provided direct evidence of the acute toxicity and sublethal, i.e. reproductive effects of newer insecticide chemistries.

Key words: organophosphates, biological control, natural enemies, pesticide impact, pears

Introduction

In the United States, organophosphate (OP) insecticides have been the key control tools in pome fruits (apples, pears) for the last 50 years. OPs were initially very effective against key and secondary pests, but, due to their broad-spectrum activity, they also created ”biological deserts” where natural enemies could not survive. In spite of 50 years of continuous use, OP insecticides and biological control have essentially remained incompatible. With the exception of predatory mites, most natural enemies have not been able to adapt to intensive OP use in orchards. The heavy reliance on OP insecticides in pome fruits in the western United States began to change during the 1990s with the registration of mating disruption for codling moth control and its successful large-scale demonstration by a federally funded project initiative (Calkins

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1998). Mating disruption gave growers a new selective control tool. However, adoption was not immediate since mating disruption was too expensive compared with conventional broad- spectrum insecticides. Also, many growers considered mating disruption as too target- specific, i.e. no control of secondary pests. There was also a perception that mating disruption might be too risky if used by itself, especially in windy areas; sloping terrain; and where codling moth pressure was too high. Because of these concerns growers began to use mating disruption at half the label rate and supplement it with OP insecticides, primarily azinphosmethyl (Guthion) and, to a lesser extent, phosmet (Imidan). Growers had no other choice since no selective insecticides were registered for codling moth control at the time. What was potentially a very selective program was made non-selective due to the supplemental OP sprays. Supplementing mating disruption with selective insecticides was not possible until the late 1990s when new insecticide chemistries became available for codling moth control (Table 2). However, little was known about the impact of these newer insecticide chemistries on the key natural enemies in pears. The focus of biological control on pears is pear psylla, Cacopsylla pyricola Foerster (Hemiptera: Psyllidae). Predaceous Hemiptera are probably the most important pear psylla predators. In northern Oregon this is Deraeocoris brevis (Uhler) (Hemiptera: Miridae), a predaceous plant bug commonly found in pear orchards and the surrounding arboreal vegetation. The following is a brief summary of research examining biological control under seasonal control programs using mating disruption supplemented by OP or non-OP insecticides. A critical objective was to assess the impact of new insecticide chemistries on natural enemies, specifically D. brevis, in field and laboratory tests, as the basis for developing pheromone-based selective IPM programs for pears.

Material and methods

This research was conducted at Oregon State University’s Mid-Columbia Agricultural Research & Extension Center and in commercial orchards of the Hood River Valley, Oregon. All field tests were carried out in Anjou pear and Red Delicious apple blocks. Trees were sprayed by either hydraulic handgun until run-off or by air-blast sprayer with a spray volume of 935 l/ha. Procedures for monitoring pests and natural enemies and for evaluating fruit damage in the various studies were similar to the ones described by Walston et al. (see paper in this volume). Rearing of D. brevis and bioassay procedures for assessing acute toxicity and sublethal effects of pesticides on D. brevis nymphs and adults were described previously (Kim and Riedl, 2005; Kim et al., 2004a,b; 2006).

Results and discussion

Impact of OPs on pests and natural enemies on pears Previous field studies indicated that use of phosmet or azinphosmethyl applied for codling moth control on pears often resulted in a steep decrease in natural enemies. This drop in natural enemies was often accompanied by an increase in pear psylla (Riedl, 1998b). Release from biological control may partially explain the increase of pear psylla populations in OP- treated pear orchards. It is possible that, in addition to natural enemy disruption, reproductive stimulation (i.e., hormolygosis) also contributes to the observed increases in pear psylla. However, this effect has not yet been proven experimentally. Selectivity of insecticides such as the OPs, which are inherently disruptive to natural enemies, could be achieved through changes in timing or by lowering the rate of application to minimize impact. On pears and 3

apples, OP applications after bloom are primarily directed against codling moth. For greatest effectiveness sprays are applied according to the phenology of codling moth and there is little flexibility to alter spray timing without jeopardizing control. Therefore, we explored in field experiments if selectivity can be achieved by lowering the rate while still maintaining acceptable control of codling moth. We chose the OP insecticide phosmet for these studies instead of azinphosmethyl since phosmet was not a restricted use pesticide and had a better chance of getting through the re-registration process with fewer label restrictions. In these tests, the rate of application for phosmet had to be reduced to ≤ ¼ of the full field rate for natural enemies to survive in substantial numbers (Riedl, 1996). Surprisingly, the ¼ rate of phosmet applied in a seasonal program of four sprays was still quite effective and reduced codling moth damage by 94% over the untreated check while the full rate provided 99% control (Riedl, 1997). This suggested that phosmet applied at ¼ field rate could provide enough supplemental codling moth control when used in combination with mating disruption while at the same time allowing natural enemies to survive and contribute to the biological control of pear psylla and other secondary pests.

Orchard trials: Impact of low rates of phosmet applied as supplemental controls to mating disruption on pests and natural enemies on pears The approach of supplementing a half-rate of mating disruption with low OP rates was tested in a commercial orchard (Riedl, 1998b). The whole orchard was treated with Isomate C+ at 500 dispensers/ha, divided into four large sections where each section was treated once by airblast sprayer at the beginning of codling moth egg hatch at the full (3.15 kg AI/ha), ½, ¼ field rate of phosmet and no phosmet, respectively. Results were similar to the observations from the small field trials. The full and ½ field rate of phosmet were most disruptive to natural enemies, primarily spiders, predaceous hemipterans, and lacewings. Survival of natural enemies in the section treated with the ¼ field rate was similar to the section where no phosmet was applied.

Table 1. Fruit damage due to codling moth, obliquebanded leafroller and pear psylla in Anjou pear blocks treated with the full and reduced rates of phosmet; whole orchard treated with Isomate C+; full rate of phosmet 3.15 kg AI/ha; Heater Orchards, Hood River, Oregon (Riedl 1998b).

Percent fruit damage due to Treatment* Codling moth Obliquebanded Pear psylla leafroller russet

Phosmet full rate 0 1.8 80.0 Phosmet ½ rate 0 0.7 20.0 Phosmet ¼ rate 0 1.0 1.7 No phosmet 0 1.3 3.0

*Isomate C+ at 500 dispensers/ha applied across all treatments.

Codling moth control was acceptable regardless of the rate of phosmet. Obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae), damage was similarly low in all four treatments. However, phosmet appeared to cause pear psylla to build up as 4

evidenced by the inverse relationship between fruit russet from pear psylla honeydew and the rate of phosmet (Table 1). Only the ¼ rate of phosmet did not induce pear psylla build-up and fruit russet was as low as in the section without a phosmet application.

Comparison of seasonal control programs on pears using mating disruption supplemented by OP or non-OP insecticides: impact on natural enemies Results of this study are described by Walston et al. (see paper in this volume). Phosmet applied in the OP section of the orchard as a supplemental spray to mating disruption significantly reduced natural enemies especially spiders, parasitic wasps, hemipteran predators, and D. brevis compared to methoxyfenozide which was applied to the non-OP section (Fig. 1). These results were consistent for the other orchards and years of the study.

0.6

0.4 Non-OP section OP Section

0.2

enemies/tray Natural

0.0 Spiders Hymenoptera Earwigs Hemiptera D. brevis Total

Figure 1. Natural enemy counts in OP and non-OP orchard sections treated with mating disruption (Isomate C+) supplemented by either phosmet or methoxyfenozide for codling moth control; Hanners Orchard, Hood River, 2003.

Registration of new insecticide chemistries for tree fruits in the United States The first mating disruption product for codling moth control was registered in the United States in 1991 (Table 2). Several other slow-release pheromone technologies have received registration since then. The only selective insecticide available at the time to supplement mating disruption was horticultural mineral oil. In addition to controlling codling moth eggs (Riedl et al., 1995), frequent applications of horticultural mineral oil during the summer also suppressed pear psylla, mites and other secondary pests (VanBuskirk et al., 2002). However, this program was not widely adopted because of concerns about phytotoxicity, especially to the fruit (Hilton et al., 2002). In the absence of OP alternatives, apple and pear growers in the western United States continued to rely on azinphosmethyl and phosmet for codling moth control until 1999 when tebufenozide, the first new and potentially selective insecticide with codling moth activity, received registration (Table 2). By comparison, fruit growers in many growing regions around the world had the benefit of using OP alternatives such as insect growth regulators (IGRs) and granulosis virus for codling moth control long before 1990 (Riedl et al., 1998; Riedl, 1998a). Several new insecticide chemistries with codling moth activity have been registered since then: the molting accelerator methoxyfenozide; the chitin-synthesis inhibitor diflubenzuron and novaluron; and the neonicotinoids acetamiprid and thiacloprid. New pyrethroid insecti- 5

cides have also become available since 1991 but are not included here as potential supplemental insecticides in combination with mating disruption because of their known broad-spectrum activity, their disruptiveness to integrated programs and high toxicity to natural enemies.

Table 2. OP alternatives registered for codling moth control in the United States between 1991 and 2005.

Active ingredient Mode of action Trade name Year registered

Codlemone (codling moth Behavioral (mating Isomate C+ 1991 sex pheromone) disruption) CpGV (granulosis virus)1 Pathogen Cyd-X 1995 tebufenozide Molting accelerator Confirm 1999 methoxyfenozide Molting accelerator Intrepid 2000 diflubenzuron Chitin-synthesis Dimilin 2002 inhibitor acetamiprid Neurotoxin Assail 2003 novaluron2 Chitin-synthesis Rimon 2005 inhibitor thiacloprid Neurotoxin Calypso 2005 rynaxypyr Neurotoxin Altacor 20083

1Not used until ca. 2000. 2Registered on apples, not on pears. 3Expected year of registration

Impact of new insecticide chemistries on the predaceous mirid D. brevis The registration of alternatives to OP insecticides over the last 10 years was an important step but little was known about the selectivity of the new insecticide chemistries to natural enemies and how well they would fit into pheromone-based selective IPM programs. A major goal of a recently completed research project among several universities and federal laboratories in the western United States was to investigate the impact of new insecticide chemistries on key natural enemies in orchards (Brunner, 2000). We focused in these studies on D. brevis because of its importance as a predator of pear psylla in northern Oregon’s pear orchards and on the newer insecticides. A few ‘old’ insecticide chemistries such as OPs and pyrethroids were also included in these studies. In addition to acute toxicity, we also assessed sublethal effects, i.e. effects on reproduction and development in laboratory bioassays. Studies included the neonicotinoids: acetamiprid, clothianidin, imidacloprid, thiacloprid, thiamethoxam, the IGRs: methoxyfenozide, novaluron, pyriproxyfen; the OP phosmet; the pyrethroid fenpropathrin; the macrocyclic lactones abamectin and spinosad; and rynaxypyr, a promising new codling moth insecticide which is still under development. Much of this work has already been published (Brooks et al., 2004a,b; Kim et al., 2004a,b; 2006). Bioassays with rynaxypyr have so far indicated low acute toxicity to D. brevis but bioassays to evaluate sublethal effects are still on-going (Riedl, unpublished data). The impact of several of the newer insecticides on D. brevis and other natural enemies were also studied in field and semi-field tests to complement the laboratory bioassays. We 6

were particularly interested in acetamiprid, a neonicotinoid insecticide which has become a widely used OP replacement for codling moth control on pears and apples in the western United States since its registration (Table 2). A single spray of acetamiprid, applied at a rate recommended for codling moth control, eliminated D. brevis for at least 5 to 6 weeks (Fig. 2a). No recolonization occurred in spite of the availability of prey on the treated trees (Fig. 2b) and the proximity of untreated trees with D. brevis. Other neonicotinoids, such as imidacloprid, thiacloprid, and thiamethoxam, had a similar impact on D. brevis in this field test.

(a) (b)

D. brevis adults and nymphs Pear psylla eggs & nymphs 4 25 acetamiprid acetamiprid 20 3 untreated check untreated check 15 2 10 1 5 No. perNo. leaf

No. per beating tray tray beating per No. 0 0

g g g g Jul Jul Jul u Jul ug u u 6- A - A A 1 23- 30- 16 23-Jul 30-Jul - 6- 13-Aug 20-Aug 27-Aug 6-A 13- 20-Au 27

Figure 2. Impact of acetamiprid applied at 167 g AI/ha on (a) the predaceous mirid D. brevis and (b) its prey pear psylla, C. pyricola; field test: Anjou pear trees treated by hydraulic handgun; Mid-Columbia Agric. Research & Extension Center, Hood River, Oregon; 2002.

In another study, exposure of young D. brevis nymphs to apple foliage from Red Delicious trees treated by an air-blast sprayer at 167 g AI/ha and a spray volume of 935 l/ha caused about 58% mortality a few hours after treatment but nymph mortality decreased to 46%, 18% and 14% after 4, 16 and 32 days, respectively (Riedl, unpublished data). Laboratory studies have shown that the OP alternatives vary considerably in terms of acute toxicity but also in terms of sublethal effects on D. brevis nymphs (Table 3). Neonicotinoid insecticides, including acetamiprid, and OPs such as phosmet had high acute toxicity to D. brevis but no sublethal effects. The widely used insecticide/miticide abamectin had high acute toxicity and major sublethal effects. Spinosad had moderate toxicity and minor sublethal effects. All IGRs tested had low acute toxicity to D. brevis. Only novaluron had major sublethal effects. One goal of this research was to provide growers with a risk rating guide for pesticides commonly used in tree fruits, especially for the newer insecticide chemistries. Table 3 summarizes the impact of some of the newer insecticide chemistries on D. brevis. This information is available now in local pest management guides for tree fruits which allow growers to choose those pesticides which have the least impact on D. brevis and other natural enemies (Anonymous 2006).

Table 3. Impact rating of new insecticide chemistries used on tree fruits on the predaceous plant bug Deraeocoris brevis.

Acute toxicity1 Sublethal effects Pesticide Low Moderate High Minor Major

abamectin √ √ acetamiprid √ methoxyfenozide √ √ novaluron2 √ √ Phosmet √ No data pyriproxyfen √ No data rynaxypyr3 √ No data Spinosad √ √ 1 L= low impact (<30% mortality); M = moderate impact (30-70%); H = high impact (>70%) 2 registered on apples only 3 not yet registered

Acknowledgements

Support for this research was provided by the Agricultural Research Foundation (Oregon), by the agricultural chemical industry, and by the USDA/IFAFS grant: Building a multi-tactic pheromone-based pest management system for western orchards.

References

Anonymous 2006. Pest management guide for tree fruits in the Mid-Columbia area. – Oregon State University Extension Pub. EM 8203: 87 pp. (http://extension.oregonstate.edu/catalog/pdf/em/em8203-e.pdf; accessed Nov 2006) Brooks, D.J., Walston, A.T., Farnsworth, A., Farnsworth, J., Smith J. & Riedl, H. 2004a. Effects of Agri-Mek, Mitac, Actara, Assail and Calypso on pear psylla and natural enemies, 2002. – Arthropod Management Tests 29: A29 Brooks, D.J., Walston, A.T., Farnsworth, A., Farnsworth, J., Smith, J. & Riedl, H. 2004b. Impact of foliar insecticides on pear psylla and natural enemies, 2002. – Arthropod Management Tests 29: A30 Brunner, J. 2000. Building a multi-tactic pheromone-based pest management system in western orchards. – Proposal funded by USDA/CSREES Program: Initiative for Future Agriculture and Food Systems (IFAFS); awarded to Washington State University (lead agency). Calkins, C.O. 1998. Review of the codling moth areawide suppression program in the western United States. – J. Agric. Entomology 15 (4): 327-333. Hilton, R., Riedl, H., VanBuskirk, P. & Sugar, D. 2002. The effect of foliar season applications of narrow-range petroleum oil on pear tree productivity and fruit quality. – In: 8

Beattie, G.A.C., et al. editors. Spray Oils Beyond 2000: Sustainable Pest and Disease Management. University of Western Sydney, Australia: 179-184. Kim, D.S. & Riedl, H. 2005. Effect of temperature on development and fecundity of the predaceous plant bug Deraeocoris brevis reared on Ephestia kuehniella eggs. – BioControl 50 (6): 881-897. Kim, D.S., Brooks, D.J. & Riedl, H. 2004a. Acute toxicity of pesticides to the mirid predator Deraeocoris brevis in laboratory tests.– Arthropod Management Tests 29: L10 Kim, D.S., Brooks, D.J. & Riedl, H. 2004b. Sublethal effects of pesticides on the mirid predator Deraeocoris brevis in laboratory tests. – Arthropod Management Tests 29: L11 Kim, D.S., Brooks, D.J. & Riedl, H. 2006. Lethal and sub-lethal effects of abamectin, spinosad, methoxyfenozide and acetamiprid on the predaceous plant bug Deraeocoris brevis in the laboratory. – BioControl 51(4). Riedl, H. 1996. Pear: selectivity of low rates of Imidan to natural enemies. – In: 1996 Annual Report of Research in Entomology, p. 45-54 (on file at Mid-Columbia Agric. Research & Extension Center). Riedl, H. 1997. Pear: seasonal codling moth control with IGRs and Imidan. – In: 1997 Annual Report of Research in Entomology, p. 77-81 (on file at Mid-Columbia Agric. Research & Extension Center). Riedl, H. 1998a. Pflanzenschutz im Obstbau in Europa und Nordamerika – ein Vergleich. – Obstbau Weinbau 35 (10): 308-309. Riedl, H. 1998b. Pear: evaluation of mating disruption in combination with low rates of Imidan for control of codling moth. – In: 1998 Annual Report of Research in Entomo- logy, p. 77-91 (on file at Mid-Columbia Agric. Research & Extension Center). Riedl, H., Halaj, J., Kreowski, W.B., Hilton, R.J. & Westigard, P.H. 1995. Laboratory evaluation of mineral oils for control of codling moth (Lepidoptera: Tortricidae). – J. Econ. Entomol. 88(1): 140-147. Riedl, H., Blomefield, T.L. & Gilomee, J.H. 1998. A century of codling moth control in South Africa: II. Current and future status of codling moth management. – J. S. Afr. Soc. Hort. Sci. 8(2):32-54. VanBuskirk, P., Hilton, R. & Riedl, H. 2002. Use of narrow-range petroleum spray oil for suppression of codling moth (Lepidoptera: Tortricidae) and secondary arthropod pests in an areawide mating disruption program. – In: Beattie, G.A.C. et al., editors. Spray oils beyond 2000: sustainable pest and disease management. University of Western Sydney, Australia: 356-361. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 9-13

Beneficial effects of hedgerow plants for insect predators in adjacent orchards – the value of pollen and nectar to Anthocoris nemorum (L.)

Lene Sigsgaard, Johannes Kollmann Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, [email protected]

Abstract: This paper reports a) on a field study comparing the spring predator community in hedgerows and in apple orchards (Malus domestica) as a measure of the potential value of different hedgerow types for conservation biological control; and b) on a laboratory study of the diet-dependent development of the predator Anthocoris nemorum (L.) (Heteroptera: Anthocoridae) on common prey types (lepidopteran eggs, aphids, collembola, pollen and nectar, and mixed diets), aiming to identify among others the value of pollen and nectar. Arthropod communities in alder hedgerows (Alnus incana, A. glutinosa), in elderberry shrubs (Sambucus nigra) in mixed hedgerows, and in the herbaceous layer under these two hedgerow types were more similar to apple orchards than insect communities in hedgerows dominated by hawthorn (Crataegus monogyna) and hazel (Corylus avellana). Total numbers of predators were highest in hazel and hawthorn followed by apple. Alder, hawthorn and herbaceous layers with stinging nettle (Urtica dioica) held the highest number of A. nemorum. At the time of the field survey, apple, elderberry and hawthorn were flowering. Though pollen and sucrose was inferior to arthropod diets for A. nemorum nymphs, they could survive on this diet in some cases over a month, showing that flowering plants can be valuable for A. nemorum survival in orchards when prey is temporarily scarce. Thus populations of A. nemorum in hedgerows may not only use this habitat as shelter or source of prey but also for collecting pollen and nectar.

Key words: spring predator community, hedgerow, pollen, nectar, prey

Introduction

Across northern Europe, spiders and predatory bugs are the most important early predators in pome fruit (Solomon et al., 2000). Anthocoris nemorum (L.) (Heteroptera: Anthocoridae) is among the most important early predatory bugs in apple orchards. It has been suggested that spiders and predatory bugs could make a significant contribution to a reduction in the number of aphids and other apple pests, but this would depend on a high density of predators in the field early in the cropping season (Symondson, 2002). In order to maintain or to attract a sufficient number of these early predators in orchards, the sites must provide sufficient food and shelter. Knowledge of habitat and dietary requirements of individual species can provide a useful tool to improve orchard design (Jervis et al., 2004). The choice of hedgerow plants and the maintenance of an adequate hedgerow structure are of great importance for how effectively natural control works in adjacent orchards and hence the possibilities to reduce pesticide input. Functional biodiversity can be a powerful tool to control insect pests (Gurr et al., 2003). It builds on the assumption that the structure and composition of adjacent habitats can be used to manipulate arthropod populations, both of pests and beneficials, in farmland. To assess the potential for conservation biological control of common hedgerows surrounding orchards we did a field study comparing the spring predator community in different hedgerow types and apple orchards (Malus domestica Borkh., Rosaceae). To sustain beneficials, hedgerows must

9 10

meet their dietary needs of spring predators. Therefore, a laboratory study was done on diet- dependent development of the predator Anthocoris nemorum (L.) (Heteroptera: Anthocoridae) on common prey types, i.e. lepidopteran eggs (Sitotroga cerealella (Olivier), Lepidoptera: Gelechiidae), aphids (Rhopalosiphum padi (L.), Homoptera: Aphididae), collembola (Fol- somia fimentaria (L.), and on a diet of bee pollen principally from fruit trees and sucrose as well as on mixed diets. The experiment aimed, among others, to provide evidence of any dietary value of pollen and nectar.

Material and methods

Study sites We chose five apple orchards with adjacent hedgerows on Zealand in eastern Denmark. The sites were on the island Fejø (54o57’N, 11o22’E; <5 m a.s.l.), near Frederikssund (55o52’N, 12o5’E; 20–40 m) on a west-facing slope overlooking Roskilde Fiord, in Jørlunde (55o50’N, 12o11’E; 10–20 m) sloping slightly towards lake ‘Burre Sø’, in Kirke Eskildstrup (55o50’N, 11o50’E; 10–20 m), and in Skibby (55o43’N, 11o57’E; <5–10 m) sloping very slightly towards Roskilde Fiord. All sites are situated on moraine soil and they are surrounded by diverse traditional farmland. The orchards in Frederikssund and Kirke Eskildstrup are under IP-management, the remaining three orchards are managed organically. Orchards are 7–30 y old, the youngest being the one in Frederikssund; the apple trees sampled were 7–20 y old.

Hedgerows Four common hedgerow species as well as the apple trees themselves were sampled in early and late May 2005. In all sites the hedgerows are separated from the orchard by a strip of uncropped grass or weeds, or in one case a narrow dirt road. The distance separating hedgerow and orchard was 2–6 m. Alder (Alnus glutinosa (L.) Gaertn., A. incana (L.) Moench, Betulaceae) was chosen in four hedgerows, elderberry (Sambucus nigra L., Caprifoliaceae) in two mixed hedgerows, hawthorn (Crataegus monogyna Jacq., Rosaceae) in three hedgerows, and hazel (Corylus avellana L., Corylaceae) in one hedgerow. In addition, the hedge bottom, i.e. the herbaceous layer under and close to the hedgerows, was sampled. Arthropods were collected from the trees and shrubs by beating, and from the herbaceous layer with a sweep net (cf. Sigsgaard, 2005). The insect data were analyzed for the effects of type of hedgerow, tree species and herbaceous layer on the population of beneficials (cf. Jackson et al., 1999; Sigsgaard et al., 2006). A Renkonen index was used to assess similarity in predator composition on apple trees and in the hedgerows. During the second sampling the botanical composition of hedgerows was recorded following the method described in Kollmann & Schneider (1999).

Diet dependent development Diet dependent development was assessed on the offspring of A. nemorum females collected in autumn. After an artificial hibernation of 1–2 months females were moved to a climate cabinet and held under conditions approaching mid-summer in Denmark at L:D 20:4 with day temperatures of 22oC and night temperatures of 12oC. Humidity was maintained at 75% RH. 1d old nymphs were moved to individual containers and offered one of the following diets: a) lepidopteran eggs (S. cerealella), b) aphids (R. padi), c) collembola (F. fimentaria), and d) a diet of bee pollen principally from fruit trees and sucrose. In addition, the nymphs were kept on mixed diets, i.e. a+b, a+c, a+d and a full mixed diet. Sucrose was used as a surrogate for nectar. The nymphs were checked daily and change of instar as well as mortality recorded. Surplus diet was provided every second day. A starvation treatment served as control. We 11

used survival, development, longevity and fecundity as measures of prey quality. Data were analyzed with ANOVA if meeting requirements for a parametric analysis, otherwise with a non-parametric analysis. Survival distributions were analysed with a non-parametric analysis, the LIFETEST-Procedure (SAS Institute, 1999).

Results

Hedgerows At the time of the study, apple, elderberry and hawthorn were flowering. All hedgerow types contained many psyllids, especially hawthorn (357) (Psylla crataegi (Schrank)). There were still few aphids, primarily in alder and hazel. Collembola were mostly found in alder and elderberry. Delphacidae and Cicadellidae (Homoptera) were more common in herbs, though we found some Cicadellidae in alder. Some Lepidopteran larvae were also found in the hedgerows, especially Geometridae in hawthorn. In the hedgerow trees, most collembola were found in alder and elderberry, however, no significant correlation with A. nemorum numbers was found for the dates of the survey. Few insect pests were found in apple. Larvae of winter moth were found in three orchards. Apple sawfly and apple psyllids in one orchard, in another we found a few apple weevils, by mainly other weevil species. The most common beneficials encountered were spiders (404), anthocorids (64), coccinellids (74) and staphylinids (104). In addition we encountered Opiliones spp. (13) green lacewing (17), earwigs (6), Cantharidae (33) and ants (65). Total numbers of predators were highest in apple and hawthorn. Alder, hawthorn and herbaceous layers with stinging nettle (Urtica dioica L., Urticaceae) held the highest number of A. nemorum. Of the spiders collected 255 were sampled from trees. On average we sampled two spiders in each beating sample in apple (10 branches of individual trees), in elderberry 1.5, in alder and hazel 1.2 and in hawthorn 1. Most common were spiders belonging to the Philodromidae, previously considered as part of the crab-spider family. This family was particularly common in apple, hazel and hawthorn. In apple Araniella sp. spiders were the second most common spider group. Araniella sp. were also common in alder. Correlation analyses of beneficials and botanical key figures showed that there were more spiders in hedgerows with some bare soil in the herbaceous layer (r = 0.3, P < 0.0001, df = 229) and with nitrogen rich soil (r = 0.2, P < 0.002, df = 229). Of the collected anthocorids, 40 were A. nemorum. This species was found in apple as well as in all hedgerow types, but most commonly in alder, hawthorn and in herbaceous layers with stinging nettle. An analysis of the occurrence of A. nemorum showed that there were significantly more in the herbaceous layer where stinging nettle occurred (F2,119 = 5.14, P < 0.007) estimated at 0.21 per sweep net sample, against only 0.03 (± 0.06, SE) where no stinging nettle occurred. Stinging nettle grows in nitrogen rich soil, and we found a significant correlation between the number of anthocorids and contents of soil nutrients (r = 0.2, P < 0.002, df = 229). Significantly more anthocorids occurred in alder and hawthorn hedgerows (F5,172= 5.31, P < 0.0001). Alder had significantly more anthocorids than apple (contrast analysis, F =13.1, df = 1, P = 0.0004). Anthocorid numbers were correlated with psyllid numbers (r = 0.17, P < 0.003, df = 298). Coccinellids were found in all hedgerows, but most numerous in hawthorn and hazel. However, the species found in hawthorn and hazel were not the same as those observed in apple. In apple there were still few coccinellids, principally C. septempunctata. Coccinellid numbers also correlated with psyllid numbers (r = 0.12, P < 0.002, df = 298). A comparison of the beneficial population in hedgerows with that of apple can provide an estimate of the expected contribution by the hedgerow type for biological control in apple. 12

We compared hedgerow trees using the Renkonen index for similarity. The index ranges from 1 (identical) to 0 (no similarity), and showed some similarity for all hedgerows. Largest similarity was found in the composition of beneficials between alder and apple (0.61), followed by elderberry-apple (0.51), hazel-apple and hawthorn-apple (0.43 and 0.42).

Diet dependent development Survival differed significantly among the diets. No anthocorids survived until the adult stage on the diet of sucrose and pollen, or on a pure collembola diet, whereas 70% survived until adult on the diets with S. cerealella combined diets, and all survived on the full mixed diet. In terms of resulting adult weight, a useful measure of predator quality, mixed diets were clearly superior (Figure 1).

30 a 25 ) a 20 b 15 c 10 d weight (mg weight d 5 0 S.S. cerealellaceralella R. padi S. S. cerealellaceralella S. cerealellaceralella, S. cerealellaceralella MixedMixed dietdiet & R. padi sucrose & & pollen collembola

Figure 1. Weight of A. nemorum adults dependent on diet during nymph development.

Discussion

Spiders and predatory bugs are key beneficials in apple early in the cropping season, and can be of major importance in control of a.o. aphids (Sigsgaard, 2004; 2005). Hedgerows are important to maintain and/or attract anthocorids to orchards, to obtain the full benefit of this efficient predator. The survey found alder and elderberry to have the highest correlation in predator composition with apple, thus being candidates as good sources of beneficials. Alder was also a good important source of anthocorids in spring, and the most common hedgerow tree to find Araniella¸ a genus which has been found to correlate with rosy apple aphid numbers, suggesting an important role in aphid control (Wyss, 1995). Hawthorn held the highest total number of beneficials together with hedge bottoms with stinging nettle. Hawthorn also hosted many anthocorids and most Philodromidae, a family known to be able to contribute to pest control in orchards (Miliczky and Horton, 2005). Anthocoris nemorum is known to be an important predator of aphids and other apple pests. Our results show that A. nemorum fitness is strongly affected by diet, so that long term performance will depend on access to a more varied diet. Sucrose and pollen has not earlier been tested for their dietary value for A. nemorum. Results show that nymphs can survive for a month or more and that some can develop to intermediate instars on a pure plant diet of sucrose and pollen. This stresses the potential value of flowers and/or honeydew for the survival of anthocorids in periods when prey is scarce. An S. cerealella diet was also 13

improved by adding pollen and nectar. Both apple and hawthorn were flowering during the study and both held many A. nemorum. Hawthorn, alder and stinging nettle in addition provided a good source of alternative prey. In conclusion A. nemorum in hedgerows may depend not only on their value as shelter or sources of prey but also on their production of pollen and nectar.

Acknowledgements

We are grateful to the growers for letting us use their orchards for the study. Special thanks go to the students Christina Krabbe and Christina Kastrup for assisting with insect handling in the laboratory. The research was supported by the ‘Fund for Research in Ecological Agriculture’ and the ‘Promilleafgiftsfonden’, a fund dedicated to support research aiming at reduced pesticide input. Both are gratefully acknowledged.

References

Gurr, G.M., Wratten, S.D. & Luna, J.M. 2003: Multi-function agricultural biodiversity: pest management and other benefits. – Basic and Applied Ecology 4: 107-116. Jackson, R.V., Kollmann, J., Grubb, P.J. & Bee, J.N. 1999: Insect herbivory on European tall- shrub species: the need to distinguish leaves before and after unfolding or unrolling, and the advantage of longitudinal sampling. – Oikos 87: 561-570. Jervis, M.A., Lee, J.C. & Heimpel, G.E. 2004: Use of behavioural and life-history studies to understand the effect of habitat manipulation. – In: Ecological Engineering for Pest Management. Advances in Habitat Manipulation for Arthropods. Eds: Gurr, G.M.; Wratten, S.D.; Altieri, M.A., Wallingford, CABI & CSIRO: 65-100. Kollmann, J. & Schneider, B. (1999) Landscape structure and diversity of fleshy-fruited species at forest edges. – Plant Ecology 144: 37-48. Miliczky, E.R. & Horton, D.R. 2005: Densities of beneficial arthropods within pear and apple orchards affected by distance from adjacent native habitat and association of natural enemies with extra-orchard host plants. – Biological Control 33: 249-259. SAS Institute. 1999: SAS/STAT User's Guide Version 8. – SAS/STAT Inc. Cary N.C. Sigsgaard, L. 2004: Oviposition preference of Anthocoris nemorum and A. nemoralis (Hetero- ptera: Anthocoridae) for apple and pear. – Entomologia Experimentalis et Applicata 111: 215-223. Sigsgaard, L. 2005: Occurrence of the anthocorids Anthocoris nemorum and A. nemoralis on apple and pear in Denmark. – IOBC/WPRS Bull. 28(7): 139-142. Sigsgaard, L., Esbjerg, P. & Philipsen, H. 2006: Experimental releases of Anthocoris nemoralis F. and A. nemorum (L.) (Heteroptera: Anthocoridae) against the pear psyllid Cacopsylla pyri L. (Homoptera: Psyllidae) in pear. – Biol. Control. In press: doi:10.1016/j.biocontrol.2006.02.008. Solomon, M.G., Cross, J.V., Fitzgerald, J.D., Campbell, C.A.M., Jolly, R.L., Olszak, R.W., Niemczyk, E. & Vogt, H. 2000: Biocontrol of pests of apples and pears in northern and central Europe – 3. Predators. – Biocontrol Science and Technology 10: 91-128. Symondson, W.O.C.; Sunderland, K.D. & Greenstone, M.H. 2002: Can generalist predators be effective biocontrol agents? – Annual Review of Entomology 47: 561-594. Wyss, E. 1995: The effects of weed strips on aphids and aphidophagous predators in an apple orchard. – Entomologia Experimentalis et Applicata 75: 43-49. 14 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 15

Adapting orchard management to re-establish earwigs in Belgian pome fruit orchards

B. Gobin1, A. Marien1, S. Davies2, H. Leirs2 1 Pcfruit (former Royal Research Station of Gorsem), Zoology Department, De Brede Akker 13, B-3800 Gorsem, Belgium, [email protected] 2 Universiy of Antwerp, Evolutionary Biology Group, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium

Abstract: Earwigs are key generalist predators to a variety of pome fruit pests, though aimed chemical control to prevent suspected fruit damage of earwigs considerably reduced populations in Belgian orchards. In recent years, integrated and organic fruit growers have tried to re-establish earwig populations, thus far with little success. We started a study linking various components of orchard management and the earwig life history to identify potential factors hazardous to earwigs. We investigate effects in both short term (e.g. knock down of pesticide use) and long term (e.g. introduction of populations). The goal of this study is to adapt management to allow optimal development of the earwig population. Studying side-effects of orchard management on this univoltine organism, especially at the population level, revealed some intrinsic problems. First of all there is a strong variation within orchards, even at the tree level at a given site within the orchard, suggesting that large sample sizes are required for valid tests. Second, there appears to be a considerable effect of niche occupation (tree and soil) during larval stages, the most sensitive life stages. Third, spatial distribution patterns seem to change during life history, from clumped nests to patchy larval distribution and continuous adult presence. Finally, there is an unprecedented persistency in effect even when we apply fast degrading products on small plot sizes (6 trees). Contrary to our expectations, we observed no re-colonisation from adjacent untreated plots. In addition to this, transplants of large earwig populations to previously unoccupied orchards are seldom successful, limiting clear-cut experimental designs. These issues need to be properly addressed to limit their impact on the outcome of side-effect testing in field tests.

Key words: Forficula auricularia, organic farming, population dynamics, beneficials

15 16 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 17-21

Abundance and seasonal distribution of natural enemies in treated vs untreated pear orchards in Lleida (NE Spain)

Ana María Jauset1,2, Miquel Artigues2, María José Sarasúa1,2 1 Universitat de Lleida, Department of Crop and Forest Science, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]; [email protected] 2 IRTA. Centre UdL-IRTA, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]

Abstract: The diversity of beneficial arthropods and the seasonal distribution of natural enemies associated with Cacopsylla pyri (L.) (Hemiptera: Psyllidae) was studied in two pear orchards (c.v. Blanquilla), one untreated and one treated with insecticides in Lleida (NE, Spain). Orchards were sampled weekly or fortnightly, using beating funnels, from March 2003 to December 2004 (except in July due to fruit maturity). In both orchards, C. pyri was the most abundant phytophagous. The three most important groups of predaceous arthropods in orchards were Coleoptera, Heteroptera and Aranea. Among heteropterans Anthocoris nemoralis, was the most abundant in both orchards. Miridae and Dermaptera appeared at the same time at the early spring. Anthocoridae were the most important pear psylla predators, showing a strong response to the psylla outbreaks in both orchards. The insecticide applications affected the diversity and density of predaceous species. Pilophorus perplexus, Hemerobius sp. and the parasitoid Trechnites psyllae were only found in the untreated orchard.

Key words: natural enemies, Cacopsylla pyri, Pyrus communis

Introduction

The conservation of natural enemies in pear orchards improves the biological control of the key pest, Cacopsylla pyri (L.) (Hemiptera: Psyllidae). This objective has an increasing interest due to the rising problems of its chemical control: availability of few insecticides and C. pyri capacity to develop resistance. The aim of this study was to know the diversity and the seasonal distribution of the beneficial arthropods in two pear orchards, one untreated and one treated with insecticides.

Material and methods

The experiment was carried out in Gimenells (Lleida). The untreated orchard (UO) had never received any insecticide nor acaricide treatments; it had a surface of 0.179 ha with 4 rows (83 trees each). The treated orchard (TO) was managed under common practices of the zone, it had a surface of 0.976 ha with 11 rows (111 trees each). The trees belonged to the cv. Blanquilla and they were 10 years old. To monitor the number of arthropods and their seasonal distribution, both orchards were sampled weekly or fortnightly, using beating funnels, from March 2003 to December 2004 (except in July due to fruit maturity). Fifty branches (one per tree) were bet in each orchard. The trees were randomly chosen at every sampling date. Arthropods were collected in a plastic bag, stored in a cool box, brought to the laboratory, killed in a freezer at -20ªC and then processed under binocular microscope.

17 18

Results and discussion

Arthropod diversity The total number and diversity of arthropods species found were higher in the UO than in the TO. The main phytophagous species in the orchards was C. pyri, 75% (of the total number of arthropods) in the TO and 68% in the UO. Natural enemies and ants were more abundant in the UO (Figure 1).

100%

75%

50%

25%

0% UO TO C. pyri Other phytophagous Natural enemies Ants

Figure 1. Abundance (%) of arthropods in the untreated orchard (UO) and treated orchard (TO).

Within the natural enemies, very few parasitoids were found. They represented 0.85% (of total natural enemies) in the UO and 0.3% in the TO. Among the parasitoids associated with pear psylla (Erler, 2004; Rieux et al., 1990), only adults of Trechnites psyllae (Ruschka) (Hymenoptera: Encyrtidae) were collected in the UO. The orders of the predaceous arthropods found during this study and their relative abundance in each orchard are shown in Table 1.

Table1. Abundance (% of total natural enemies) of predaceous orders in the untreated (UO) and treate (TO) pear orchards.

ORDER UO TO Coleoptera 44.5% 35.8% Heteroptera 24.0 % 28.4% Aranea 23.0 % 12.1% Neuroptera 5.5 % 21.8% Dermaptera 1.6 % 0.3 % Acari 1.2 % 1.6 % Diptera 0.2 % 0.0 %

The families of predators collected are represented in Figure 2. Families of spiders were not determined. Coleoptera and Heteroptera were the main predaceous groups in both orchards followed by spiders. The role of spiders in pear psylla biological control is less known that the role of insect predators and parasitoids. 19

40 COCCINELLIDA E SPIDERS ANTHOCORIDAE MIRIDA E 30 CHRY SOPIDA E E DERMA PTERA TROMBIDIIDA E HEMEROBIIDA E 20 STA PHY LINIDA E PERCENTAG SY RPHIDA E CA NTHA RIDA E NA BIDA E 10 LYGEIDAE

0 UO TO

Figure 2. Abundance of the main predaceous arthropods families (%) in the untreated (UO) and the treated (TO) orchards.

Coccinelids were the most common coleopterans found and their abundance was very similar, 97.5% (of total Coleoptera) in the UO and 95,1% in the TO. The most common species was Stethorus sp. Heteroptera was the second important predaceous group. The anthocorids (85.5 % of total Heteroptera in the UO and 75.5 % in the TO) were more abundant than the mirids (14 % of total Heteroptera in the UO and 17.7 % in the TO). Among heteropterans, Anthocoris nemoralis (Fabricius) was the most abundant species collected during the study, followed by Campylomma verbasci (Meyer), Orius sp.and Pilophorus perplexus (Douglas-Scott), the last one only present in the UO as others authors have also found (Artigues et al., 1996; Erler, 2004). Anthocorids, and mainly A. nemoralis are the most effective predators of C. pyri in the pear orchards of the area of Lleida, but only when populations of psylla are high, and too late to prevent damage (Artigues et al., 1996; Sarasúa et al., 1994). Chrysopidae (Chrysopaerla carnea (Stephens), Forficulidae (Forficula auricularia (L)) in both orchards and Hemerobiidae (Hemerobius sp.) in the UO were less abundant but they still are important components of biological control systems in orchards. These predators are polyphagous and have been reported as predators of C. pyri (Erler, 2004; Matias et al., 1990).

Seasonal distribution The evolution during this study of the main phytophagous, C. pyri and its natural enemies is shown in the Figure 3. Independently of the year, the pear psylla population was low in the TO in the first half of years due to the insecticide applications but it rose when applications stopped (Figure 3). Adults of the parasitoid T. psyllae were collected in May, June and August of the year 2003 only in the UO. 20

700 100 UNTREATED ORCHARD 600

80 500

60 400

300 40 Mean number of 200

20 100

0 0 C.pyri per 50 trays 30 300 TREATED ORCHARD 250

20 200 Mean number of predators per 50 trays

150

10 100

0 0 M A My Jn Au S O N D J F M A My Jn Au S O N D 2003 2004

MIRIDAE FORFICULIDAE Trechnites psyllae COCCINELIDAE NEUROPTERA C. Pyri ANTHOCORIDAE

Figure 3. Monthly mean numbers of C. pyri and natural enemies (adults+nymphs/larvae) from March 2003 to December 2004, in the untreated and treated pear orchard; arrows show insecticide applications.

The density of coccinelids was very different between years, higher in the year 2003 than 2004. They were present at all the sampling times showing a similar pattern of fluctuation in both orchards. Their fluctuations were not related with pear psylla populations. The density of anthocorids rose with the increase of the psylla populations, and were the only group of predators that followed the psylla populations independently of the year and season, as described in Erler (2004) and Sarasúa et al. (1994). They showed a strong response to summer and early autumn psylla outbreaks than to spring ones (Figure 3). Their presence in 21

the orchards in the late winter was related with the prey abundance of the previous year (Sarasúa et al., 1994), this explain that at the beginning of the year 2004 the density of anthocorids was higher in the TO than in UO. Mirids and dermapterans appeared before the anthocorids, in early spring. Mirids were more abundant in the year 2003 (low psylla population) than in the year 2004. The densities of both predaceous insects were lower than anthocorids and did not show a direct relation with the psylla population. Their role could be important on the first pear psylla generation. After insecticide applications they decreased or disappeared in the TO. The highest density of neuropterans was found in October in both orchards and years. The results of this study showed that insecticide applications reduce the diversity and density of beneficial arthropods and that the effectiveness of the natural enemies is not sufficient to control the pear psylla. Others strategies to improve the biological control of the pear psylla should be used during the whole season.

References

Artigues, M.; Avilla, J.; Jauset, A.M. & Sarasúa, M.J. 1996: Predators of Cacopsylla pyri in NE Spain. Heteroptera: Anthocoridae and Miridae. – IOBC/wprs Bulletin 19(4): 231- 235. Erler, F. 2004: Natural enemies of the pear psylla Cacopsylla pyri in treated vs untreated pear orchards in Antalya, Turkey. – Phytoparasitica 32 (3): 295-304. Matias, C. ; Bouyjou, B.; Avelar, J. & Domingues, V. 1990: Faune prédatrice et proies potentielles en vergers de poiriers dans deux situations (Lutte chimique et lutte intégrée au Portugal). – IOBC/wprs Bulletin 13(2): 11-16. Rieux, R.; Armard, E.; Lyoussoufi, A. & Faivre d’Arcier, F. 1990: Etude des populations des parasitoides du psylle du poirier Psylla pyri (L.) (Homoptera: Psyllidae) et de leur hôte en vergers de la région d’Avignon_Montfavet: Evolution de la prévalence au cours d’un cycle annuel et intérêt de certaines caractéristiques de ce parasitisme. – IOBC/wprs Bulletin 13 (2): 22-25. Sarasúa, M.J.; Solá, N.; Artigues, M. & Avilla, J. 1994: The role of Anthocoridae in the dynamics of Cacopsylla pyri populations in a commercial orchard without pesticides. – IOBC/wprs Bulletin 17(2): 138-141.

22 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 23

Pressure of predation in treated vs untreated pear orchards in Gimenells (Lleida, Spain) during spring and summer 2004

Miquel A. Díaz 1,2, Miquel Artigues2, María José Sarasúa2,3 1 Balasch-Joval S.L., Nou 1, 25331 – Tornabous, Lleida, Spain, [email protected] 2 IRTA. Centre UdL-IRTA, Rovira Roure 191, 25198 – Lleida, Spain, miquel.artigues @irta.es 3 Universitat de Lleida, Department of Crop and Forest Science, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]

Abstract: Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest in pear orchards in Lleida (Spain). This pest is difficult to control because of its ability to develop resistance to pesticides and the few available and effective products that can be used. Biological control by conservation and increase of predators can contribute to its control it must be included in the integrated pest management of pear psylla. Our objective was to estimate the level of activity of the predators during the spring and summer generations of C. pyri, with and without chemical control. We offered as prey eggs of Ephestia kuehniella Z (Lepidoptera: Pyralidae) in two orchards of Pyrus communis cv. Blanquilla, one orchard under chemical control (TO) and the other orchard untreated against insects and mites since plantation (UO). From January until the first insecticide treatment (April, 14th), we observed in the TO a slightly higher predation activity than in the UO, significant in some weeks. The psylla population in the previous autumn was higher in the TO and this favoured that the predators remained during winter, the anthocorids were present in TO already in January. From May onwards the level of predation was higher in the UO, mainly due to the predators with chewing activity. These predators were mainly earwigs, not detected in TO. During this year the activity of the predators with sucking activity was low in both orchards because the population of the psylla did not increase enough to attract Anthocoris nemoralis (F.) (Heteroptera: Anthocoridae). However in the UO the sucking activity was underestimated, as we have detected when we have separated the diurnal and nocturnal activity, because the eggs partially sucked during the day were exposed to the earwigs that have nocturnal activity.

Key words: Predation, biological control, predators, pear psylla

23 24 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 25-30

Impact of alternative insect management programs on the predatory arthropod complex of green apple aphids

James F. Walgenbach1, Raul T. Villanueva2 1 Department of Entomology, North Carolina State University, Mountain Horticultural Crops Research and Extension Center, 455 Research Drive, Fletcher, NC 28732, USA, [email protected] 2 Department of Plant Pathology, NC State University, Don Ellis Laboratories, 1320 Varsity Drive, Raleigh NC 27695, USA, [email protected]

Abstract: The North Carolina apple industry has relied on organophosphate (OP) insecticides as the primary pest management tool for >40 years. Concern about the safety of these pesticides to human health and the environment has led to regulatory actions that have reduced the availability of these pesticides to growers. Hence, studies were conducted from 2002-2005 to assess the impact of reduced- risk (RR) pest management strategies on pest and beneficial arthropod populations. In nine commercial apple orchards, plots ranging in size from 2-5 ha were used to compare RR vs. conventional (OP) based pest management programs targeting 6-10 key pests. Reduced-risk strategies included mating disruption and new insecticides classified as reduced-risk. Comparatively similar numbers of the aphids (Aphis pomi and A. spiraecola) were found in the RR and conventional programs A diversity of generalist predators that preyed primarily on aphids, including predators in the families Coccinellidae, Chrysopidae, Syrphidae, Cecidomyidea, were found in slightly higher numbers in RR vs. conventional plots, but these differences were not significant. Results suggest that as apple growers switch from OP to RR based IPM programs, generalist predator populations common in North Carolina apple systems should not be adversely affected.

Key words: Aphids, reduced-risk pest management, predators, biological control.

Introduction

Organophosphate (OP) insecticides have been an important component of North Carolina (USA) pome fruit pest management programs for >40 years (Croft and Bode 1983). Implementation of the Food Quality Protection Act of 1996 (Anonymous 1966) by the US Environmental Protection Agency has resulted in the reduced availability of OP’s to tree fruit growers. In recent years a wide array of alternative pest control tools have become available to the apple industry, including mating disruption and new-chemistry insecticides with novel modes of actions, many classified as reduced-risk insecticides. While we have increased our understanding of how to properly use many of these tools to manage pest populations (Borchert et al., 2004, Kovanci et al., 2005), there is relatively little knowledge of their effects on natural enemy populations when used in whole-farm systems. The reduction of natural enemies has been a recurring problem in agriculture, because natural enemies are generally more susceptible to insecticides than pests (Horn and Wadleigh 1986). This has been most evident when outbreaks of indirect pests occurred. Aphids are common indirect pests in apple orchards, and are most important in newly planted or smaller, high density plantings (Kaakeh et al., 1993, McVay et al., 1994). Two morphologically similar aphid species are common pests of apples in western North Carolina – the green apple aphid (Aphis pomi DeGeer (Hemiptera: Aphididae)) and spirea aphid (Aphis spiraecola Patch)

25 26

– and are abundant on summer water sprout shoots in May and June (Filajdic et al., 1995). Aphis spriraecola has displaced A. pomi as the most dominant aphid species on apple in several states in the eastern USA and Israel due to resistance of the former to azinphosmethyl (Hogmire et al., 1992). During periods of peak aphid activity, there is a diversity of natural enemies that can help to reduce the potential for aphid outbreaks (Brown, 2004). The abundance and diversity of natural enemy populations can serve as indicators of the environmental impact of insecticides used in apple orchards. In this study, we monitored aphid and generalist natural enemy populations in blocks of apples managed with OP vs. reduced-risk insecticides at nine locations over a four-year period.

Material and methods

Study sites were located in three counties in western North Carolina, USA; Henderson, Lincoln, and Polk, which had six, one and two test sites, respectively. At each site, arthropod pests in a block of apples ranging in size from 2 to 5 ha was managed using mating disruption and reduced risk (RR) insecticides, and an adjacent block of similar size was managed using conventional OP insecticides. The diversity of pest control tools used in RR and Conventional blocks is shown in Table 1, and the description of orchards can be found in Villanueva and Walgenbach (2003).

Table 1. Insecticides and pheromone products used in reduced risk and conventional IPM programs from 2002 to 2005.

Products used 1 Growth Target Pests Reduced-Risk Conventional Stage Dormant to oil, thiamethoxam, oil, dimethoate, SJS, RAA, Pink pyriproxyfen, or chlorpyrifos PC, PB ERM or indoxacarb Petal fall to indoxacarb, azinphosmethyl, PB, PC, second cover methoxyfenozide, esfenvalerate, OFM, TABM, OFM and CM pheromones or permethrin CM, LR Third to acetamiprid, azinphosmethyl, AA/SA, fourth cover methoxyfenozide, esfenvalerate, endosulfan, OFM, CMB, OFM pheromone methoxyfenozide, LH, ERM imidacloprid, thiacloprid Fifth to sixth acetamiprid, thiacloprid, azinphosmethyl, phosmet, CM, AM, cover OFM pheromones esfenvalerate, thiacloprid OFM, LR, ERM Seventh to Spinosad, azinphosmethyl, phosmet, OFM, TABM, eighth cover methoxyfenozide, esfenvalerate, CM OFM pheromones fenpropathrin, methoxyfenozide 1AA/SA = apple aphid/spirea aphid, AM = apple maggot, CM = codling moth, CMB = Comstock mealybug, ERM = European red mite, LH =leafhoppers, LR = leaf rollers, OFM = oriental fruit moth, PC = plum curculio, PB = plant bug, RAA = rosy apple aphid, SJS = San Jose scale, TABM = tufted apple bud moth.

Not all RR products listed in Table 1 were used at each study site, because the selection of products to use and frequency of application were based on the diversity and abundance of pest populations at each site. Individual growers made decisions as to the type of insecticides to apply and frequency of application in the conventional treatments. Aphid populations were monitored in RR and Conventional blocks during periods of high aphid activity (May – June). Aphid counts were made by observing 20 (2002) or 10 (2003-2005) water sprout shoots per tree on 20 trees per treatment and recording the number of leaves (per shoot) infested with >1 apterous aphid. Natural enemy populations were monitored on each shoot used to sample aphids, and included individuals from the families Cecidomyiidae, Chrysopidae, Coccinelidae, Miridae, and Syrphidae families. Data from each orchard was treated as a replicate, and numbers were transformed using ()x ± 0.05 and subjected to two-way ANOVA. Results are presented as back transformations.

Results

The average amount of insecticide from different classes of pesticides applied to the RR and Conventional treatments is shown in Table 2. The total kg of active ingredient applied to Conventional blocks was about 8X greater than that in RR blocks.

Table 2. Class of products used and mean amounts of active ingredients used (kg /ha) on the reduced risk and conventional IPM programs from 2002 to 2005.

IPM Program Class of products used Reduced risk Conventional Organophosphates (Guthion, Imidan, Lorsban) 0.13 7.29 Carbamates (Sevin: not for thinning) 0.00 0.10 Cyclodiene (Endosulfan, Thiodan) 0.04 0.16 Pyrethroid (Asana, Danitol) 0.00 0.19 Antibiotic (SpinTor) 0.06 0.01 Miticide (Vendex, Acramite, Pyramite, Zeal) 0.08 0.06 IGR (Intrepid, Esteem, Rimon, Applaud) 0.41 0.26 Oxadiazine (Avaunt) 0.10 0.00 Nicotinoid (Actara, Assail, Calypso, Provado) 0.23 0.07 Sprayable Pheromone (3M-OFM, Checkmate) 0.03 0.00 Total 1.09 8.14

There were no significant differences in the number of aphid-infested leaves in RR versus Conventional treatments throughout the duration of the study (Fig 1a). Although populationsnumerically inhigher both RRnumbers and Convent of predatorsional weretreatments found were in the lower RR versusthan other Conventional years. In 2002, program the firston most year sample of the dates study, (Fig. 1b),the thesemost differences abundant werepredator not significant. was Aphidoletes In 2003, naturalsp. (Diptera: enemy Cecidomyiidae), but numbers of these predators appeared to decline in the following years. The most dominant group of predators in 2004 was the syrphids (Diptera: Syrphidae), while 28

coccinellids (Coleoptera: Coccinellidae) were the dominant group in 2005. The latter was represented predominately by Harmonia axyridis Pallas.

Fig. 1a 7 Red.Risk 6 Conv.

2 1

No. infested leaves / 10 shoots 0 7 J 13 J 26 J 26 M 1 J 11 J 18 J 17 M 18 J 7 J 25 J 2002 2003 2004 2005

Fig. 1b 5 Orius sp. 4 Coccinelid Chrysopid 3 Syrphid Cecidomyid 2

0 RR RR RR RR RR RR RR RR RR RR RR Con Conv Conv Conv Conv Conv Conv Conv Conv Conv Conv No. of naturalenemies/10 shoots 7 J 13 J 26 J 26 M 1 J 11 J 18 J 17 M 18 J 7 J 25 J

2002 2003 2004 2005

Figure 1. Mean (±SEM) number of aphid infested leaves per 10 shoots (top), and mean number of natural enemies per 10 shoots (bottom) in reduced risk and conventionally managed apple orchards in North Carolina. 29

Discussion

Populations of A. pomi and A. spiraecola did not differ between the RR and Conventionally managed treatments during this four-year of this study. Neither were there differences in the abundance of natural enemies between the RR and Conventional treatments, although predator populations were numerically slightly higher in RR blocks. It should be noted that the predatory arthropod complex in North Carolina apples has been exposed to OP insecticides for at least 40 years, and it is probable that many of the species sampled in this study have developed a certain level of tolerance to OP insecticides. In contrast, many of the insecticides used in RR blocks represent new chemistry with novel modes of action to which natural enemy populations have not been previously exposed. Despite the term “reduced risk,” many of these new compounds are toxic to predatory arthropods. For example the biologically based insecticide spinosad is toxic to the phytoseiid mite Neoseiulus fallacis Garman (Acari: Phytoseiidae) (Villanueva and Walgenbach, 2005); the insect growth regulator novaluron affects the predatory bug Podisus maculiventris (Say) (Hemiptera: Pentatomidae) (Cutler et al., 2006); and the neonicotinoids imidacloprid and thiamethoxam can adversely affect Micromus tasmaniae (Walker) (Neuroptera: Hemerobiidae) (Cole and Horne, 2006)). Nonetheless, the RR insecticides used in these studies did not adversely affect the predatory arthropod complex in comparison to the conventionally based OP programs, as evidenced by the lack of significant differences between the two treatments and by the fact that predator populations were consistently higher in our RR than Conventional blocks. Studies in West Virginia, USA, (Biggs et al., 2000) and Australia (Nicholas et al., 1999) that compared predatory populations in apples managed with alternative IPM programs to OP-based programs have documented high predator numbers in alternative systems. In addition, the during our four-year study, N. fallacis and Agistemus fleschneri (Acari: Stigmaeid), key predators of European red mite, Panonychus ulmi Koch (Acari: Tetranychidae), were found in significantly higher numbers in the RR compared with Conventional program (RTV and JFW, unpublished data). These results suggest that, at least in the short term, generalist predatory arthropods that are common regulators of aphid populations North Carolina apple systems will not be adversely affected as the industry transitions away OP insecticides in favor of more targeted, narrow spectrum insecticides. However, as additional new products become registered on apples, it will be important to assess their impact on predatory-prey relationships.

Acknowledgments

We thank the apple growers of North Carolina for their cooperation in conducting these studies. This research was supported, in part, by USDA-RAMP grant no. 2001-1681, Gerber Products Company, and the North Carolina Agricultural Research Service.

References

Anonymous. 1996: Food Quality Protection Act. – U.S. Congressional Record. 142: 1489- 1538. Biggs, A.R.; Hogmire, H.W. & Collins, A.R. 2000: Assessment of an alternative IPM program for the production of apples for processing. – Plant Disease 84: 1140-1146. Borchert, D.M.; Stinner, R.E.; Walgenbach, J.F. & Kennedy, G.G. 2004: Oriental Fruit Moth (Lepidoptera: Tortricidae) Phenology and Management with Methoxyfenozide in North Carolina Apples. – J. Econ. Entomol. 97: 1353-1364. 30

Brown, M.W. 2004: Role of the aphid predator guild in regulating spirea aphid populations on apple in West Virginia, USA. – Biol. Control 29: 189-198. Cole, P.G. & Horne, P.A. 2006: The impact of aphicide drenches on Micromus tasmaniae (Walker) (Neuroptera: Hemerobiidae) and the implications for pest control in lettuce crops. – Australian J. Entomol. 45: 244-48. Croft, B.A. & Bode, W.M. 1983: Tactics for deciduous fruit IPM. – In: B.A. Croft & S.C. Hoyt [eds.], Integrated management of insect pests of pome and stone fruits. Wiley & Sons, NY: 454 pp. Cutler, G.C.; Scott-Dupree, C.D.; Tolman, J.H. & Harris, C.R. 2006: Toxicity of the insect growth regulator novaluron to the non-target predatory bug Podisus maculiventris (Heteroptera: Pentatomidae) – Biol. Control 38: 196-204. Filajdic, N.; Sutton, T.B.; Walgenbach, J.F. & Unrath, C.R. 1995: The influence of green apple aphid/spirea aphid complex on intensity of Alternaria blotch of apple and fruit quality and yield characteristics. – Plant Disease 79: 691-694. Hogmire, H.H.; Brown, M.W.; Schmitt, J.J. & Winfield, T.M. 1992: Population development and insecticide susceptibility of apple aphid and spirea aphid (Homoptera: Aphididae) on apple. – J. Entomol. Sci. 27: 113-119. Horn, D.J. & Wadleigh, R.W. 1986: Resistance of aphid natural enemies to insecticides. – In World crop pests. Aphids. Their biology, natural enemies and control. Vol 2B: A.K Minks and P. Harrewijn (Eds.), Elsevier: 337-347. McVay, J.R.; Walgenbach, J.F.; Sikora, E.J. & Sutton, T.B.. 1994: Apple insects and diseases in the Southeast. – Alabama Cooperative Extension Service. ANR-838. Kaakeh, W.; Pfeiffer, D.G. & Marini, R.P. 1993: Effect of Aphis spiraecola and A. pomi (Homoptera: Aphididae) on the growth of young apple trees. – Crop Protection 12 (2): 141-147. Kovanci, O.B.; Schal, C.; Walgenbach, J.F. & Kennedy, G.G. 2005: Comparison of mating disruption with pesticides for management of Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) in North Carolina apple orchards. – J. Econ. Entomol. 98: 1248-1258. Nicholas, A.H.; Thwaite, W.G. & Spooner-Hart, R.N. 1999: Arthropod abundance in an Australian apple orchard under mating disruption and supplementary insecticide treatments for codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). – Australian Journal of Entomology 38: 23-29. Villanueva, R.T. & Walgenbach, J.F. 2003: North Carolina risk and mitigation program - 2002. In: Reduced-Risk Management Programs for Eastern Tree Fruits. - USDA Risk Avoidance and Mitigation Program (RAMP) Summary of year 1 Results: 2002. – CD available at Rutgers Agricultural Research and Extension Center, NJ. Villanueva, R.T. & Walgenbach, J.F. 2005: Development, oviposition and mortality of Neoseiulus fallacis (Phytoseiidae) in response to reduced-risk insecticides. – J. Econ. Entomol. 98: 2114-2120.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 31-35

Presence of the common earwig Forficula auricularia L. in apple orchards and its impact on the woolly apple aphid Eriosoma lanigerum (Haussmann)

Herman Helsen1, Marc Trapman2, Matty Polfliet3, Jan Simonse1 1 Applied Plant Research, Wageningen UR, P.O. Box 200, 6670 AE Zetten, The Netherlands, [email protected]. 2 BioFruitAdvies, Dorpsstraat 32, 4111 KT Zoelmond, The Netherlands, [email protected]. 3 FruitConsult, P.O. Box 70, 6670 AB Zetten, The Netherlands, [email protected].

Abstract: Apple growers encounter increasing problems to control woolly apple aphid Eriosoma lanigerum (Hausm.). Various authors have shown that the common earwig Forficula auricularia L. preys on aphids and thus plays a role in controlling aphid pests. It was assumed that earwig numbers in orchards had decreased in recent years. Therefore an inventory was made to assess the number of earwigs in apple orchards and to identify factors that determine their presence. In 125 apple orchards shelter traps were used to measure earwig densities. In addition, a questionnaire regarding characteristics and management of the orchards was filled in by the growers. In 93 of these orchards the density of woolly apple aphid was measured. The average number of earwigs in cardboard traps varied between 0 and 34 per trap, but in half of the orchards, average trap catches were less than 1 earwig per trap. There was a strong negative correlation between the numbers of earwigs and woolly apple aphid infestation, which shows that earwigs play an essential role in controlling woolly apple aphid in commercial orchards. Several factors seem to affect the presence of earwigs in the orchards, the most important one being the drainage of the soil. Organic orchards had slightly more earwigs than IPM treated orchards.

Key words: Apples, Integrated pest management, Biological control, Forficula auricularia, Eriosoma lanigerum

Introduction

The common earwig, Forficula auricularia L. (Dermaptera: Forficulidae) is considered to be both beneficial and a pest in orchards. On apple, earwigs are known to feed on the fruits, but mainly at points where the fruit is russeted or cracked. Besides this secondary damage, earwigs sheltering in apple clusters contaminate the fruits with their frass. On the other hand, earwigs are important predators of several orchard pests (Phillips, 1981; Nicholas et al., 2005; Lahusen et al., 2006). Woolly apple aphid, Eriosoma lanigerum (Hausm.) (Homoptera: Aphididae), is considered the most problematic pest in apple in recent years. Mueller et al. (1988) and Mols (1996) demonstrated the relationship between earwig density in trees and the extermination of woolly apple aphid (WAA) colonies. In recent years, fruit growers in the Netherlands and Belgium have observed a decline in earwig numbers in their orchards. Although no systematic counts of earwig densities have been done, growers often state that they hardly find dead earwigs when opening their cold storage chambers after having stored their harvest. The earwigs are brought into the chambers with the harvested fruits. In the past, thousands of dead earwigs could be found near doors of storage chambers, having tried to escape the low oxygen regime inside.

31 32

The earwig overwinters as an adult, mostly in subterranean nests. Females oviposit in the nests in early spring and show parental care for the eggs and young nymphs. The nymphs usually remain in the nest with the female until after the first moult. During the nesting phase, the female, and later her nymphs, may forage outside at night, but always spend the day inside the nest. From the second instar onwards, during the so-called free foraging phase, nymphs do not return to the nests, but they forage and shelter in the trees instead. Some females will produce a second brood after raising the first (Lamb and Wellington, 1975). The presented work is part of a survey in apple and pear orchards carried out in 2004. The objectives of this survey were: • to determine the relative numbers of earwigs in commercial orchards; • to investigate the relationship between the numbers of earwigs and insect pest densities as seen in small scale experiments; • to identify factors in the orchard management that affect earwig numbers. In this paper we focus on the effect of earwigs on the WAA infestation.

Material and methods

Earwig sampling Earwig samples were taken in commercial apple and pear orchards in the Netherlands and Belgium. Two types of earwig traps were used. Shelter traps made from corrugated cardboard (after Phillips 1981) were placed in 51 IPM apple orchards and in 42 organic apple orchards. The traps consisted of 50 by 5 cm strips of corrugated cardboard which were rolled up and attached to the tree branches. In each orchard 20 rolls were used. A further 32 IPM apple orchards were sampled using black polyethylene bags filled with straw. Five straw bags per orchard were used. Both types of traps are easily accepted by the earwigs as a shelter during the day time. The traps were left in the trees for at least 10 days. After this period the traps were collected and the earwigs were counted. As earwigs often need some years to colonise young orchards, only orchards that were at least 5 years old were selected. To minimize the effect of the surrounding vegetation, traps were applied at a minimum distance of 20 m from the orchard border. Trapping took place between mid-June and mid-August, when the majority of the earwig population in orchards is dwelling in the tree crown (Helsen et al., 1998).

WAA sampling The WAA density was determined in all orchards where cardboard rolls were used as earwig traps. On the same day the earwigs were counted, the WAA density was measured on those 20 trees that had an earwig trap. A score from 0 to 8 was given to each tree, 0 signifying no WAA infestation and 8 signifying a severe infestation (Stäubli and Chapuis, 1987)

Questionnaire In a questionnaire, the growers gave their opinion on the severity of WAA infestation and the need to control it in their orchard. The growers of 83 IPM orchards were asked to assign their orchard to one of the following groups: 1) no WAA; 2) some WAA, no control needed; 3) moderate WAA, control sometimes needed; 4) severe WAA infestation, control necessary at least once a year. These groups were related to the earwig density. In this analysis the earwig catches in cardboard traps and in straw bags were pooled. Before pooling, the catches in straw bags were corrected with a factor 0.54 (= the average number of earwigs in cardboard traps divided by the average number of earwigs in straw bags in all IPM orchards in the survey). The questionnaire also included questions on characteristics and management of the orchard, 33

such as soil type, soil organic matter, soil drainage, weed control, and the use of some pesticides during the previous five years.

Results

In the organic orchards (N=42) an average of 7.1 (± 1.3 SE) earwigs per cardboard roll was found, while the IPM orchards (N=51) had only 1.5 (± 0.5 SE) earwigs per cardboard trap. Plastic straw bags had on average 2.5 (± 1.2 SE ) earwigs in 32 IPM orchards. Especially in the few orchards with high earwig densities the straw bags accommodated more earwigs than the much smaller cardboard traps. Figure 1 shows the frequency distribution of earwig numbers per cardboard trap in organic and IPM orchards. Orchards are grouped into 5 classes, based on the average number of earwigs per trap: 0; 0.1-1.0; 1.1-5.0; 5.1-15; >15 earwigs per trap. In 33% of the IPM orchards no earwigs were found in the traps, compared to 12% of the organic orchards. The other eye-catching difference can be seen in the fraction with the highest earwig density: 14% of the organic orchards had more than 15 earwigs per trap, compared to only 2% of the IPM orchards.

40 Organic IPM

% of orchards 10

0 0 0,1-1,0 1,1-5,0 5,1-15,0 > 15 number of earwigs per cardboard trap

Figure 1. Frequency distribution of average earwig nubers in cardboard traps in organic (N=42) and IPM (N=51) apple orchards.

Most of the orchards had at least some WAA infestation: In 79% of the organic orchards and 88% of the IPM orchards WAA could be found on the inspected trees. A strong negative correlation was found between the number of earwigs and the level of WAA infestation in an orchard (Figure 2). In orchards with more than 6 earwigs per trap, the WAA infestation never exceeded an infestation level 3, with WAA only present in low densities (< 5 colonies) on the stem or on the older branches. In orchards with low earwig numbers WAA infestation often reached high and harmful levels, although a large variation in WAA densities was observed. Figure 3 shows the relationship between the earwig density and the level of WAA infestation according to the growers in 83 IPM orchards. It demonstrates that the relationship from Figure 2 is also reflected in the opinion and the spraying behaviour of the growers. Low numbers of earwigs are associated with moderate or severe WAA infestation and a necessity of chemical control. In orchards with more than 5 earwigs per trap none of the growers considered it necessary to treat against WAA. 34

8 7 organic IPM 6 5 4 3 2

woolly aphid infestation aphid woolly 1 0 0 5 10 15 20 25 30 35 number of earwigs per trap

Figure 2. Relationship between number of earwigs per trap and relative infestation level of woolly apple aphid in organic orchards (N=42) and IPM orchards (N=51).

100 no WAA 80 some moderate 60 severe 40 20 % of orchards 0 0 0,1-1,0 1,1-5,0 >5.0 number of earwigs per trap

Figure 3. Percentage of orchards with no, some, moderate or severe WAA infestation as indicated by the fruit growers, in relation to the average number of earwigs per trap in IPM orchards (N=83).

Discussion and conclusions

This survey clearly demonstrates that earwigs play a major role in controlling WAA in commercial apple orchards. Severe WAA infestations were always associated with low densities of earwigs. This relationship was strongly reflected in the opinion and the spraying behaviour of the growers. IPM growers did not need to control WAA chemically in orchards with a relatively high earwig density. Organic orchards had on average more earwigs than IPM orchards. Several authors have shown the adverse effects of chemical pesticides on earwigs (Ravensberg, 1981,; Nicholas and Thwaite 2003; Lahusen et al., 2006) and it is likely that these effects play a role in the IPM orchards in this survey. But pesticides and fertilisers of natural origin, as used in organic orchards, might also have such adverse effects. 35

Some other factors seem to affect the earwig densities as well. On soils that in the opinion of the growers had bad drainage, earwigs were absent (data not shown here). This corresponds with the observations of some growers that earwigs are absent on wet spots in the orchard, locally resulting in higher WAA infestation. The influence of drainage might be explained by the inability of earwigs to make their nests or reproduce in a water-saturated soil during winter and early spring. In many organic orchards weed is controlled mechanically, theoretically resulting in a disturbance of the nests. In these orchards earwig numbers were slightly lower than in orchards where no tillage was carried out under the trees. We have to conclude that variation in earwig densities between orchards is high and could not always be explained by management or characteristics of the orchard. For many pesticides and fertilisers used in the IPM system, but also in organic orchards, the side-effects on earwigs are unknown so far. For a better use of natural control agents in apple growing it is of high importance that these side-effects will be systematically investigated.

References

Helsen, H., Blommers, L. & Vaal, F. 1998: Phenology of the common earwig Forficula auricularia L. (Dermaptera: Forficulidae) in an apple orchard. – International Journal of Pest Management 44(2): 75-79. Lahusen, A., Hoehn, H. & Gasser, S. 2006: Der Birnenblattsauger und ein in Vergessenheit geratener Gegenspieler. – Schweiz. Z. Obst-Weinbau 2006: 10-14. Lamb, R.J. & Wellington, W.G. 1975: Life history and population characteristics of the European earwig, Forficula auricularia (Dermaptera: Forficulidae), at Vancouver, British Columbia. – Canadian Entomologist 107: 819-824. Mols, P.J.M. 1996: Do natural enemies control woolly apple aphid? – IOBC/WPRS Bulletin 19 (4): 203-207. Mueller, T.F., Blommers, L.H.M. & Mols, P.J.M. 1988: Earwig (Forficula auricularia) predation on the woolly apple aphid, Eriosoma lanigerum. – Entomologia Experimentalis et Applicata 47: 145-152. Nicholas, A.H. & Thwaite, W.G. 2003: Toxicity of chemicals commonly used in Australian apple orchards to the European earwig Forficula auricularia L. (Dermaptera: Forficulidae). – General Applied Entomolgy 32: 9-12. Nicholas, A.H., Spooner-Hart, R.N. & Vickers, R.A. 2005: Abundance and natural control of the woolly aphid Eriosoma lanigerum in an Australian apple orchard IPM program. – Biocontrol 50: 271-291. Phillips, M.L. 1981: The ecology of the common earwig Forficula auricularia in apple orchards. – PhD thesis, University of Bristol: 246 pp. Ravensberg, W. 1981: The natural enemies of the woolly apple aphid Eriosoma lanigerum (Homoptera: Aphididae), and their susceptibility to diflubenzuron. – Mededelingen van de Faculteit der Landbouwwetenschappen Rijksuniversiteit Gent 46/2: 437-441. Stäubli, A. & Chapuis, Ph. 1987: Problèmes posés par le puceron lanigère, Eriosoma lanigerum Hausm., dans le contexte de la protection intégrée des vergers de pommiers. – Revue suisse Vitic. Arboric. Hortic. 19 (6): 339-347 36 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 37-42

Biology and management of woolly apple aphid, Eriosoma lanigerum (Hausmann), in Washington state

E. H. Beers1, S. D. Cockfield1, G. Fazio2 1 Tree Fruit Research & Extension Center, Washington State University, 1100 N. Western Ave., Wenatchee, Washington 98801 USA. [email protected] 2 USDA-ARS, Plant Genetic Resources Unit, Cornell University, 630 W. North Street, Geneva, NewYork 14456 USA. [email protected]

Abstract: Woolly apple aphid, Eriosoma lanigerum (Hausmann) (Homoptera: Aphididae) has become a more severe pest in Washington apple production in the past few years. Milder winters have promoted overwintering survival on the aerial parts of the tree. A very low percentage of the current apple acreage is planted on resistant rootstocks, nor are such rootstocks used for new plantings. The transition from organophosphate insecticides to either insect growth regulators or neonicotinyl insecticides may also be contributing to higher pressure. In addition, this pest became one of quarantine concern in 2006. Alternatives to organophosphate pesticides have been tested for several years. Of these, petroleum oil shows some promise, as does a particle film used for sunburn protection. A neem-based insecticide provided temporary suppression, as did several neonicotinyl insecticides. A second approach to management, that of controlling the root colonies, was explored for the first time in this region. In potted tree assays, several compounds including imidacloprid, spirotetramat and oxamyl showed good root and systemic activity; in field trials, however, results were more variable. A greenhouse test of 8 clonally propagated rootstocks and 2 seedling rootstocks demonstrated that several of the new Geneva rootstocks to have virtual immunity to a Washington strain of woolly apple aphid, whereas the older Malling-Merton rootstocks had a lesser degree of antixenosis.

Key words: woolly apple aphid, host plant resistance, selective insecticides

Introduction

Woolly apple aphid, Eriosoma lanigerum (Hausmann) (Homoptera: Aphididae), is a pest of apple world wide. It is native to eastern North America, where it used American elm (Ulmus americanum L.) as the primary host. This pest was spread to other growing regions throughout the globe beginning as early as the mid-1700s, before quarantine measures were conceived, let alone implemented. Early American entomologists originally imputed this species to the Old World, until the association with American elm was found, identifying it as a New World species (Patch, 1912). Its reputation for devitalizing trees earned it the nickname of “American Blight” by European entomologists. The case of woolly apple aphid and its primary parasitoid, Aphelinus mali Haldeman, is one of the notable early examples of classical biological control (DeBach ,1964; Asante and Danthanaryana, 1992). A. mali is also native to eastern North America. Parasitized aphids were shipped to many of the countries around the world where woolly apple aphid had become established. In most cases, establishment of A. mali was also successful, although the degree of control it exerted was more variable. Woolly apple aphid has been classed as a serious pest in Washington since the industry began in the late 1800s. According to early writers, the introduction of A. mali in 1931

37 38

effectively demoted its status to a minor pest (Yothers, 1947). Early efforts at biological control with A. mali were considered a success, and the occasional outbreak treated with nicotine compounds. DDT disrupted biological control, and problems became more severe after World War II. The transition to the organophosphates (primarily for codling moth), probably provided a significant degree of suppression of woolly apple aphid. The current transition away from organophosphate insecticides may be part of the reason for the increasing incidence and severity of woolly apple aphid populations in Washington. In addition, milder winters may have allowed more survival in the aerial parts of the trees, and more rapid establishment in the post-dormant period. While the increase in woolly apple aphid incidence would be a minor concern to pest management programs, its status as a quarantine concern has elevated it to a more serious level. In February of 2006, China stopped shipments from two Washington packing houses because of the presence of woolly apple aphids in either the stem or calyx end of the apples. The interest in controlling this pest rose proportionately. Woolly apple aphid control in Washington is currently a combination of chemical and biological control. The reasons for success or failure of biological control are not well understood; however, more recent work in Washington (Walker, 1985) indicated that the predator complex may play a greater role than previously thought. Resistant rootstocks played somewhat of a role in the past, but the Malling-Merton 100 series are no longer planted. Control has been aimed exclusively at the aerial colonies; the reservoir on the roots, both its role in perpetuating populations and its effect on the trees, has been largely ignored.

Material and methods

Phenological studies The phenology of 1st instar (crawlers) of woolly apple aphid migration was studied in 2005 and 2006. Commercial orchard blocks were selected on the basis of a previous history of infestation and the use of a selective insecticide program. Crawler movement was studied using two sticky bands on the main tree trunk. The lower band was about 15 cm above the soil surface and trapped the crawlers moving upward from root colonies. The upper band was about 1 cm above the lower band, and trapped crawlers moving downward. However, since both bands interfered with normal establishment of colonies in the aerial portions of the tree, presumably the data on downward movement is less representative than that of upward movement. Bands were constructed of a 3-cm strip of aluminum foil held in place with spray- on adhesive. A thin bead of Tree Tanglefoot was applied to the center of the band. Bands were checked ca. weekly from bloom until frost by removing them from the trees, placing them in plastic bags, and counting the crawlers under a stereomicroscope.

Rootstock evaluations Apple rootstock liners, 6-10 mm diameter, were planted in a soil mixture of equal parts peat, pearlite, and vermiculite on 21 April. Ten rootstock types were used: The Geneva line 4210, Geneva 41, Geneva 202, Bud (Budagovsky) 9, Bud 118, M.9 (Malling 9), M.26, MM.111 (Malling-Merton 111), seedlings from Washington (Willow Drive Nursery), and seedlings from New York. Ten replicates of of each rootstock were planted. Trees had approximately 6 cm of new shoot growth before infestation. Insects were collected from a commercial orchard in East Wenatchee, WA. Stem sections 4-6 cm long, each with 50-200 aphids, were placed at the base of each tree on 19 May. First instars were observed on the trees the following day. Fresh stem sections were collected on 22 May and placed on any trees that appeared to have a low number of first instars. This included 39

all of Geneva 41, Geneva 202, 4210, and about half of the other trees. Trees were arranged on a greenhouse bench in a randomized complete block design (10 rootstocks × 10 replicates). Aphids had matured by 8 June and had begun to produce new first instars Trees were evaluated on 16 June using a numeric rating system on a scale of 0 to 4, where 0=no infestation, 1=very few (1-2) small colonies; 2=few (3-8), small colonies; 3=moderate number of normal-sized colonies; 4=large, coalesced colonies. The rating was done by unaided visual inspection of the whole tree.

Insecticide tests, aerial colonies Airblast tests were conducted in commercials orchards in central Washington. Plots consisted of 5-10 trees in single rows, with buffer rows separating treated rows. Three woolly apple aphid shoot colonies per plot were tagged. Trees were sprayed with an airblast sprayer at 100- 200 gallons per acre. Live and parasitized aphids were counted in the tagged colonies pre- and post-treatment until densities in the check were low. Handgun tests were conducted in a similar manner to the airblast tests, except the plot size was smaller (1-2 trees), and trees were sprayed to drip with a handgun sprayer operated at 200 psi. Data were analyzed using the Statistical Analysis System (SAS 1988). Data were tested prior to analysis for homogeneity of variance using Levene’s (1960) test. Variances found to be non-homogeneous were transformed [ln(y+0.5)] before analysis. PROC GLM was used to conduct an analysis of variance, and treatment means were separated using the Waller- Duncan k-ratio t-test.

Potted tree bioassays 2005 Bioassays. Two bioassays were conducted in a greenhouse on potted apple trees (September and December). Seedling apple trees (12 mm diam) were planted in 15-cm diam pots. Soil was gathered from a commercial orchard near Quincy, WA. Uncultivated soil was taken from the side of block. The soil was primarily sand and silt (Shano series [mixed, mesic Xerollic Camborthids]). Trees were potted in 1.5 liters of one part soil and one part pearlite. After trees had grown shoots approximately 15 cm long, twigs from infested trees were placed on the branches. After a few weeks, aphids were well established and had formed shoot colonies. At that time about 0.5 liters of soil in the pots was removed to expose part of the roots to the new mobile aphids. Crawlers settled on the roots by crawling down from shoots or from infested twigs. Twenty-four trees were selected for each bioassay. Woolly apple aphid were counted on all shoots. All exposed aphids on the roots were counted, then the missing soil mixture was replaced. Trees were distributed into four replicate blocks based on the population of woolly apple aphid on the roots, then treatments were randomly assigned within each block. Spirotetramat was applied to the foliage (2 times, 2 wk apart) to run-off with a 3.78-liter sprayer. Water for this treatment was first acidified to a pH of 6-7 with a few drops/liter of 1N HCl. All other products were applied as soil drenches. Trees were fully watered three days before application, then 250 ml of insecticide solution was poured onto the soil. This volume completely saturated the soil in the pots. Starting three days after treatment, trees were watered regularly, but minimal water was lost through the drainage holes of the pots. Shoot colonies were assessed periodically for 4 wk after the second application, then all trees were lifted, the soil gently washed from the roots, and root colonies assessed. 2006 Bioassay: This bioassay was done as described above except trees were potted in a mixture of equal parts peat, pearlite, and vermiculite.

Insecticide tests, edaphic colonies Three commercial orchard blocks in central Washington were chosen for a previous history of woolly apple aphid infestation. One block (PR) had made a post-harvest treatment for aphids in the fall of 2005, and had no aerial colonies in May of 2006. However, viable aphid colonies were found regularly on root suckers. A second orchard (MV) consisted of ca. 25-yr-old ‘Fuji’ apples, and had a large population of aphids overwintering on the aerial parts of the tree. The third block (BT) had had heavy infestation of aphids for the past several years, but no visible colonies at the time of application. Treatments were applied in mid-May with a boom sprayer calibrated to deliver ca. 100 gallons per acre. Insecticides were applied to the herbicide-treated strip under the trees in a band ca. 1.2 m wide on either side of the trunk. While all orchards received regular herbicide applications, the degree of weed infestation in the herbicide strip varied among the blocks. The day before application, the trees were given a full irrigation set (ca. 12 h) and allowed to drain overnight. After the insecticides were applied, irrigation water was turned on for about 45 min to help move the insecticides into the soil profile. Woolly apple aphid populations crawler movement was evaluated using a single sticky band (described previously). Based on previous experience, the single band was likely most indicative of upward crawler movement, thus providing a measure of control of the root colonies. Three trees per plot were banded. Crawlers were counted and fresh bands were applied at 2-3 wk intervals from May through August. In addition, numbers of woolly apple aphid aerial colonies were assessed with a 3-min count at intervals throughout the season. The timed search spanned the entire plot, omitting the banded trees.

Results and discussion

Phenological studies Numbers of upward-moving crawlers was higher than downward movement in most of the orchards studied. In 2005 (when only one orchard was studied), migration began in early May, peaked in early June, and returned to low levels by mid-July. In 2006 (three orchards studied), the peak of upward crawler movement was shifted later in the season by 4-6 wk, peaking in late June to mid-July. The timing of downward movement of crawlers was roughly the same as upward movement, but about 75% few individuals were caught. However, as mentioned previously, the interference of the bands in the establishment of aerial colonies could be partly responsible for the weaker downward movement.

Rootstock evaluation Differences among the different rootstocks in degree of woolly apple aphid infestation were apparent within a few week of artificial infestation. After 4 wk, the susceptible rootstocks (including M.9, M.26, Bud 9, Bud 118, and seedlings from New York and Washington) were heavily infested. Colonies had coalesced on the worst trees, producing copious amount of woolly filaments. On MM.111, the ‘Northern Spy’ derived resistant rootstock, colonies were small and poorly developed, but woolly apple aphids had established successfully. On the Geneva ‘Robusta 5’ derived resistant rootstocks (G.202, G41, and 4210), the majority of the replicates were free from infestation; on the remainder, a few colonies, usually consisting of a very limited number of aphids, had established. This experiment demonstrated that the East Wenatchee strain of woolly apple aphids used in this test has not overcome the ‘Northern Spy’-based resistance, as has been noted in other areas of the world (Gilliomee et al., 1968; Rock and Zeiger, 1974; Sen Gupta and Miles, 1975). This is likely a moot point since it is probable that only a small percentage of 41

Washington’s apple acreage is planted on a resistant rootstock. Both of the most popular rootstocks in the Malling-Merton 100 series (MM.111 and MM.106) have been discarded for more dwarfing and productive rootstocks (e.g., M.9 and M.26). Some of the Geneva series of rootstocks offer a higher level of woolly apple aphid resistance than the Malling-Merton series, and may also have more desirable horticultural characteristics. A highly resistant rootstock may greatly facilitate management of this pest, and possibly improve the likelihood of biological control.

Insecticide tests, aerial colonies Of the materials tested to date, diazinon has provided the most consistent level of control. Endosulfan has also been quite effective, while the systemic material dimethoate is less so. All of these materials (two of which are organophosphates) are older compounds, and their continued registration on tree fruits is in question. The increase in woolly apple problems, coupled with possible withdrawal of effective materials, makes the search for replacement compounds more urgent. As a class, the neonicotinyls are not as active on woolly apple aphid as they are on other tree fruit aphid species. Thiamethoxam, imidacloprid and acetamiprid have been tested as foliar sprays; and although they provide a degree of suppression, they do not give control at the same level as the organophosphates. Azadirachtin (neem) provided temporary suppression, but populations rebounded in a few weeks. Of the non-registered materials tested, flonicamid and NNI-0101 showed promise for control of woolly apple aphid. Petroleum oils continue to show promise as a lower-cost, low-impact alternative for woolly apple aphid. However, they also only provide suppression. Best results will likely be obtained with higher rates and higher spray volumes, and possibly repeated applications.

Potted tree bioassays 2005 Bioassays. All treatments, whether applied foliarly (spirotetramat) or as a drench (imicloprid and oxamyl) provided good control of the shoot colonies. Spirotetramat and imidacloprid tended to be a little slower than oxamyl in reducing shoot colonies. At the end of the experiment, all treatments also provided excellent control of root colonies. The unique feature of spirotetramat is its bidirectional translocation within the plant, thus root colonies may be control with foliar sprays. 2006 Bioassay. This bioassay provided more extensive testing of spirotetramat. The material was tested at two rates and with two different adjuvants (methylated seed oil and an organosilicone). An additional material, dinotefuran (a neonictinyl) was also tested as a soil application, with imidacloprid as a standard. All materials provided excellent control of shoot colonies, although the organosilicone adjuvant appeared to provided somewhat faster control.

Insecticide tests, edaphic colonies Imidacloprid provided the most consistent suppression of crawler movement and mid-summer establishment of aerial colonies. These results echo those of Pringle (1998), who saw several years residual effect from a single imidacloprid application. Oxamyl, which looked equivalent to imidacloprid in potted tree bioassays, failed to show significant control in field trials. A May application of spirotetramat did not affect crawler movement, however, it did suppress aerial colonies in mid-summer. Dinotefuran gave a lesser degree of control. Two experimental treatments, NNP-731 and NNP-732, gave an intermediate degree of control. Two non- insecticidal treatments (sticky bands and insulation foam banded around the trunk) provided no reduction in the numbers of aerial colonies. In several instances, the information from the bands did not correspond well to the degree of shoot infestation. 42

Acknowledgements The authors gratefully acknowledge the Washington Tree Fruit Research Commission for funding of these studies. Funding was also provided by Bayer CropScience, Cerexagri, DuPont, Gowan, and Nichino America.

References

Asante, S.K. & Danthanaryana, W. 1992: Development of Aphelinus mali, an endoparasitoid of woolly apple aphid, Eriosoma lanigerum, at different temperatures. – Entom. Exp. Appl. 65: 31-37. DeBach, P. 1964: Biological control of insect pests and weeds. – Reinhold Pub., N.Y. Gilliomee, J.H., Strydom, D.K. & Van Zyl, H.J. 1968: Northern spy, Merton and Malling- Merton root-stocks susceptible to woolly aphid, Eriosoma lanigerum, in the western Cape. – S. Afr. J. Agric. Sci. 11: 183-186. Patch, E.M. 1912: Elm leaf curl and woolly apple aphid. – Bull. 203, Maine Agricultural Experiment Station, Orono, ME. Pringle, K.L. 1998: The use of imidacloprid as a soil treatment fo the control of Eriosoma lnaigerum (Hausmann) (Hemiptera: Aphididae). – Journal of South African Society for Horticultural Science 8: 55-56. Rock, G.C. & Zeiger, D.C. 1974: Woolly apple aphid infests Malling and Malling-Merton rootstocks in propagation beds in North Carolina. – J. Econ. Entomol. 67: 137-138. Sen Gupta, G.C. & Miles, P.W. 1975: Studies on the susceptibility of varieties of apple to the feeding of two strains of woolly aphis (Homoptera) in relation to the chemical content of the tissues of the host. – Aus. J. Agric. Res. 26: 157-168. Walker, J.T.S. 1985: The influence of temperature and natural enemies on population development of woolly apple aphid, Eriosoma lanigerum (Hausmann). – Ph.D. Thesis, Washington State University, Pullman. Yothers, M.A. 1947: DDT and the woolly apple aphid parasite Aphelinus mali. – J. Econ. Entomol. 40: 934. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 43-50

Effects of exclusion or supplementary honey feeding of the common black ant, Lasius niger (L.), on aphid populations and natural enemies on apple

Csaba Nagy1, Viktor Markó2, Jerry Cross1 1 East Malling Research, New Road, East Malling, Kent ME19 6BJ UK, [email protected] 2 Corvinus University of Budapest, 1118-Budapest, Ménesi u. 44, Hungary, [email protected]

Abstract: Two replicated experiments were conducted in an unsprayed apple orchard (cv Discovery) at East Malling Research in 2006 to evaluate the effects of the common black ant, Lasius niger (L.), on populations of rosy apple aphid, Dysaphis plantaginea (Passerini), and green apple aphid, Aphis pomi DeGeer. Ants were either excluded from trees by a sticky barrier band round the base of the trunk (experiment 1) or provided with honey baits at the base of the trunk or in the canopy (experiment 2). Trees where ants had free access and trees without artificial baits were provided for experimental controls. Exclusion of ants resulted in increased populations of predators (Coccinellidae adults and larvae, predatory Heteroptera, Syrphidae larvae, Dermaptera, Neuroptera larvae and Araneae) and rapid decreases in the populations of both aphid species. In comparison, populations of both aphids increased rapidly on control trees where ants had not been excluded and where predator populations were lower. Thus, exclusion of ants greatly reduced crop damage due to both aphid species. Provision of artificial baits, either at the base or in the canopy of the trees, also caused reductions in D. plantaginea numbers and their tending ants, but the effects were weaker. The influence on A. pomi and their tending ants was not remarkable, and effects on predators were unclear. On trees where no aphids were present or where aphid numbers were small, ants fed on the artificial baits allowing aphid colonies, where present, to increase in size. On trees with numerous aphids, ants showed a preference for feeding in aphid colonies and visited the artificial baits in smaller numbers having limited impact on aphid populations. The implications of these results for management of aphid and other pest populations in apple orchards are discussed.

Key words: Lasius niger, Dysaphis plantaginea, Aphis pomi, predators, exclusion, supplementary feeding

Introduction

Mutualism between honeydew-producing homopterans and ants is a well-known phenomenon (Way, 1963; Stadler and Dixon, 1999). Ants benefit from this mutualism by getting honeydew from aphids. The honeydew is a complex mixture of sugars, free amino-acids, amides, proteins, minerals and B-vitamins, composing the main part of the food of the Lasius genus (Way, 1963). Aphids benefit from this association by many ways. El-Ziady (1960) showed that Lasius niger (L.) has direct effect on Aphis fabae Seop. by tranquilizing what enhanced the aphid`s rates of feeding, assimilation, growth and reproduction. Anyway it caused a delay in alate production and dispersal as well (El-Ziady, 1960; Skinner, 1983). Sanitary behaviour by attendant ants reduces aphid mortality by decreasing the number of aphids drowning or

43 44

becoming immobilized in honeydew (Nixon, 1951; Skinner, 1983). In the absence of attending ants, the accumulation of honeydew raised leaf rot and encouraged the growth of various sooty moulds (Capnodiaceae) (Hill and Blackmore, 1980; Skinner, 1983) and fungi of the genus Entomophthora (Skinner, 1983). El-Ziady and Kennedy (1956) and Banks (1958) described denser, cleaner and generally more healthy A. fabae colonies tended by L. niger. Additionally, the honeydew accumulations can be attractive to aphid predators (Carter and Dixon, 1984; Sutherland et al., 2001; Choi et al., 2004) and aphid parasitoids (Budenberg, 1990). Furthermore tending ants have a direct effect on aphid colonies by protecting them from predators (El-Ziady and Kennedy, 1956; Banks, 1962; Addicott, 1979; Tilles and Wood, 1982; Skinner, 1983; Buckley, 1987; Stadler and Dixon, 1999; Yao, 2000; Kaneko, 2003) and aphid parasitoids (Völkl, 1992; Müller et al., 1999). On one hand by protection from predators, and on the other hand by the possibility of direct transportation of aphids (Collins and Leather, 2002), dispersal of aphids can be quicker by the effect of the ants. In contrast, aphids can suffer some costs when attended by ants such as prolonged development times, smaller body size, delayed offspring production, proportionally smaller gonads and fewer well developed embryos (Stadler and Dixon, 1998 and 1999; Yao et al., 2000). Additionally, some parasitoids specialized in parasitizing aphids attended by ants (Völkl, 1992; Stadler and Dixon, 1999; Völkl and Mackauer, 2000; Kaneko, 2003). Furthermore, Bird et al. (2004) reported L. niger can be a vector of the entomopathogen fungus Lecanicillium longisporum (Zimmerman) to Dysaphis plantagynea. Finally, the ants also prey on their homopteran partners (Cherix, 1987; Rosengren and Sungstrom, 1991; Sakata, 1994, 1995; Offenberg, 2001). The rosy apple aphid, Dysaphis plantaginea (Passerini) and the green apple aphid, Aphis pomi DeGeer are two important aphid pests of apple in Europe. These two aphid species are commonly attended by the common black ant, Lasius niger (L.) (Skinner, 1983). The aim of this study was to examine the effects of exclusion and supplementary honey feeding of the L. niger on aphid and their predator populations.

Material and methods

Experiment 1: Ant exclusion Forty randomly chosen apple trees of cultivar Discovery were used in an unsprayed apple orchard at East Malling Research (EMR), East Malling, Kent, UK. From twenty trees the ants were excluded (EXCL) by a band of cloth tape, while the other twenty trees were left untreated controls (CONT). Cloth tape barriers were applied to the bases of the tree trunks on 18th of April 2006. The abundances of aphids, ants and predators were assessed about weekly for 5 weeks from 12 May until 9 June 2006. Statistical procedure of the data was performed with Ministat 3.3 using Fligner-Policello test with Welch-like degree of freedom (FPW-test) (Delaney and Vargha, 2002). In case of abundance of ants, statistical difference from 0 was tested with sign test.

Experiment 2: Supplementary feeding Forty randomly chosen Discovery apple trees were used in the same unsprayed apple orchard at EMR. Thirteen of them were treated with honey sources at the trunk (FTRUNK), the sources were applied in the canopy (FCANOPY) in case of thirteen other trees. The remaining fourteen trees were controls (CONT) without honey sources. Twenty baits were applied on every trees with about 0,5 ml. of honey in each of them. 45

The baits were applied to the trees on 25th of April 2006. They were checked 2 times per week and refilled if some of them were empty of honey. The abundances of aphids, ants and predators were assessed approximately weekly for 7 weeks from 19 May until 1 July 2006. The data were statistically elaborated using a Tukey-Kramer pairwise comparison of rank means after testing the hypothesis of stochastic homogeneity (with James test on ranks).

Results

Experiment 1: Ant exclusion Exclusion of ants resulted in a rapid slow down in the increase of the populations of D. plantaginea. During the experiment, the abundance of D. plantaginea increased 64-fold relative to the initial numbers on the CONT trees but by just a bit more than 3-fold on the EXCL trees (Fig. 1). Meanwhile, the number of the ants tending D. plantaginea colonies on CONT trees rose in the first three weeks of the experiment, but this increase stopped in the last week. During the experiment the number of tending ants increased 7.6-fold relative to the initial number on the CONT trees, so their increase was less than of the proportional increase in aphid numbers. The initial number of A. pomi was 7 times greater on the randomly chosen EXCL trees, but this difference decreased during the experiment. At the end of the experiment, the number was almost 25 times greater on the CONT trees (Fig. 2). The number of ants tending A. pomi colonies increased throughout the sampling period. At the end of the experiment, the number of A. pomi was 6 times greater (Fig. 2) while the number of ants tending A. pomi colonies was 15 times greater than it was at the beginning. The total number of predators (Coccinellidae adults and larvae, predatory Heteroptera, Syrphidae larvae, Dermaptera, Neuroptera larvae and Araneae) was significantly greater on the EXCL trees at the start of the experiment, but later the number equalised. By the end of the experiment, predator numbers were numerically greater on the CONT trees, but this difference was not significant (Fig. 3). CONT EXCL 1600 A

1280 /tree 960 A 640 D. plantaginea 320 A A AAAA B B 0 12.05. 17.05. 23.05. 31.05. 09.06.

Figure 1. The effect of ant exclusion on the abundance of D. plantaginea (control trees: CONT; ant excluded trees: EXCL; A-A: n.s.; A-B: p<0,01) 46

15 CONT EXCL

12 B

9 A tree

6 A A. pomi/ A A A

3 A A A B 0 12.05. 17.05. 23.05. 31.05. 09.06.

Figure 2. The effect of ant exclusion on the abundance of A. pomi (control trees: CONT; ant excluded trees: EXCL; A-A: n.s.; A-B: p<0.05)

7.5 CONT EXCL

6 A

4.5

B A 3

predators/tree A A A 1.5 B A A A 0 12.05. 17.05. 23.05. 31.05. 09.06.

Figure 3. The effect of ant exclusion on the abundance of predators on the canopy of apple trees (control trees: CONT; ant excluded trees: EXCL; A-A: n.s.; A-B: p<0.05)

Experiment 2: Supplementary feeding Provision of artificial bates, either at the base or in the canopy of the trees, caused reductions in the numbers of D. plantaginea (Fig. 4) and their tending ants (Fig. 5), but the effects were weaker than in the case of exclusion. There was no significant difference between the ants tending D. plantaginea on FTRUNK and FCANOPY trees (Fig.5). The influence of ant feeding on A. pomi and on their tending ants was not remarkable. The abundance of A. pomi was similar on the FCANOPY and CONT trees, but there were numerically less A. pomi on FTRUNK trees (Fig. 6). Numbers of the tending ants followed the numbers of A. pomi. Their numbers were similar on the CONT and FCANOPY trees, but there were numerically less tending ants on FTRUNK trees (Fig. 7). There were no significant differences of total predator (Coccinellidae adults and larvae, predatory Heteroptera, Syrphidae larvae, Dermaptera, Neuroptera larvae, Aphidoletes aphidimyza larvae, Cantharidae and Araneae) numbers between the treatments, but in the 47

middle of June the predator number was numerically higher on CONT trees. Their abundances followed the aphid numbers.

CONT FCANOPY FTRUNK 2000 A A a 1600 a A /tree 1200 A a a A a 800 AB B AB A A b b b A b A B D. plantaginea a B b AB 400 A A b A b b A A A b a a b a a a 0 19.05. 25.05. 02.06. 08.06. 15.06. 22.06. 29.06.

Figure 4. The effect of ant feeding on the abundance of D. plantaginea (control trees: CONT; trees where ants were fed at the trunk: FTRUNK; trees where ants fed in the canopy: FCANOPY; A-A, B-B, a-a, b-b: n.s.; A-B: p<0.05; a-b: p<0.10)

CONT FCANOPY FTRUNK 35 A A a a

/tree 28

A A 21 a A a a A D. plantagineaD. A 14 a a B A b b A AB A AB B A B b B b 7 B a b B B b B a b b ants tending tending ants b b b b 0 19.05. 25.05. 02.06. 08.06. 15.06. 22.06. 29.06.

Figure 5. The effect of ant feeding on the abundance of ants (L. niger) tending D. plantaginea colonies (control trees: CONT; trees where ants fed at the trunk: FTRUNK; trees where ants fed in the canopy: FCANOPY; A-A, B-B, a-a, b-b: n.s.; A-B: p<0.05; a-b: p<0.10)

Discussion

Exclusion of ants resulted in rapid decreases in the populations of both aphid species compared to control trees, but increased the populations of predatory insects and spiders. In contrast, populations of both aphid species increased rapidly on control trees where ants had not been excluded and where predator populations were smaller at the beginning. These results are partly similar to the results of some previous experiments (Skinner, 1983; Stewart- Jones et al., in prep.). 48

The effect of the supplementary feeding on D. plantaginea is clear. The extra food (honey) reduced the number of ants tending the D. plantaginea colonies probably giving a chance for predators to destroy the aphid colonies. Reduction of aphid tending by ants after honey feeding was shown in a laboratory experiment (Offenberg, 2001), and our study suggests this phenomenon to be present in field conditions also. The effect on A. pomi is not so clear, but the FTRUNK treatment seems to have a similar effect to exclusion, but much weaker. For clear results more experiments are necessary. The honey is probably not as attractive and useful food for L. niger as aphid honeydew and probably there are some components in honeydew which are important or essential for the ants. Further examination of these components is important.

CONT FCANOPY FTRUNK A 450 a

360 A ab 270 /tree A A b A 180 a A. pomi a A a A A 90 a A A a A A A A A A A A A A A a a a a a a a a a a a a a 0 19.05. 25.05. 02.06. 08.06. 15.06. 22.06. 29.06.

Figure 6. The effect of ant feeding on the abundance of A. pomi (control trees: CONT; trees where ants fed at the trunk: FTRUNK; trees where ants fed in the canopy: FCANOPY; A-A, a-a, b-b: n.s.; a-b: p<0.10)

CONT FCANOPY FTRUNK 10

A 8 a AB /tree ab 6 A A. pomi A. a A 4 a A a B A A a b ants tending tending ants a 2 A A A A A A A A A A a A A a a A a a a a a a a a a a 0 19.05. 25.05. 02.06. 08.06. 15.06. 22.06. 29.06.

Figure 7. The effect of ant feeding on the abundance of ants (L. niger) tending A. pomi colonies (control trees: CONT; trees where ants fed at the trunk: FTRUNK; trees where ants fed in the canopy: FCANOPY; A-A, B-B, a-a, b-b: n.s.; A-B: p<0.05; a- b: p<0.10) 49

Both the D. plantaginea and A. pomi colonies benefit from the presence of ants, so their populations may conversely affect each other not only through the food plant, but also through the ants and predators. Insecticide treatments could reduce numbers of ants, so they may also influence aphid numbers indirectly through the ants and aphidophagous predators. Further research is needed to describe the role of aphid tending in the population increase of D. plantaginea and A. pomi and to work out methods which, through reducing the abundance of ants on the aphid colonies, would enhance the efficacy of aphidophagous predators in apple orchards.

Acknowledgements

This study was funded by the UK Department of Environment Food and Rural Affairs. Viktor Marko’s work was partly supported by grant OTKA 46380. We thank Chelsea Eby, Dr. Michelle Fountain, Lia McKinnon and Péter Sipos for their help in field work.

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The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in UK pome and stone fruit orchards

Michelle T. Fountain, Jerry V. Cross East Malling Research, Kent, ME19 6BJ, UK, [email protected]

Abstract: The light brown apple moth (LBAM), Epiphyas postvittana (Walker), is one the most important pests of tree fruits in Australia and New Zealand. It was unknown in Europe until 1936 when it was found breeding on ornamental spindle at Newquay, Cornwall, England. It established on a wide variety of plants in Devon and Cornwall and spread east but was not known to attack fruit crops. A pheromone trap survey in 10 commercial apple orchards in Kent, Hereford, Essex, Oxfordshire and Somerset (England) in 1994 revealed no LBAM. However, in 2005, a severe and extensive attack of the pest occurred in a commercial cherry orchard at Yalding, Kent, causing considerable fruit losses. Larvae were reared to adult and identification confirmed. It is suspected that other reported attacks on cherry in 2003 and 2004, believed to be caused by summer fruit tortrix moth, Adoxophyes orana Fischer von Rösslerstamm, at the time, may well have been caused by LBAM as the larvae are difficult to distinguish. Specialist lepidopterists report that the LBAM has spread throughout England where it has become common. In recent years, the pest has become a significant pest problem in hardy ornamental nursery stock throughout England. In 2006 a pheromone trap survey aimed at determining the flight dynamics and occurrence of E. postvittana on commercial fruit trees (apple, pear, plum and cherry) in England was established, so that pesticide applications might be used more effectively. The growers (with 13 x apple, 10 x pear, 13 x plum and 12 x cherry orchards) in the counties of Buckinghamshire, Essex, Gloucestershire, Hereford, Kent, Oxfordshire, Somerset, Suffolk, Surrey and Sussex recorded the number of male moths caught in traps each week from May to the end of September. In the UK E. postvittana has been reported to have two generations. Here we report the occurrence of three generations of the moth in 2006. Traps in a cherry orchard at East Malling Research showed that the peak flights were the end of May, the middle of September, and towards the end of October in this year. The greatest number of moths were found in cherry (compared to apple, pears and plums), probably because cherry crops are seldom sprayed with insecticides post blossom. Adjacent habitats (crops or natural) may also be involved in the incidence of LBAM on fruit crops.

Key words: light brown apple moth, Epiphyas postvittana, pest monitoring, pheromone trap survey

Introduction

The tortricid, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), commonly known as the light brown apple moth (LBAM), is one of the most important pests of apple and other tree fruits in the countries that it occurs. A native of Australia, the moth has been introduced into New Zealand (where it is now the dominant leafroller in some districts (Shaw et al.; 1994)), New Caledonia, Hawaii and the British Isles (Geier and Briese, 1981). The moth is of great economical importance to Australian and New Zealand pome fruit exports to northern America and Japan, as consignments of fruit are rejected if they contain LBAM. Research into the control of the spread of LBAM have included treatment of the crop postharvest to extremes of temperature (e.g. Whiting et al., 1999; Smith and Lay-Yee, 2000) and the

51 52

addition of substances such as alkanes (e.g. Taverner et al., 1999), ethanol vapour (e.g. Jamieson et al., 2003) and sodium bicarbonate (Lewthwaite et al., 1999). The moth was unknown in Europe until 1936 where it was found breeding on ornamental spindle established in Devon and Cornwall (England). It then spread east, but was not known to attack fruit crops at that time. A pheromone trap survey in 10 commercial orchards in Kent, Hereford, Essex, Oxfordshire and Somerset (England) in 1994 revealed no LBAM populations at this time (Cross, 1996). However, in 2005, a severe and extensive attack of the pest occurred in a commercial cherry orchard at Yalding in Kent, causing considerable fruit losses. Larvae were reared to adult and identification confirmed as LBAM. It is suspected that other reported attacks on cherry in 2003 and 2004, believed to be caused by the summer fruit tortrix moth, Adoxophyes orana, may well have been caused by LBAM as the larvae are difficult to distinguish. In Australia the moth can have 4-5 generations per year. Specialist lepidopterists in the UK report that the LBAM has spread throughout England where it has become common and in recent years and become a significant pest problem in hardy ornamental nursery stock throughout England. The moth is able to adapt different strategies for different local climates (phenotypic plasticity) enabling it to transfer to cooler climates (Gu & Danthanarayana, 2000). The identification of LBAM larvae is problematical as they are very similar to the larvae of other leaf rollers. In the adults the forewing is characteristically curved. Male moths are 6- 10 mm long, with the anterior part of the forewing generally much lighter than the posterior, which is rusty dark red/brown (Figure 1, Bradley et al., 1973). Much lighter forms may also be found. The females are larger than the males (7-13 mm long) and are more difficult to identify as colour varies from a uniform light yellowish brown with almost no distinguishing marks (www.hortnet.co.nz). However, they do have a small dark spot over centre of body, on the forewings when at rest (Figure 2).

Figure 1. Male light brown apple moth Figure 2. Female light brown apple moth reared from cherries. reared from cherries.

Females usually mate with one male depending on the number of females a male has mated with before (Foster and Ayres, 1996), the female then shutting down the production of the sex pheromone (Foster, 1993; Foster and Roelofs, 1994). Males, on average, mate with 6.6 females in a lifetime (Foster and Ayres, 1996). The sex pheromone titre is highest in 2 day old females and the greatest proportion of copulations occurs in 3 day old females (Danthanarayana and Gu, 1991). Likewise, the greatest response to female pheromone was recorded in 3 day old males (Foster et al., 1995). The maximum distance that the LBAM flies is estimated to be around 600 m although 100 m was a more typical flight distance in 24 hours. Moths were found to fly at air temperatures of 10 – 30°C with the longest flight 53

duration at 20°C for both sexes. Flight duration is also affected by humidity and weather conditions, such as, wind (Danthanarayana and Gu, 1992). Epiphyas postvittana is highly polyphagous and although the moth is believed to have evolved as a feeder on herbaceous plants (Danthanarayana et al., 1995), the species is known to feed on over 120 dicotyledonous plant species (e.g. poplar, willow, alder, clover, gorse, broom, dock, plantain (Venette et al., 2003; Suckling et al., 1998)), including various fruits (e.g. apples, pears, apricots, kiwifruit, cirus fruits, grapevines and cherries). The moth is also a pest on cut flowers (Karunaratne et al., 1997) and in 2005–2006, on a hop plot (Kent, England), an average of 1.2% of the 12.5% of hop cones showing caterpillar feeding damage, was caused by E. postvittana (pers comm. C. Campbell). Female moths are stimulated to oviposit in the presence of a foodplant (Foster and Howard, 1999a) by plant volatiles, such as, hexanal, linalool, nonanol, octanol and nonanal (Suckling et al., 1996). Tactile cues are a strong driver of oviposition (Foster and Howard, 1998) with females prefering to lay eggs on smooth rather than rough surfaces (Foster et al., 1997). Larvae react to chemicals cues and colour from fruit and leaves, actively moving towards favoured foodplants before ‘spinning down’ and beginning to feed (Suckling and Ioriatti, 1996; Harris et al., 1999; Foster and Howard, 1999b). Damage to the foliage by early feeding larval instars is caused by feeding on the leaf mesophyll under silken webs (Harris et al., 1995). Later larvae construct feeding niches between adjacent leaves and/or fruit (Lo et al., 2000), in the developing bud, or on a single leaf (leaf rolling). Late stages feed on all leaf tissue except for the main veins. The fruit suffers from superficial damage particulary in compact cluster apple varieties. Larvae may spin silk, binding leaves to the fruit. Internal damage to fruit is less common, but a young larva may enter fruit through the calyx of pome fruit (Van der Geest and Evenhuis, 1991). The moth can also complete its lifecycle on oranges but the larval survival rate is very low (<20%) compared to non-citrus host plants (Mo et al., 2006a). Larvae developed faster on young compared to older citrus leaves (Mo et al., 2006a). In experiments examining the distribution of larvae on fruits there was a high mortality in the first instar stage and most of the surviving larva were found in the calyx, followed by the stem and the cheek of apples. The 3rd and 5th instars also prefered the calyx and stem cavity and by day 7 both instars could be found in the core of the fruits (Whiting et al., 1997). Fruit cultivars resistant to LBAM attack are being investigated (pears (Chervin, et al., 2000), raspberries (Wilde et al., 1991)). On 38 apple cultivars only three varieties showed reduced survival of LBAM larvae (Nevis 1, A40R04T119 and Sir Prize) compared to other varieties (Wearing et al., 2003). Royal Gala and Liberty varieties seem to be more prone to attack than Jonafree and Fiesta apple varieties (Wearing, 1998). Epiphyas postvittana has several natural enemies (MacLellan, 1973), which include parasitoids (Danthanarayana, 1980a; 1980b; Munro, 1998; Suckling et al., 2001; Rundle and Hoffman, 2003; Paull and Austin, 2006) and predators (Harris, 1994). Some studies have shown that manipulating the floral under story of crops can increase parasitism of LBAM larvae (Begum et al., 2006; Berndt et al., 2006; Irvin et al., 2006). Other studies have demonstrated that the commercial release of parasitoids can be beneficial (Glenn and Hoffman, 1997; Glenn et al., 1997). Bacillus thuringiensis (Bt) has been used to control LBAM although some populations have become resistant (Harris et al., 2006) and there is some evidence of avoidance of Bt contaminated food by the larvae of the moth (Bailey et al., 1996). LBAM populations are also vulnerable to the E. postvittana nucleopolyhedro virus (EppoNPV) and have a LT50 (lethal time of 50% of individuals) of 11 days in third instar larvae (Markwick et al., 2002). The LBAM sex pheromone released by the females is a mixture of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadianyl acetate (Bellas and Bartell, 1983; Newcomb et al., 54

2002). This pheromone is not only used for monitoring purposes, but has been successfully applied in mating disruption trials in fruit orchards (e.g. Suckling and Clearwater, 1990; Suckling and Shaw, 1992; Suckling and Shaw, 1995; Suckling and Angerilli, 1996; Mo et al., 2006b) and trials with combinations of insecticides and mating disruption (McLaren et al., 1998; Nicholas et al., 1999) aimed at reducing pesticide usage in fruit crops (Suckling and Shaw, 1995). No resistance to pyrethroids has been observed. However, resistance to pesticides such as azinphos-methyl has occurred in LBAM (Armstrong and Suckling, 1988; Suckling et al., 1989). Azinphos-methyl resistant LBAM were also cross-resistant to phosmet, chlorpyrifos and carbaryl (Suckling and Khoo, 1990). It is suspected that the moth may be a particular problem on cherry but not other tree fruits in the UK because insecticides that are active against caterpillar pests are not used on cherry in the UK. Currently, no insecticides, other than Bt, pirimicarb and thiocloprid are approved for use post blossom on cherry in the UK. The work presented here are the results of a 2006 pheromone trap survey of commercial tree fruit orchards (apple, pear, plum and cherry) in England. It was anticipated that the survey would provide information on relative abundance in different fruit crops of the LBAM and the flight dynamic through the growing season. Here we present the flight dynamics of the population at East Malling Research (Kent, England) and the number of moths caught on each crop type in English orchards.

Material and methods

Traps were deployed by growers in May 2006 on 48 orchards on 17 farms (with 13 x apple, 10 x pear, 13 x plum and 12 x cherry orchards) in the counties of Buckinghamshire, Essex, Gloucestershire, Hereford, Kent, Oxfordshire, Somerset, Suffolk, Surrey and Sussex. At each farm one trap was placed in the centre of each crop type where available, monitored weekly by the growers until end of September and the pheromone lures changed every 6 weeks as per manufacturers’ instructions (Agrisense BSC Ltd). At East Malling two traps were placed in mixed variety cherry orchards, one in a net covered plot and one in an uncovered plot. The numbers of moths caught per week were recorded.

Results

The data suggests that the peak flight for the first generation in 2006 was at the end of May in cherry (Figure 3). There appeared to be two second generation flight peaks at East Malling, the first in mid August and the second in mid September. However, on closer inspection it can be seen that there was a rise in the temperature at the end of August causing the increase in male moth numbers at this time. At 20 oC the development time from egg to adult can take around 55 days and is longer at reduced temperatures. It is likely that the 4th flight peak in October is a third generation (from adults which mated in August). This is more generations than previously reported for the UK and it is possible that increasing average annual temperatures associated with climate change may increase the number of generations per year in the region. The lower temperature threshold for the moth to develop is 7-7.5 oC (Van der Geest, and Evenhuis, 1991), so it unlikely, at present, that the moth will be capable of continued development throughout the winter in the UK. It appears that the LBAM is more abundant on cherry crops compared to apple, pear and plum (Figure 4). However, there is a high variation between crop type and farms and reasons for this effect need to be investigated further.

30 7 day mean temp Uncovered crop

C 25 Covered crop o

Number of moths/ temperature 5

0 04-Jul 11-Jul 18-Jul 25-Jul 06-Jun 13-Jun 20-Jun 27-Jun 03-Oct 10-Oct 17-Oct 24-Oct 01-Aug 08-Aug 15-Aug 22-Aug 29-Aug 05-Sep 12-Sep 19-Sep 26-Sep 30-May

Figure 3. Number of male moths caught weekly (solid lines) in pheromone traps in cherry orchards (covered crop - ▲, uncovered crop – ●). The mean air temperature of the 7 days prior to the recording of male moth numbers (■ dashed lines) is also shown for each data point.

160

140

120

100

40 Mean number of moths trapped moths of number Mean

0 Apple Plum Pear Cherry

Figure 4. Mean number of light brown apple moth caught in pheromone traps on different fruit crops in England. Data from April – August 2006.

Conclusions

• The LBAM in England had three peak flights in 2006 compared to the 2 flights previously reported and the 4-5 generations in Australia and New Zealand. • LBAM is abundant on cherry. However, the variation between sites is high and more work is needed to identify the causes of this variation, such as, surrounding topography, vegetation and spray regimes on each plot. It is possible that pesticide spray records may reveal that cherry was more susceptable due to the differences in spray applications compared to the other pome and stone fruit crops.

Acknowledgements

We would like to thank the Horticultural Development Council for funding this study and Peter Shaw for his help in monitoring the traps at East Malling at the beginning of the survey.

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Jamieson, L E., Meier, X., Smith, K.J., Lewthwaite, S.E., & Dentener, P.R. 2003: Effect of ethanol vapour treatments on lightbrown apple moth larval mortality and 'Braeburn' apple fruit characterization. – Postharvest Biology and Technology 28: 391-403. Karunaratne, C., Moore, G.A., Jones, R., & Ryan, R. 1997: Phosphine and its effect on some common insects in cut flowers. – Postharvest Biology and Technology 10: 255-262. Lewthwaite, S.E., Dentener, P.R., & Connolly, P.G. 1999: Mortality of Epiphyas postvittana (Lepidoptera: Tortricidae) after exposure to sodium bicarbonate at elevated temperatures or combined with emulsifiers. – New Zealand Journal of Crop and Horticultural Science 27: 83-90. Lo, P. L., Suckling, D.M., Bradley, S.J., Walker, J.T.S., Shaw, P.W., & Burnip, G.M. 2000: Factors affecting feeding site preferences of lightbrown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae), on apple trees in New Zealand. – New Zealand Journal of Crop and Horticultural Science 28: 235-243. MacLellan, C.R. 1973: Natural enemies of light brown apple moth, Epiphyas postvittana, in Australian Capital Territory. – Canadian Entomologist 105: 681-700. Markwick, N., Graves, S., Fairbairn, F.M., Docherty, L.C., Poulton, J., & Ward, V.K. 2002: Infectivity of Epiphyas postvittana nucleopolyhedrovirus for New Zealand leafrollers. – Biological Control 25: 207-214. McLaren, G.F., Fraser, J.A., & Suckling, D.M. 1998: Mating disruption for the control of leaf rollers on apricots. – New Zealand Journal of Crop and Horticultural Science 26: 259- 268. Mo, J.H., Glover, M., Munro, S. & Beattie, G.A.C. 2006a: Development of Epiphyas postvittana (Lepidoptera: Tortricidae) on leaves and fruit of orange trees. – Journal of Economic Entomology 99: 1321-1326. Mo, J.H., Glover, M., Munro, S., & Beattie, G.A.C. 2006b: Evaluation of mating disruption for control of lightbrown apple moth (Lepidoptera: Tortricidae) in citrus. – Journal of Economic Entomology 99: 421-426. Munro, V.M.W. 1998: A retrospective analysis of the establishment and dispersal of the introduced Australian parasitoids Xanthopimpla rhopaloceros (Krieger) (Hymenoptera: Ichneumonidae) and Trigonospila brevifacies (Hardy) (Diptera: Tachinidae) within new Zealand. – Biocontrol Science and Technology 8: 559-571. Newcomb, R.D., Sirey, T.M., Rassam, M., & Greenwood, D.R. 2002: Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus. – Insect Biochemistry and Molecular Biology 32: 1543-1554. Nicholas, A.H., Thwaite, W.G., & Spooner-Hart, R.N. 1999: Arthropod abundance in an Australian apple orchard under mating disruption and supplementary insecticide treatments for codling moth, Cydia pomonello (L.) (Lepidoptera: Tortricidae). – Australian Journal of Entomology 38: 23-29. Paull, C., & Austin, A.D. 2006: The hymenopteran parasitoids of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) in Australia. – Australian Journal of Entomology 45: 142-156. Rundle, B.J., & Hoffmann, A.A. 2003: Overwintering of Trichogramma funiculatum Carver (Hymenoptera: Trichogrammatidae) under semi-natural conditions. – Environmental Entomology 32: 290-298. Shaw, P.W., Cruickshank, V.M., & Suckling, D.M. 1994: Geographic changes in leafroller species composition in Nelson orchards. – New Zealand Journal of Zoology 21: 289-294. Smith, K.J., & Lay-Yee, M. 2000. Response of 'Royal Gala' apples to hot water treatment for insect control. – Postharvest Biology and Technology 19: 111-122. 59

Suckling, D.M., Burnip, G.M., Gibb, A.R., Daly, J.M., & Armstrong, K.F. 2001: Plant and host effects on the leafroller parasitoid Dolichogenidia tasmanica. – Entomologia Experimentalis et Applicata 100: 253-260. Suckling, D.M., & Angerilli, N.P.D. 1996: Point source distribution affects pheromone spike frequency and communication disruption of Epiphyas postvittana (Lepidoptera: Tortricidae). – Environmental Entomology 25: 101-108. Suckling, D.M., & Clearwater, J.R. 1990: Small-scale trials of mating disruption of Epiphyas postvittana (Lepidoptera, Tortricidae). – Environmental Entomology 19: 1702-1709. Suckling, D.M., & Ioriatti, C. 1996: Behavioural responses of leafroller larvae to apple leaves and fruit. – Entomologia Experimentalis et Applicata 81: 97-103. Suckling, D.M., & Khoo, J.G.I. 1990: Cross resistance in the lightbrown apple moth Epiphyas postvittana (Lepidoptera, Tortricidae). – New Zealand Journal of Crop and Horticultural Science 18: 173-180. Suckling, D.M., & Shaw, P.W. 1992: Conditions that favour mating disruption of Epiphyas postvittana (Lepidoptera, Tortricidae). – Environmental Entomology 21: 949-956. Suckling, D.M., & Shaw, P.W. 1995: Large-scale trials of mating disruption of lightbrown apple moth in Nelson, New-Zealand. – New Zealand Journal of Crop and Horticultural Science 23: 127-137. Suckling, D.M., Burnip, G.M., Walker, J.T.S., Shaw, P.W., McLaren, G.F., Howard, C.R., Lo, P., White, V., & Fraser, J. 1998: Abundance of leaf rollers and their parasitoids on selected host plants in New Zealand. – New Zealand Journal of Crop and Horticultural Science 26: 193-203. Suckling, D.M., Karg, G., Gibb, A.R., & Bradley, S.J. 1996: Electroantennogram and oviposition responses of Epiphyas postvittana (Lepidoptera: Tortricidae) to plant volatiles. – New Zealand Journal of Crop and Horticultural Science 24: 323-333. Suckling, D.M., Khoo, J., & Rogers, D.J. 1989: Dynamics of azinphosmethyl resistance in Epiphyas postvittana (Lepidoptera, Tortricidae). – Journal of Economic Entomology 82: 1003-1010. Taverner, P., Bailey, P., Hodgkinson, M., & Beattie, G.A.C. 1999: Post-harvest disinfestation of lightbrown apple moth, Epiphyas postvittana Walker (Lepidoptera: Tortricidae), with an alkane. – Pesticide Science 55: 1159-1166. Van der Geest, L.P.S, & Evenhuis, H.H. (eds.) 1991. World Crop Pests; Tortricid pests their biology, natural enemies and control. – Elsevier, Amsterdam. Venette, R.C., Davis, E.E., DaCosta, M., Heisler, H., & Larson, M. 2003: Mini risk assessment light brown apple moth, Epiphyas postvittana (Walker) [Lepidoptera: Tortricidae]. – Department of Entomology, University of MinnesotaSt. Paul, MN 55108 CAPS PRA: Epiphyas postvittana 1: 1-38. Wearing, C.H. 1998: Bioassays for measuring ovipositional and larval preferences of leafrollers (Lepidoptera: Tortricidae) for different cultivars of apple. – New Zealand Journal of Crop and Horticultural Science 26: 269-278. Wearing, C.H., Colhoun, K., Attfield, B., Marshall, R.R., & McLaren, G.F. 2003: Screening for resistance in apple cultivars to lightbrown apple moth, Epiphyas postvittana, and greenheaded leafroller, Planotortrix octo, and its relationship to field damage. – Entomologia Experimentalis et Applicata 109: 39-53. Whiting, D.C., Jamieson, L.E., & Connolly, P.G. 1999: Effect of sublethal tebufenozide applications on the mortality responses of Epiphyas postvittana (Lepidoptera: Tortric- idae) larvae exposed to a high-temperature controlled atmosphere. – Journal of Economic Entomology 92: 445-452. 60

Whiting, D.C., O’Connor, G.M., & Maindonald, J.H. 1997: Density and time effects on distribution and survival of lightbrown apple moth (Lepidoptera: Tortricidae) larvae on Granny Smith apples. – Environmental Entomology 26: 277-284. Wilde, G., Hall, H., & Thomas, W. 1991: Resistance to raspberry bud moth (Lepidoptera, Carposinidae) in raspberry cultivars. – Journal of Economic Entomology 84: 247-250. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 61-66

Ecology and management of the obliquebanded leafroller, Choristoneura rosaceana, in New York apple orchards

H. Reissig, M. Sarvary, J. Nyrop Department of Entomology, New York State Agricultural Experiment Station, Geneva, NY, 14456 USA, [email protected]

Abstract: During the last 20 years, the obliquebanded leafroller (OBLR), Choristoneura rosaceana Harris (Lepidoptera: Tortricidae) has caused more economic damage of apples in New York than any other insect pest. Outbreaks of this pest occur in localized areas and persist in the same locations year after year. OBLR are resistant to organophosphates, carbamates, pyrethroids, and methoxyfenozide. Tests of reduced-risk insecticide programs without organophosphates, synthetic pyrethroids, or carbamates were set up in small plots, 2.5 ha in commercial apple orchards throughout NY. These studies showed that prophylactic sprays of methoxyfenozide at petal fall for overwintering larvae followed by summer sprays of spinosad for the summer generation provided excellent control when these programs were applied in the same plots for 3 consecutive years. During the last year of this 4- year study in 2005, monitoring programs were initiated in all blocks for overwintering and summer generations of larvae to determine if sprays were needed. Levels of overwintering OBLR larvae were very low in all of the monitored plots and no control sprays were recommended at petal fall for control of this generation. However, sampling during the summer showed that levels of infestation of terminals by the subsequent summer generation of larvae exceeded the recommended action threshold of 3% in most of the plots. Studies were also conducted to compare the effects of parasitoids and predators in unpsrayed hedgerows, plots treated with the reduced-risk insecticide programs for multiple seasons and growers’ orchards treated with standard insecticides, using implanted larvae on various types of mobile media. These studies showed that the total parasitism and predation levels were similar throughout the season in all three locations. The overall results of these studies shown that although OBLR can be effectively managed by reduced-risk insecticides there is no evidence that seasonal programs of these materials enhances opportunities for conservation biological control. Orchards, at least relatively small areas can be re-infested by the summer generaion of OBLR and continuous use of insecticides may be necessary to insure adequate control. Future research should be conducted to test area-wide or whole farm management of OBLR, and improvement of current sampling protocols to determine if this pest can be managed in problem blocks without applying propylactic applications of insecticides.

Key words: Choristoneura rosaceana, reduced-risk insecticides, parasitoids, predators, biological control

Introduction

The obliquebanded leafroller (OBLR), Choristoneura rosaceana Harris (Lepidoptera: Tortricidae) has been a serious pest in NY apple orchards since the early 1970’s. During the last 30 years, OBLR has caused more economic damage of apples in NY than any other insect pest. Outbreaks of this pest occur in localized areas throughout apple production regions of NY and elsewhere (100-2,000 ha). OBLR problems persist in the same orchards and in the same localized areas year after year, regardless of how many insecticide sprays are applied. Field populations of OBLR in NY are resistant to organophosphates, carbamates, synthetic pyrethroids, and methoxyfenozide.

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Research has been conducted in NY to develop a management program with selective insecticides that will provide stable control of OBLR at low levels for multiple seasons. It was originally assumed that the long term use of selective materials would facilitate the buildup of indigenous natural enemies for conservation biological control. The long term goal of our research is to develop an integrated management system that would allow stable control of OBLR by natural enemies in commercial orchards without the use of special leafroller sprays.

Material and methods

Seasonal programs of reduced-risk insecticides Reduced risk insecticide programs were tested in 17 orchards throughout all major apple growing regions throughout NY State. Insecticides were tested in 2.5 ha plots. One-half of each plot was treated with pheromone ties, Isomate CTT for codling moth or Isomate M-100 for oriental fruit moth. Throughout the season, reduced risk insecticides were applied to the entire plot. Insect and mite populations in each of these reduced risk plots were compared to those in nearby growers’ standard plots treated with conventional insecticide programs. All sprays were applied by the growers. No broad spectrum insecticides such as organophosphates, synthetic pyrethroids, and carbamates, except carbaryl applied for thinning fruit were used in the research plots. The following selective pesticides were applied in the research plots. Clofentezine, hexythiazox or prebloom petroleum sprays were used for early season control of mites, followed by pyridaben in the summer if necessary. At pink and/or petal fall, thiamethoxam, or indoxacarb were used to control the plum curculio, rosy apple aphid, spotted tentiform leafminer, tarnished plant bug, and European apple sawfly. At petal fall through the first cover spray, methoxyfenozide, and Bacillus thuringiensis were used against overwintering OBLR and indoxacarb and acetamiprid were used against codling moth. From June-July, imidacloprid was used if needed to control aphids and mating disruption was set out in half of the plots for the second and subsequent broods of codling moth, oriental fruit moth, and lesser appleworm. Spinosad was applied against the summer generation of OBLR and apple maggot and indoxacarb, and acetamiprid was applied against oriental fruit moth and apple maggot during July and August. A program of Strobilurin fungicides was applied early in the season and no post bloom Ethylene Bis-dithiocarbamates were used to avoid potential negative effects against predator mites.

Reduced-risk insecticides for control of OBLR During the first three years of the study from 2002-2004, prophylactic sprays were recommended for control of both the overwintering and summer generations of OBLR larvae in all research plots because most of the blocks had previously had severe infestations of this pest. Tebufenozide or methohxyfenozide was applied at petal fall for control of overwintering larvae. Spinosad was applied at the estimated first hatch of eggs of the summer generation of larvae (usually during the first week in July), followed by another spray 7-10 days later. At the end of the first 3 years of the study, fruit damage was very low in all of the research plots (< 1.5%). Therefore, monitoring programs were initiated in all of the research plots for overwintering and summer generations of larvae to determine if special control sprays were necessary. During bloom, 2000 flowering clusters were sampled for overwintering larvae, and sprays were not recommended unless 60 live larvae were observed. During the summer, 1000 actively growing terminals were sampled for the summer generation of larvae, and sprays were not recommended unless terminal infestations averaged 3% or greater. 63

Comparison of natural enemies in unsprayed hedgerows, reduced risk insecticide plots, and grower’s standard orchards Tests were set up in 2003 and 2004 in five commercial orchards to compare populations of natural enemies of OBLR in three types of habitats: unsprayed hedgerows bordering commercial apple orchards, 2.5ha plots in grower’s orchards that had been treated with seasonal programs of reduced risk insecticides during 2002-2004, and nearby growers orchards treated with standard insecticides. OBLR larvae that were similar in size to natural populations of larvae during the summer were allowed to establish feeding sites on unsprayed apple leaves in various types of media (small potted trees, leaves placed in waterpicks, and a branch placed in a PVC pipe filled with water). Fifty-100 larvae were set out in each habitat three times during the summer. These larvae were re-collected after 48 hours. Then, they were brought into the laboratory and reared individually on artificial diet in small plastic cups so that the incidence of parasitism could be recorded. Predation of these larvae in the field was estimated by adjusting the recorded loss of larvae by a predetermined estimate of dispersal loss from the various types of media in the absence of natural enemies.

Results and discussion

Control of OBLR with reduced risk insecticides OBLR fruit damage at harvest in all of the plots treated with reduced risk (RAMP) insecticides declined during each consecutive season from 2002-2004 when prophylactic insecticide treatments were applied against both generations, and averaged less than 1.5% during the 2004 growing season (Figure 1). However, when the prophylactic treatments of Methoxyfenozide were eliminated during the beginning of the 2005 growing season, the average damage in the reduced risk (RAMP) plots increased to 3.5%.

Obliquebanded Leafroller Damage - NY 4

3.5 RAMP Comparison

3 Eliminated Intrepid for 2.5 OW brood

1.5 Mean % Damage 1

0.5

0 2002 2003 2004 2005

Figure 1. Comparison of OBLR fruit damage at harvest in RAMP (Reduced Risk) plots and grower’s comparison plots treated with conventional insecticides in NY apple orchards from 2002-2005. 64

As expected during the start of the 2005 growing season, populations of overwintering OBLR larvae were very low in all of the reduced risk insecticide plots. Only 1 larva was found in 3 of the 17 orchard sites/2000 sampled fruit clusters, and no larvae were detected in the other sites (Table 1). Since the treatment action threshold for insecticide treatment of overwintering larvae is 3% infested flower clusters, 60 live larvae would have been necessary in each monitored orchard to initiate a spray. Consequently, no treatment was recommended in any of the 17 monitored orchards. However, when the orchards were sampled again in July to monitor subsequent populations of the summer generation of larvae, infestation levels exceeded the recommended treatment threshold for summer larvae of 3% infested terminals in 12 of the 17 research orchards (Table 1). Apparently, these relatively high levels of summer generation larvae resulted from OBLR invasion of the test plots by gravid females immigrating from nearby external sources of infestation, probably from neighboring commercial orchards in which control of overwintering larvae was inadequate.

Table 1. Populations of overwintering and summer generation larvae in individual orchard plots treated with reduced-risk insecticides in 2005

Orchard B B A S C R O L B F D T C F H K W P S A R B # OW 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 larvae1 % Inf. 13 16 0 5 7 4 5 3 7 9 0 0 1 7 0 6 18 Term.2 1 2000 fruit clusters/plot were sampled during bloom; OW = overwintering OBLR larvae. 2 1000 actively growing terminals were sampled during July and considered infested if live larvae were found; Inf. Term. = Infested Terminals.

Table 2. Comparison of OBLR fruit damage at harvest (average % OBLR damaged) in individual orchards treated with reduced risk insecticides plots (RR) and grower’s standard plots (Std.), 2005.

Orchard Plot R O L B B F D T B A C F H K S W C P S A R B RR 11 2 1 2 2 1 5 1 3 3 3 2 6 3 <1 3 5 Std. 5 1 <1 <1 1 <1 10 4 4 32 <1 1 1 2 0 0 <1

The overall average levels of fruit damage were actually slightly lower in the Reduced Risk plots than in the growers’ standard orchards (Figure 1). However, this average in the standard plots was somewhat skewed by the high damage that occurred in one of the orchards (AA), which had 32% OBLR damage (Table 2). OBLR damage was higher in the reduced risk plots in 9 of the sites (76%) than in the corresponding grower’s standard orchards even 65

though sometimes the differences were small. Better control was obtained in the reduced risk plots at only 4 sites (24%). This reduced efficacy of control of damage from the summer generation of OBLR observed in 2005 was probably caused by delays in treatments by growers that occurred because sprays were not recommended until after blocks were sampled. This problem commonly occurs when treatment decisions for summer larvae are based on current sampling protocols because larval infestations can not be accurately estimated until substantial numbers of eggs from the summer generation have hatched.

Comparison of natural enemies in unsprayed hedgerows, reduced risk insecticide plots, and grower’s standard orchards Parasitism and predation levels in all three sites, hedgerows, reduced risk plots, and grower’s standard orchards were similar during both 2003 and 2004 (Figs 2,3). The estimated levels of predation on OBLR larvae were generally higher than those for parasitism, particularly during 2004. The effects of parasites and predators in this study were relatively low compared to estimated percentages of OBLR larvae attacked by natural enemies in previous trials, which sometimes reached levels of 40-60%, when larvae were placed in unsprayed apple orchards or isolated sites of a feral host, gray dogwood (Sarvary, Unpublished data).

Figure 2. Comparison of parasitism and predation of OBLR larvae in unsprayed hedgerows, educed risk insecticide plots, and standard grower’s orchards, 2003.

These results showed no evidence that the use of reduced risk insecticides for several consecutive seasons in these relatively small plots enhanced the effectiveness of natural enemies. It is difficult to explain why the levels of natural enemies in the unsprayed hedgerows were not higher than those in the commercial orchards that were regularly treated with insecticides. Perhaps the proximity of the hedgerows to relatively large areas of pesticide treated orchards combined with the mobility of natural enemies masked some the potential 66

differences among these treatments. Ideally, it would have been desirable to have set up an additional treatment in a feral area of gray dogwood plants that was isolated from any pesticide treatments during both years of this study to determine if levels of natural enemies were similar to those in the treatments and unsprayed hedgerows near the commercial orchards.

Figure 3. Comparison of parasitism and predation of OBLR larvae in unsprayed hedgerows, reduced risk insecticide plots, and standard grower’s orchards, 2004.

In conclusion, our recent studies have shown that OBLR can be effectively managed by using seasonal programs of reduced risk insecticides. However, seasonal treatments of these materials apparently did not enhance the effects of indigenous natural enemies. Our research also suggests that orchards, at least relatively small blocks of apple trees, can be re-infested by the summer generation of OBLR when gravid females move in from outside sources and therefore continuous use of insecticides may be necessary to insure adequate control in areas afflicted with chronic infestations of OBLR. In the future it may be desirable to investigate the effects of area wide or at least whole farm management programs for OBLR to determine if more stable management systems can be developed that will minimize insecticide inputs. It may also be necessary to develop improved monitoring or forecasting techniques for OBLR to determine when and if special sprays are necessary, particularly against the summer generation of larvae. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 67

Temperature effect on egg-laying timing in Cydia pomonella (L.)

Daniel Casado1,4, Peter Witzgall2, César Gemeno3,4, Jesús Avilla3,4, Magí Riba1,4 1 Universitat de Lleida, Department of Chemistry, Rovira Roure 191, 25198 – Lleida, Spain, [email protected] 2 Department of Crop Science, Swedish University of Agricultural Sciences, Box 44, 23053 – Alnarp, Sweden 3 Universitat de Lleida, Department of Crop and Forest Science, Rovira Roure 191, 25198 – Lleida, Spain, [email protected] 4 IRTA. Centre UdL-IRTA, Rovira Roure 191, 25198 – Lleida, Spain

Abstract: Temperature is an important factor in the modulation of the timing of insect rhythms, such as calling or egg-laying. We studied egg-laying timing of Cydia pomonella (L.) (Lepidoptera: Tortricidae), a key pest of pome fruits world wide, and how temperature affects this behavior. The results on attraction of C. pomonella females to host volatiles reached in laboratory bioassays have been usually poor, and our study aimed to determine better conditions to carry out these experiments. Semi-field as well as laboratory experiments were conducted. Semi-field assays were made in summer 2005 in Sweden and in Spain. In these assays, couples of C. pomonella adults were individually placed on glass tubes on an apple orchard. Mating activity and number of eggs laid were checked at different times of evening and night. In the laboratory, groups of 9 to 12 females and 10 to 15 males were placed in mating boxes, and kept at different constant temperatures, from 12 to 32ºC. The number of eggs laid was counted every hour during late-photophase and early-scotophase. In semi-field assays, egg-laying began when temperature fell below 25ºC and continued until temperature was below 21ºC. In the laboratory, a delay on egg-laying onset and peak was observed as temperature increased. At the highest temperatures (27 and 32ºC) a small percentage of the total number of eggs was laid before light off, and the number of eggs laid peaked during the first hour of the scotophase. On the other hand at mild temperatures (17 and 22ºC), egg-laying activity was more constant through the studied period, and for some groups it peaked several hours before light off. At 12ºC no egg-laying occurred.

Key words: Cydia pomonella, egg-laying, oviposition, timing, temperature

67 68 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 69-73

Biological interactions between the apple leaf curling midge, Dasineura mali (Kieffer), and its inquiline, Macrolabis mali Anfora

Gianfranco Anfora1, Nunzio Isidoro2, Claudio Ioriatti3 1 SafeCrop Centre, IASMA, Via E. Mach 1, 38010, San Michele all’Adige (TN), Italy, [email protected] 2 Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Via Brecce Bianche, 60131, Ancona, Italy, [email protected] 3 IASMA Research Centre, Plant Protection Department, Via E. Mach 1, 38010, San Michele all’Adige (TN), Italy, [email protected]

Abstract: The apple leaf curling midge, Dasineura mali (Kieffer) (Diptera Cecidomyiidae), damages apple trees by leaf galling. Recently, a new species, Macrolabis mali Anfora (Diptera: Cecidomyiidae), was described and hypothesized to be an inquiline of D. mali. We studied the biology of M. mali and its interaction with D. mali by a laboratory bioassay and field observations. This work confirmed that M. mali females lay eggs in young galls caused by their host insect and the larvae dwell as inquilines. The inquiline population increases during the summer; at the end of the season it becomes larger than that of the host insect and may cause the withering of larvae of the host insect. Therefore the presence of M. mali could contribute to regulation of the population of the apple leaf curling midge.

Key words: Cecidomyiidae, Diptera, gall midges, Malus domestica.

Introduction

The apple leaf curling midge, Dasineura mali (Kieffer) (Diptera: Cecidomyiidae), is widespread and damages cultivated apple trees. The larvae feed on fluids of tissue of young developing leaves and cause the edges to roll tightly inwards towards the central nervure. Leaves swell (reddish galls) and drop prematurely. D. mali control requires pesticide applications, especially in nurseries and young plantations (Antonin and Baggiolini, 1972). During studies on D. mali, another midge species emerged from apple leaf galls collected in Trento Province (Italy). This new species was described as Macrolabis mali Anfora (Diptera: Cecidomyiidae) (Anfora et al., 2005a). Following preliminary investigations, it was hypothesized that M. mali is unable to make galls and dwells as an inquiline in those of D. mali (Anfora et al., 2005b). Further studies on its biology were necessary aiming at clarification of the behaviour and biological role of the Macrolabis species. The inquiline egg-laying behaviour and development was investigated by a laboratory bioassay using plastic cages with potted apple trees. Field monitoring was carried out in order to understand biology and interactions between the populations of the two species.

Material and methods

Insect rearing Leaf galls infested with midge larvae were collected from organic Malus domestica Borkh. (Rosaceae, cv. Golden, Stark, Fuji) orchards in Trento Province in spring and summer 2005. To rear adult midges, some galls were placed in cubic plastic cages (length 45 cm) with a

69 70

3-cm layer of horticultural soil, where the larvae of both species could pupate. Cages were housed in a climatic chamber (26 ± 2°C, 60 ± 5% R.H., L16:D8 photoperiod).

Laboratory bioassay Potted apple plants (cv. Golden) presenting young growing leaves were isolated individually in cylindrical plastic cages (height 26 cm; diameter 12 cm). Six newly emerged pairs of D. mali adults were placed in each cage and allowed to lay eggs on the buds; after one day only plants with visible eggs were used (T0). Six newly emerged pairs of M. mali adults were placed in the cages at T0 and then, in different cages, every other two days after T0 (T0 + 2; T0 + 4; T0 + 6; T0 +8; T0 + 10), namely during the subsequent stages of gall growth (N=5). Five apple plants were caged only with M. mali adults as controls. Gall contents were examined at T0 + 15 days, before the pupation of both species. Insect behaviour in the cages was observed. The experiment was replicated three times. Numbers of galls with M. mali were compared across the intervals using a one-way ANOVA, followed by Duncan’s test for posthoc comparison of means. Before statistical analysis, data (%) were subjected to the Bliss angular transformation.

Field trials An established apple orchard (> 1ha.; cv. Golden) located at San Michele all’Adige (200 m a.s.l.; Trento, Italy) with a large population of D. mali and treated regularly with broad- spectrum insecticides was selected. Larval population monitoring was carried out during the whole midge flying season. Ten reddish and swollen leaf galls were collected weekly and the number of D. mali and M. mali larvae contained was counted and expressed as in percentages of each species. Twice a week the egg-laying D. mali and M. mali females were observed and counted in 100 growing shoots randomly chosen in the centre of the orchard.

Results

Table 1. Mean percentage ± standard deviation of M. mali larvae present in galls induced by D. mali and caged with the inquiline adults at different growth stages in the laboratory bioassay. Values followed by the same letter are not statistically different (Duncan’s test: P<0.05).

Days after D. mali Galls with egg-laying M. mali larvae (%) T0 0 a T0 + 2 9.1 ± 7.3 b T0 + 4 36.8 ± 12.0 c T0 + 6 47.6 ± 2.3 c T0 + 8 5.9 ± 0.9 b T0 + 10 0 a

Control 0 a

Laboratory bioassay About 80 % of the plants caged with D. mali adults showed eggs after 2 days and leaf galls after 2 weeks. In the plants caged with M. mali adults only, no galls were observed. 71

The first galls containing M. mali larvae were found when their females were released in the cages at T0 + 2 (9.1 ± 7.3 %). Plants used at T0 + 4 and T0 + 6 showed the highest percentages of galls containing M. mali larvae, 36.8 ± 12.0 and 47.6 ± 2.3 respectively, with no statistical differences between them; at these stages the leaf edges are not completely rolled inwards and swollen. M. mali females were observed to lay their eggs along the central nervure of the swelling leaves. At T0 + 8 days the infestation level decreased to 5.9 ± 0.9 %. No inquiline larvae were found in the galls when M. mali adults were released after 10 days of D. mali egg-laying (T0 + 10). Results are reported in Table 1. In a considerable number of galls only M. mali larvae were found.

Field trials We did not find M. mali larvae in galls inhabited by D. mali larvae at the beginning of the season. First inquiline larvae were recorded in May. The presence of M. mali larvae in galls increased over time reaching more than 90 % in the late season. Results are reported in Figure 1. In a considerable number of galls only M. mali larvae were found out, mainly at the end of the season. The first egg-laying by M. mali females corresponded with of the second D. mali flight (Figure 2). During the last flight the number of M. mali egg-laying females was higher than that of D. mali. The peak of the egg-laying inquiline females was shifted some days later than that of their host-insects.

100

60 larvae (%) larvae

40 M. mali

0 4-Jul 6-Jun 1-Aug 8-Aug 5-Sep 11-Jul 18-Jul 25-Jul 2-May 9-May 11-Apr 18-Apr 25-Apr 13-Jun 20-Jun 27-Jun 15-Aug 22-Aug 29-Aug 12-Sep 19-Sep 26-Sep 16-May 23-May 30-May

Figure 1. Mean percentage of M. mali larvae present in galls induced by D. mali, in an apple orchard located at San Michele all’Adige (Italy) during 2005. Vertical bars indicate the standard deviation.

12 D. mali M. mali

6 N° egg-laying females females egg-laying N° 4

r l g pr ay u u -J -Ap -A -M 4 8-Jul -Aug -Sep 1 9 6-Jun 0-Jun 1 1-A 2-Sep 1 25 23-May 2 15 29-Aug 1 26

Figure 2. Number of egg-laying D. mali and M. mali females observed in an orchard located at San Michele all’Adige (Italy) during 2005.

Discussion

Our experiments confirmed that M. mali is an inquiline species; its females laid eggs on apple trees with young galls induced by D. mali. The inquiline species seems to be able to occupy the host galls only during a short time span, before the complete closure of the gall edges. In the laboratory and in the field M. mali females were observed to lay eggs along the central nervure of leaves with the edges partially rolled inwards by D. mali larvae. This was confirmed by the fact that the peak of the egg-laying M. mali females in the field is shifted some days after compared to that of D. mali. Because of morphological characters and behavioural aspects M. mali can be considered similar to Macrolabis luceti Kieffer and Macrolabis alatauensis Fedotova, which both live as inquilines in galls of Wachtliella rosarum (Hardy) (Diptera: Cecidomyiidae) on various species of Rosa (Rosaceae) (Fedotova, 2000). In the field, the first adults and larvae of M. mali were recorded at the same time as the second D. mali flight. The inquiline population increased over time and at the end of the season became larger than that of the host insect. The large number of galls occupied only by inquiline larvae indicates that they continue to develop in the galls after the host-insect pupation. According to our observations we hypothesized also that the larvae of the gall inducer may die because of the competition with the large number of inquilines as it is known in M. luceti larvae of which may cause the withering of larvae of the gall causer, W. rosarum on Rosa spp. (Robbins, 2000). Therefore the presence of the inquiline species could contribute to regulation of the population of the apple leaf curling midge.

Acknowledgements

Research supported by Autonomous Province of Trento (Research Projects SEDAMA and SafeCrop Centre). We thank Elisabetta Leonardelli (IASMA) for the technical assistance.

References

Anfora, G.; Isidoro, N.; De Cristofaro, A. & Ioriatti, C. 2005a: Description of Macrolabis mali sp. nov. (Diptera Cecidomyiidae), a new inquiline gall midge species from galls of Dasineura mali on apple in Italy. – Bulletin of Insectology 58 (2): 95-99. Anfora, G.; Ioriatti, C.; Moser, S.; Germinara, G.S. & De Cristofaro, A. 2005b: Electro- physiological responses of two different species of apple gall midges (Diptera Cecido- myiidae) to host plant volatiles. – IOBC/wprs Bulletin 28 (7): 413-417. Antonin, P. & Baggiolini, M. 1972: La cécidomyie des feuilles du pommier (Dasineura mali Kieffer). – Revue Suisse de Viticulture Arboriculture Horticulture 4 (2): 51-53. Fedotova, Z.A. 2000: A review of gall midges of the genus Macrolabis Kieffer (Diptera, Cecidomyiidae) with description of new species from Kazakhstan. – Entomological Review 80 (6): 620-634. Robbins, J. 2000: Some observations on Macrolabis luceti Kieffer: (Diptera, Cecidomyiidae). – Cecidology 15 (1): 76.

74 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 75

Effect of different pest control strategies on phytophagous and predatory mites in apple orchards of Girona (NE of Spain)

Adriana Escudero1, Mariano Vilajeliu1, Josep Lluis Batllori2, Francisco Ferragut3 1 IRTA - Estació Experimental Agrícola Mas Badia, Canet de la Tallada, 17134 - La Tallada d’Empordà, Girona, Spain, adriana [email protected]; [email protected] 2 Servei de Sanitat Vegetal, DARP, Aiguamolls de l’Empordà, 17486 - Castelló d’Empúries, Girona, Spain, [email protected] 3 Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain, [email protected]

Abstract: The European red mite Panonychus ulmi (Koch) (Acari: Tetranychidae) is a pest of apple orchards in Girona (NE Spain). Control strategies consist on acaricide sprayings or biological control by the effective conservation of the predatory mite Amblyseius andersoni (Chant) (Acari: Phytoseiidae). In this survey, three apple pest management methodologies were compared for their impact on tetranychid and phytoseiid mite populations. Specie diversity and differences among orchards were evaluated under: i) Integrated Fruit Production methodology as is normally used in the area (IFP conventional), ii) IFP in which alternative to chemical pest control methods were used (less summer insecticide sprayings) (IFP APRI), and iii) organic fruit production system (OFP). The IFP conventional system only used chemicals for pests control; the IFP APRI did not spray any insecticide since post-bloom till harvest, because of the use since 2001 to 2005, of mating disruption for codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) and leopard moth Zeuzera pyrina L. (Lepidoptera: Cossidae), and mass trapping for Mediterranean fruit fly Ceratitis capitata Wied. (Diptera: Tephritidae). The OFP system had been managed according to the 2092/91 CEE rules. Results from the two year survey (2004-2005) reflect differences in alpha diversity among apple orchards and OFP system showed the highest level. The IFP APRI management throughout five years was not enough to increase significantly tetranychid and phytoseiid diversity in relation to IFP conventional. Species of tetranychids as a potential food for A. andersoni were found. Functional diversity (A. andersoni / P. ulmi relationship) in all of the orchards was kept. With regard to the results, the future objectives of the IFP APRI system are pointed and interest in increasing it throughout the Girona fruit area are discussed.

Key words: apple, Integrated Fruit Production, Tetranychidae, Phytoseiidae, diversity

75 76

Behaviour and behavioural control

78 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 79-83

Effect of flat anti-hail nets on Cydia pomonella (L.) reproductive behaviour

Marco Tasin1 , Camilla Ryne2, Vittorio Veronelli3, Anna-Carin Bäckman1, Claudio Ioriatti4 1 SafeCrop Centre, IASMA, Via E. Mach 1, 38010 San Michele a/A, Italy, [email protected] 2 Lund University, Department of Ecology, SE-223 62 Lund, Sweden 3 CBC Europe Via E. Majorana 2, 20054 Nova Milanese, Italy 4 IASMA Research Center, Plant Protection Department, Via E. Mach 1, 38010 S. Michele a/A, Italy

Abstract: In this study we investigated the effect of the flat anti-hail nets on the behavior of the codling moth Cydia pomonella (L.). We hypothesized that the net interferes with male flight behavior and acts as an enclosure, reducing pheromone losses to the sky in orchards treated with pheromone. To test these hypotheses, experiments were carried out in net-covered and uncovered apple plantations treated with mating disruption or with conventional insecticides. In a re-trapping experiment, the number of males recaptured in the net-covered orchards was significantly lower than in the uncovered plantations. Damage caused by codling moth larvae was found less severe in the net-equipped plantations. In addition, inhibition of mating under the net was demonstrated by exposing tethered virgin females. A portable electroantennograph device was employed to measure the concentrations of pheromone in the atmosphere of mating disrupted orchards. However, the same male antennal response was measured in both uncovered and net-covered plots as well as in a control orchard without mating disruption. Because of the reductions in male trap catch and in larval damage as well as the reduced female mating frequency, we conclude that the flat anti-hail net has a disruptive effect on the reproductive behavior of the codling moth.

Key words: codling moth, pest management, mating disruption, pheromone

Introduction

In Northern Italy the occurrence of hailstorms causes large economic losses for the fruit yield. The use of anti-hail net is an active defence against hailstorms and the coverage of apple orchards with anti-hail nets is becoming more and more cost-effective (Borin and Saoncella, 2000). However, research on possible effects of the nets on the fauna of the orchards is far from complete (Drescher, 2004; Graf et al., 1999; Orts et al., 2002; Vaissèrie et al., 2000). In this study we investigated the effect of flat anti-hail nets on the behavior of the codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae), a major pest of pome fruit worldwide. Experiments were carried out both in orchard treated with conventional insecticides and with mating disruption. We hypothesized that the net both interferes with male flight behavior and acts as an enclosure, reducing pheromone losses to the sky.

79 80

Material and methods

Apple orchards Two apple plantations (termed “MD” and “Conventional”) located in Trento were selected for this study. Orchard “MD” was treated with mating disruption and orchard “Conventional” was treated with conventional insecticides. In both plantations 3.5 m high apple trees (cv. Golden delicious grafted on rootstock EM 9) were grown according to the spindle system. Rows of trees were 3.5 m apart and distance between trees on the row was 1.0 m. Half of the orchards were equipped with a gray flat type anti-hail net (Borin and Saoncella, 2000). Rows of trees were covered with a 3.8 m wide stripe of polyethylene net (3 x 7 mm, Agrinova, Italy). Stripes were connected to each other in the centre of the inter-row lane by mean of plastic clips. Distance between clips along the line was 1.3 m. Isomate C plus (Shin Etsu, Tokyo, Japan) dispensers loaded with 190 mg of a mixture containing E8,E10-dodecadien-1- ol, dodecan-1-ol and tetradecan-1-ol were hung in the “MD” plantation at a rate of 1000 dispensers / hectare. Dispensers were twisted around apple branches at a height of 3.0 m from the soil and distance between them on the row was 2.9 m. The conventional plot was treated with five insecticides per year.

Insects Codling moth pupae were purchased from Andermatt Biocontrol (Grossdietwill - CH) and kept in plastic cages until emergence at 22° 70% RH.

Field EAG A portable electroantennograph device (Syntech/VDP Laboratories, Hilversum, The Netherlands) was employed to measure the concentrations of pheromone in the atmosphere. The apparatus was used to monitor pheromone concentration and relative doses in the two mating disrupted apple orchards, with net and without net in year 2005. Recordings were made in a grid system, nine positions in each orchard. Positions were chosen in both periphery and central parts of the orchards. Twenty male antennae were tested in each orchards during morning and evening of the same day.

Tethered females After removing the scales from its pronotum, a 24-old virgin codling moth female was tethered with a 20 cm tulle thread glued to its pronotum and to the center of a white delta trap (Chemia, Italy). Females were exposed in covered and uncovered plots to determine the net effect on mating. Experiments were conducted in the “Conventional” orchard and two treatments (net and no net) with two level (1.5 and 2.5 m above ground) were compared.

Recaptured of released males A total of 1600 laboratory-reared males were released into the two orchards. A fluorescent green (non-covered plots) or yellow (covered plot) powder was used to mark the males before their release in the field. Delta traps baited with either 3 mg or 10 mg of codlemone, the main sex- pheromone component of codling moth, were hung in the orchards at a height of 1,5 and 3,5 m above ground.

Fruit injury assessment Fruit injury has been checked throughout the season by inspecting 1000 fruit per plot. The identity of the larvae bearing into the apples was assessed by a microscope examination.

Results and discussion

Field EAG In the mating disrupted orchard the same male antennal response was measured in both uncovered and net-covered plots. These responses did not significantly differ from those recorded in the conventional orchard.

Tethered females A lower number of the exposed females was mated in the net-equipped plots compared with those in the uncovered orchards (F = 32.16; df = 1, 39; P = 0.03). This effect varied with tethered female height (treatment by height interaction: F = 20.39; df = 1, 39; P = 0.046) and only the proportion of females placed at 2.5 m was significantly different among the coverages. Averaged mortality of tethered females was 12%.

Recapture of released males Trapping with 3 mg traps (Figure 1). A higher number of males was recaptured in the uncovered than in the net-covered orchards (GLM, F=15.882; df=1,23; P<0.001) as well as in the conventional compared with the pheromone treated plots (GLM, F=35.816; df=1,23; P<0.001). The number of recaptured males significantly decreased with height in the net- covered orchards (P<0.001). There were no height effects in uncovered plots (P=0.5526).

0 15

6.7 17

0.3 0

1.0 0.7

Figure 1. Codling moth males re-trapped in pheromone traps in conventional (empty trees) and mating disrupted (gray trees) apple orchards. Trees on the left were covered with a hail- net. Traps were baited with 3 mg of main sex pheromone component, E8-E10- dodecadien-1-ol (codlemone).

Trapping with 10 mg traps (Figure 2). The number of males caught did not differ among coverage of the orchards (GLM, F=1.1489; df=1,23; P=0.2837). A higher number of males was recaptured in the conventional compared with the mating disrupted orchards (GLM, F=18.912; df=1,23; P<0.001) . Overloaded traps caught a significantly higher number of males in the lower part of the canopy in the net-equipped plots (GLM, F=41.966; df=1,23; P<0.001). In the uncovered plots the number of males recaptured in the upper part of the canopy was significantly higher, but only in the conventional orchards (control method by trap position interaction: GLM, F=4.083; df=1,11; P=0.043). 82

1.3 9.7

15.6 0.3

0 0.3

1.3 1

Figure 2. Codling moth males re-trapped in pheromone traps in conventional (empty trees) and mating disrupted (gray trees) apple orchards. Trees on the left were covered with a hail- net. Traps were baited with 10 mg of main sex pheromone component, E8-E10- dodecadien-1-ol (codlemone).

Fruit injury The number of fruits injured by codling moth larvae was significantly higher in the uncovered than in the net-covered plots in years 2004-2006 (Figure 3).

Figure 3. Number of damaged apples (on 1000 fruits) (±SEM) by Cydia pomonella L. in net covered and uncovered apple plantations during years 2004-2006 (t-test, F1,13; F=5.053; P=0.0442).

Discussion

The net was affecting the flight activity of the males in their courtship zone. The percentage of mated tethered females was significantly lower in the upper foliage of plots equipped with the net. In addition, the number of males recaptured in the net-covered orchards was significantly lower than in the uncovered plantations. The number of fruits injured by codling moth larvae was significantly higher in the uncovered plots. EAG recordings performed in the field did not detect any difference in the pheromone atmospheric concentration between covered and uncovered plots. As responses elicited by pheromone and environmental odours 83

were found not to be independent from each other (Rumbo et al. 1995), we cannot exclude that any difference in pheromone level between the orchards could be indistinguishable in our recordings. We show here that the anti-hail nets can create a disturbance on the flight activity of codling moth males. Males may therefore become less capable of finding and mating calling females. As a consequence, the use of anti-hail nets can contribute to the control of C. pomonella in apple orchards.

Acknowledgements

This study has been funded by SafeCrop Center Project, funded by Provincia Autonoma di Trento (Italy) and by CBC (Milan, Italy). We are thankful to Marco Fanti, Ferruccio Pellegrini and Dario Chilovi for helping during field experiments.

References

Borin, M. & Saoncella, C. 2000: Anti-hail net plantings, technical and economic aspects. – Informatore Agrario 56 (29): 64-68. Drescher, C. 2004: Radiotracking of Myotis myotis (Chiroptera, Vespertilionidae) in South Tyrol and implications for its conservation. – Mammalia 68(4): 387-395. Graf, B.; Höpli, H.; Rauscher, S. & Höhn, E. 1999: Hagelnetze beeinflussen das Migrations- verhalten von Apfel- und Schalenwickler. – Schweiz. Z. Obst-Weinbau 12: 289-292. Orts, R.; Branchereau, D. & Laude, G. 2002 : Use of trapping to monitor codling moths: the impact of anti-hail nets and mating disruption. – Infos-Ctifl 180: 31-35. Rumbo, E.R.; Suckling, D.M. & Karg, G. 1995: Measurement of airborne pheromone concentration using electroantennograms: interactions between environmental volatiles and pheromone. – Journal of Insect Physiology 41: 465-471. Vaissèrie, B.; Morison, N.; Crété, X.; Ferre, G.; Matti, M. & Vilain, J. 2000: Incidence des filets paragrêle sur les abeilles et la pollinisation des pommiers. – Arboriculture Fruitière 544: 19- 25. 84 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 85-93

Competitive attraction as a primary mechanism of moth mating disruption in tree fruit crops

Larry J. Gut, James R. Miller, Lukasz L. Stelinski, David L. Epstein Department of Entomology, Michigan State University, East Lansing, MI 48824; [email protected]

Abstract: Over the past few years we have been exploring ways of achieving "high-performance" moth mating disruption. The foundation for this work has been a series of studies examining the mechanisms underlying pheromone-based mating disruption in tortricid moth pests of fruit. Collectively, our results support competitive attraction as an important, if not essential, component of communicational disruption of tortricid moths. Four main lines of evidence support this conclusion. Males are attracted to high-dosage dispensers in the field. Disruption efficacy is pest density- dependent. Effective mating disruption using high-dosage dispensers occurs in the field despite overall atmospheric concentrations not reaching levels high enough to desensitize moths by adaptation or habituation. Finally, the theoretical properties of competitive-attraction phenomena have proved consistent with the majority of disruption profiles obtained in the field. Key traits of competitive attraction are: concave profiles on untransformed axes, with an asymptotic approach to zero catch; a straight line with positive slope when 1/catch is plotted against dispenser density; and a straight line with negative slope when catch is plotted against dispenser density * catch. Key traits of non- competitive disruption profiles include: an initial linear disruption profile on untransformed axes; a concave inverse plot; and a re-curving complex plot. Of 13 published disruption profiles with sufficient data for analysis, 11 were a better fit to the predictions of competitive attraction than a non- competitive disruption mechanism. If competitive attraction plays a major role in achieving mating disruption, the following practical implications should guide us in developing high-performance approaches and formulations: 1) attractiveness should be competitive with females, 2) dispenser density should be high, and 3) distribution should be uniform rather than clumped.

Key words: mating disruption mechanisms, percent disruption, false-trial following, pheromone

Introduction

Mating disruption has been shown to be a feasible control tactic for several tortricid pests of tree fruits, the most notable being codling moth, Cydia pomonella (L.) and Oriental fruit moth, Grapholita molesta (Busck). An estimated 73,000 hectares worldwide were treated with pheromone to control these key pests in 2002 (Gut et al., 2004). Although substantial, this only represents ca. 25% of the world's apple and stone fruit acreage impacted by codling moth or Oriental fruit moth. Further adoption of this promising approach to manage these and other fruit pests has likely been impeded by the high cost of disruption and the lack of consistency in the level of control achieved. The difficulty encountered in controlling moderate to high population densities has been particularly troublesome. To minimize the risk of failure, growers typically have applied companion insecticides to reduce pest densities. Mating disruption for tree fruit pests is at a juncture where its efficacy and reliability needs to be significantly improved if it is to increase its share of the pest-control market. We propose that the next generation of formulations need to provide "high-performance" mating disruption. They should provide nearly complete inhibition of male captures in monitoring

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traps and be effective against higher population densities. In many instances they should yield excellent control as a stand-alone program. Over the past few years we have been exploring ways of raising the bar and achieving “high-performance" mating disruption of tortricid moth pests of fruit. The foundation for this work is a series of studies examining the mechanisms underlying pheromone-based mating disruption. Our reasoning was that different release devices, distributions, and active ingredients may be called for (Gut et al., 2004) depending upon the mechanism(s) of disruption to which a particular pest species or population size is most vulnerable. Although others have previously stressed that optimizing mating disruption will be aided by understanding the effects of particular formulations on moth behavior (Sanders, 1997; Cardé et al., 1998), few have tackled this crucial area of investigation. The work presented herein summarizes our efforts over the past five years to better understand how mating disruption is achieved in the field. Our studies have focused on discerning the relative importance of one disruption mechanism over another. Four main lines of evidence have been examined: 1) direct observations of male attraction to high-dosage dispensers in the field, 2) examination of the requirements for desensitizing males, 3) analysis of the effect of population density on mating disruption outcomes, and 4) differentiating between properties of mating disruption profiles operating via a competitive vs. non-competitive means.

Major disruption mechanisms

Four main mechanisms have been proposed to explain communicational disruption (Cardé, 1990): 1) false-plume following; 2) camouflage; 3) desensitization – including adaptation and habituation and 4) sensory imbalance. To differentiate between the four mechanisms, we first propose that by definition only a single mechanism, false-plume following, involves attraction to the release device; the other three do not (Figure 1). As it further entails competition between authentic and false females, we prefer the term competitive attraction when referring to this mechanism. For clarity in the discussions that follow, competitive attraction to taken to mean that the frequency with which male moths find calling females or monitoring traps (proxy for calling females) in a crop under disruption is sub-normal because males are diverted from orienting to females or traps due to preoccupation with nearby attractive plumes from dispensers of synthetic pheromone (Miller et al., 2006a). The three remaining means of achieving disruption can be collectively classified as non-competitive mechanisms (Figure 1). All of them are envisioned as operating by interfering with the male's ability to sense and respond normally to pheromone. Interference is thought to occur either through impairment of the sensory system (desensitization), or some other means that does not involve sensory impairment (camouflage and sensory imbalance). Desensitization is proposed as the explanation for mating disruption where exposure of males to synthetic pheromone released from dispensers causes males to become less sensitive to pheromone at the level of the antennae (adaptation) or level of the brain (habituation). We recognize that desensitization and competitive attraction could each contribute to mating disruption. In this scenario, exposure of males approaching close to a dispenser may result in sensory impairment. Camouflage entails a compromise in the spatial integrity of the female signal; a calling female cannot be recognized and located by males because the guiding boundaries of her pheromone plume are obscured by ubiquitously present synthetic pheromone. Sensory imbalance entails a compromise in the chemical composition of the female signal; the active ingredient released from dispensers distorts the normal perception of the signal.

MAIN DISRUPTION MECHANISMS

Attraction Not attraction

COMPETITIVE NON-COMPETITIVE ATTRACTION MECHANISMS

Impaired sensory Not impaired system sensory system

Desensitization Camouflage

Sensory imbalance

Figure 1. A dichotomousFigure 1. A dichotomous key to the keymain to thedisr mainuption disruption mechanisms mechanisms based based on on male response to the deliverymale response device to theand delivery pheromonal device imandpact pheromonal on the maleimpact sensory on the male system sensory system.

Attraction to dispensers

The most direct way to differentiate whether competitive-attraction vs. a non-competitive mechanism of disruption is operating under field conditions is to determine whether males orient to and closely approach pheromone sources. Such events should rarely occur if a non- competitive disruption is the main mechanism operating, but are required by competitive attraction. Field observations (Stelinski et al., 2004a, 2005a) have revealed that Oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), redbanded leafroller (Argyrotaenia velutinana) and codling moth (Cydia pomonella) readily approach their respective polyethylene tube dispensers of pheromones. All four species visited their respective dispenser deployed singly in an otherwise non-pheromone treated orchard. Three of the four species also were attracted to dispensers in a fully pheromone-treated orchard. Codling moth males were often observed visiting dispensers even in orchards treated with 10 ropes per tree or ca. five times the label rate (Stelinski et al., 2006). Oriental fruit moth and codling moth males have also been observed readily approaching paraffin wax dispensers (0.1 ml drops containing 5% pheromone), which provided >98% disruption when deployed as 100 point sources per tree (Stelinski et al., 2005b; Epstein et al., 2006). In all cases, males generally approached to within 0-60 cm of the dispensers. Most visits lasted less than 10 s, after which the majority of moths departed by flying upwind. If the nightly observations made at a single dispenser reflect what is taking place at the majority of dispensers in a treated plot, then the overall visitation rate to this type of source could be high.

Concentrations of pheromone required for desensitizing males

Considerable attention has been directed toward understanding the role adaptation and habituation may play in the disruption of mating. Our own work has demonstrated that confinement in pheromone-permeated air causes long-lasting peripheral adaptation in some 88

key tortricid fruit pests (Stelinski et al., 2005c). Additionally, pre-exposure to polyethylene rope dispensers in a flight tunnel can have an effect on subsequent orientation and flight behaviors 24 hours later (Stelinski et al., 2004b). Despite such evidence that exposures to high dosages of pheromone in the lab or flight tunnel can lead to desensitization; there is substantial evidence in the literature against it occurring in the field. The major inconsistency is that the atmospheric concentrations of pheromone required to desensitize males far exceed that achieved under mating disruption in the field (Schmitz et al., 1997; Judd et al., 2005). For example, reduction in the numbers of male grapevine moths (Lobesia botrana) captured in traps in disrupted vineyards was obtained only when males were pre-exposed in the laboratory to pheromone at concentrations that were orders of magnitude greater than those that have been measured in the field (Schmitz et al., 1997; Koch et al., 1997). Long-lasting peripheral desensitization as measured for obliquebanded leafroller (Choristonuera rosaceana) in the lab occurred in the field only after a full day's confinement a few cm from polyethylene-tube dispensers (Stelinski et al., 2003b).

100 Non-Competitive Disruption 80 (Subtraction)

Male Catch Competitive Attraction 20 (Division)

0 0 20 40 60 80 100 Pheromone Dispensers Density (DD) Figure Figure2. Untransformed 2. Untransformed plots plotsof simulated of simulated communicational communicational disruptio disruptionn profilesprofiles for competi- fortive competitive attraction attraction or non-competitive or a non-competitive disrupt disruptionion mechanism mechanism operating operating under the con- under the conditions detailed in Miller et al., (2006) . ditions detailed in Miller et al. (2006)

Graphical analysis of mechanisms

Competitive and non-competitive disruption mechanisms differ fundamentally in how they operate. Competitive attraction functions primarily by division; the attention of males is divided between a given female and nearby dispensers. On the other hand, non-competitive disruption operates mainly by subtraction; moths in pheromone-treated areas are effectively removed from the pool of potential mates. Recognizing this, Miller et al., 2006a developed a set of comparative graphical plots based on simple mathematical simulations of the two phenomena. Profiles of simulated male-visitation rates to pheromone-baited traps deployed in pheromone-treated crops were graphed against density of pheromone dispensers using various types of axes. For disruption by competitive attraction, a graphical plot of male catch in traps (on the y-axis) against density of synthetic pheromone dispensers (on the x-axis) generates a 89

concave profile (Figure 2). Male visitation rate initially falls sharply with small incremental increases in dispenser density. However, the additional declines in visitation rate become progressively smaller as dispenser density continues to increase; approach to zero is asymptotic. Plotting 1 / male visitation rate to a given attractant source on the y-axis against dispenser density on the x-axis yields a straight line with positive slope. Plotting male visitation rate to a given attractant source on the y-axis against (dispenser density x visitation rate) on the x-axis yields a straight line with negative slope. Non-competitive phenomena do not to share this set of properties. Outcomes for simulations of mating disruption by purely non-competitive mechanisms were largely straight lines on non-transformed graphical axes in plots of disruptive effect vs. dispenser density and curved lines on both transformed plots (Miller et al., 2006a). The most striking feature of non-competitive profiles was a distinct recurve in the compound plot, with its inflection point occurring near the midpoint of the y- axis. Impact of pest density

There is general agreement within the tree-fruit research community that higher density populations in many instances are more difficult to control using the mating disruption technique than less dense populations. For example, the best disruption of codling moth has been achieved where pest pressure is low. Maximum application rates are more efficacious than reduced rates where codling moth pressure is moderate, while attempts to control high- pressure populations have been problematic regardless of the number of dispensers deployed (Gut et al., 2004). In an effort to mitigate the effects of population density, growers typically apply one or more companion insecticide sprays to reduce pest pressure.

100 Moths per plot 80 1000 60

20 100 30 0 % Catch (relative to control) 10 0 50 100 150 200 250 300 Dispenser Density per 72 Tree Plot

FigureFigure 3. Outcome 3. Outcome of simulated of simulated communicational communicational disruption disruption by competitive by competitive attraction as attractioninfluenced as influenced by moth density by moth unter density the conditions under thedetailed conditions in Milleret detailed al. (2006a) in Miller et al. (2006a).

The strong impact of pest density on disruption efficacy is consistent with competitive attraction as the major mechanism mediating mating disruption. In contrast, if disruption is principally achieved through a non-competitive mechanism there should be little to no effect 90

of pest density. A common percentage of males should be prevented from locating females irrespective of pest density. The exception would be under circumstances of extremely high pest densities where mate finding could occur without the need for long-range attraction to calling females. The principle that competitive-attraction outcomes vary severely with density of the target pest, as established by Miller et al. (2006a), is illustrated in Figure 3. Under conditions of applying 50 dispensers to a 72-tree plot, an acceptable level of disruption is only predicted for a low moth density of 10/plot. The outcome deteriorates substantially as the density increases to only 30 moths/plot. Higher moth densities result in less than 50% communicational disruption.

Disruption profiles of field data

The analytical procedures of Miller et al. (2006a) were to a large extent developed with the aim of determining whether one category of disruption was more common than the other in the pheromone literature. The approach was to graph published profiles of male catch by pheromone-baited traps in disrupted plots against density of point sources on the various types of axes (Miller et al. 2006b). Thirteen studies were found with sufficient data for graphical analysis. They encompassed disruption trials for eight pest species, and included several types of hand- or machine-applied dispensers.

3.5 8000 3 a. 7000 b. 2.5 6000 5000 2 4000

1.5 Catch Catch Catch 3000 1 2000 0.5 1000

0 nights) traps/ha/20 (males/40

(males/3.33 traps/ha/night) 0 0 1000 2000 3000 4000 0 2000 6000 10000 14000 18000 Dispensers per Hectare Wax Drops per Hectare

45 7000 40 c. 6000 d. 35 5000 30 25 4000 Catch 20 Catch 3000 15 2000 10 1000 5

0 traps/ha/season) (males/50 0 (males/30traps/ha/20 nights) 0 100 200 300 400 500 600 0 200 400 600 800 1000 1200 Ropes per Hectare Ropes per Hectare FigureFigure 4. 4. Examples Examples of disruptiondisruption profiles profiles that that fit thefit competitivethe competi attractiontive attraction model: model:a) model a) data, model b) Oriental fruitdata, moth b) Oriental data (Stelinski fruit moth et al., data2005), (Stelinsk c) Orientali et fruit al., moth2005), da tac) (Rothschild, Oriental fruit 1975), moth d) data obliquebanded(Rothschild, leafroller 1975), data (Lawson d) Obliquebanded et al., 1996). Plotsleafro adaptedller data from (Lawson Miller et al.,et al.,( 2006a). 1996). Plots adapted from Miller et al. (2006a).

Of the 13 published disruption profiles with sufficient data for such analyses, 11 were a better fit to the predictions of competitive attraction than a non-competitive disruption mechanism (Miller et al., 2006b). Plots of the untransformed data produced curvilinear rather than linear profiles (Figure 4). Profiles from the published data consistently resembled the predicted competitive attraction profile of a concave curve that approaches zero asymptotically (Figure 2 and Figure 4d). Semi-inverse and compound plots of the data from these 11 cases produced fairly linear profiles, again as predicted for competitive attraction. Two data sets generated profiles better fitting the predictions of a non-competitive disruption mechanism. Most telling for these cases were the profiles generated from the compound plots, which re-curved significantly and according to the signature of non- competitive disruption (Figure 5b,c). In both cases, pheromone release rate per dispenser was extraordinarily high; neither case involved a commercialized product.

4500 4500 1.2 a. b. c. 4000 4000 1.0 3500 3500 3000 3000 0.8 2500 2500

Catch 0.6 Catch 2000 2000 1500 1500 0.4 1000 1000

Male Visits/Female/Night Male 0.2 (males/20 traps/ha/50 nights) traps/ha/50 (males/20 (males/20 traps/ha/50 nights) traps/ha/50 (males/20 500 500 0 0 0.0 0 50 100 150 200 250 300 0 20000400006000080000100000120000 0 5 10 15 20 Wax Dollops per Hectare Wax Dollops per Ha * Catch Dispenser Density * Male Visits/Female

FigureFigure 5. Profiles of of Oriental Oriental fruit mothfruit disruptionmoth disr foruption plots treated for plots with waxtreated dollops: with a) untransformedwax dollops: a) plot, b) compound plot, and c) compound plot of model data. Adapted from Miller et al., (2006a) . untransformed plot, b) compound plot, c) compound plot of model data. Adapted from Miller et al. (2006a).

Practical implications

Collectively, our findings support competition between pheromone dispensers and females as an important, if not essential, component of communicational disruption of tortricid moths in the field. If competitive attraction plays a major role in achieving mating disruption, the following practical implications should guide us in developing high performance approaches and formulations. First and foremost is the recognition that it will not be an easy task. Conceptualizing the competitive attraction profile in two phases reveals how difficult achieving high performance disruption may be (Figure 6). Deployment of relatively few dispensers has a large effect on mate finding - this is the "easy phase" of disruption. For economical reasons, considerable effort has been directed toward operating within this area of the disruption profile. Not surprisingly, significant levels of communicational disruption have been achieved using very few release devices (Shorey et al., 1996). However, whether high performance disruption can be achieved is arguable. Recall that we have set the standard as nearly complete inhibition of moth captures in monitoring traps. For this to occur using very few dispensers, population densities must be extremely low. In the difficult phase, each additional dispenser provides only a small increase in disruption. Ramping up from 80-90% to 98-100% inhibition of moth captures in monitoring traps will be difficult, especially without the use of companion insecticides to drive down adult numbers. The next generation of 92

disruption formulations needs to surpass the formulations currently on the market. They will need to be highly attractive and amenable to delivery at high point source densities uniformly dispersed through a crop. If disruption primarily involves attraction, formulations designed to provide high performance disruption should be competitive with females. This may require optimizing the blend or release rate so as to generate plumes matching or somewhat exceeding those of females. The active ingredients should be well protected from degradation through various chemical processes so as to maintain their high level of attractiveness. As pointed out by Miller et al. (2006a), an off-blend may be the appropriate choice if attractiveness of dispensers releasing abnormally high rates of a less expensive pheromone component can match attractiveness of females releasing a blend containing costly minor components. If disruption is largely achieved via competition between females and false females, attractive point sources should be applied at high densities. Additionally, such dispensers should be uniformly distributed in the crop so as to avoid pheromone-free areas. This is particularly important when the area treated is small. Treating large areas many allow for some reduction in dispenser density, provided pest density is low. When pest densities are high, the number of point sources will likely need to exceed the numbers affordable to manual application, i.e., formulations will need to be amenable to machine-application.

100 90 80 70 Easy Phase 60 50 40 Difficult Phase Male Catch 30 20 10 0 012 4 816 32 Pheromone Dispenser Density (D D)

Figure 6. TwoFigure phases 6. Twoof disruption phases of disruption as they as occurthey occur under under a a competitivecompetitive attraction attraction model model .

References

Cardé, R.T. 1990: Principles of mating disruption. – In: R.L. Ridgway and R.M. Silverstein (eds.). Behavior-Modifying Chemicals for Pest Management: Applications of Phero- mones and other Attractants. Marcel Dekker, New York: 47-71. Cardé, R.T., Staten, R.T. & Mafra-Neto, A. 1998: Behavior of pink bollworm males near high-dose, point sources of pheromone in field wind tunnels: insights into mechanisms of mating disruption. – Entomol. Exp. Appl. 89: 35-46. Epstein, D.L., Stelinski, L.L., Reed, T.P., Miller, J.R. & Gut, L.J. 2006: Higher densities of distributed pheromone sources provide disruption of codling moth (Lepidoptera: Tortric- idae) superior to that of lower densities of clumped sources. – J. Econ. Entomol. 99: 1327-1333. 93

Gut, L.J., Stelinski, L.L., Thomson, D.R., & Miller, J.R. 2004: Behaviour-modifying chemicals: prospects and constraints in IPM. – In: O. Koul, G.S. Dhaliwal & G.W. Cuperus (eds.). Integrated Pest Management: Potential, Constraints, and Challenges. CABI Publishing, Cambridge, MA: 73-121 Judd, G.J.R., Gardiner, M.G.T., Delury, N.C., & Karg, G. 2005: Reduced sensitivity, behavioral response and attraction of male codling moths, Cydia pomonella, to their pheromone (E,E)-8,10 dodecadien-1-ol following various pre-exposure regimes. – Entomol. Exp. Appl. 114: 65-78. Koch, U.T., Lüder, W., Clemenz, S., & Cichon, L.I. 1997: Pheromone measurement by field EAG in apple orchards. – IOBC/WPRS Bull. 20(1): 181-190. Lawson, D.S., Reissig, W.H., Agnello, A.M., Nyrop, J.P., & Roelofs, W.L. 1996: Interference with the mate-finding communication system of the obliquebanded leafroller (Lepido- ptera:Tortricidae) using synthetic sex pheromones. – Environ. Entomol. 25: 895-905. Miller, J.R., Gut, L.J., de Lame, F.M. & Stelinski, L.L. 2006a: Differentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone: (Part 1) theory. – J. Chem. Ecol. 32(10): 2089-2114. Miller, J.R., Gut, L.J., de Lame, F.M. & Stelinski, L.L. 2006b: Differentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone: (Part 2) case studies. – J. Chem. Ecol. 32(10): 2115- 2143. Rothschild, G.H.L. 1975: Control of oriental fruit moth (Cydia molesta) (Busck) (Lepido- ptera:Trotricidae) with synthetic female pheromone. – Bull. Ent. Res. 65: 473-490. Sanders, C.J. 1997: Mechanisms of mating disruption in moths. – In: Cardé, R.T. and Minks, A.K. (eds.). Insect Pheromone Research, New Directions, Chapman and Hall, New York: 333-346. Schmitz, V., Renou, M., Roehrich, R., Stockel, J., & Lecharpentier, P. 1997: Disruption mechanisms in the European grape moth Lobesia botrana Den & Schiff. III. Sensory adaptation and habituation. – J. Chem. Ecol. 23: 83-95. Shorey, H.H., Sisk, C.B., & Gerber, R.G. 1996: Widely separated pheromone release sites for disruption of sex pheromone communication in two species of Lepidoptera. – Environ. Entomol. 25: 446-451. Stelinski, L.L., Gut, L.J., Pierzchala, A.V., & Miller, J.R. 2004a: Field observations quantifying attraction of four tortricid moth species to high-dosage pheromone rope dispensers in untreated and pheromone-treated apple orchards. – Entomol. Exp. Appl. 113: 187-196. Stelinski, L.L., Gut, L.J., Vogel, K.J. & Miller, J.R. 2004b: Behaviors of naïve and pheromone pre-exposed leafroller moths in plumes of high-dose pheromone dispensers in a sustained-flight wind tunnel: implications for pheromone-based mating disruption of these species. – J. Insect Behav. 17: 533-553. Stelinski, L.L., Gut, L.J., Epstein, D., & Miller, J.R. 2005a: Attraction of four tortricid moth species to high dosage pheromone rope dispensers: Observations implicating false plume following as an important factor in mating disruption. – IOBC/WPRS Bull. 28(7): 313- 317. Stelinski, L.L., Gut, L.J., Mallinger, R.E., Epstein, D., Reed, T.P., & Miller, J.R. 2005b: Small plot trials documenting effective mating disruption of Oriental fruit moth, Grapholita molesta (Busck), using high densities of wax-drop pheromone dispensers. – J. Econ. Entomol. 98: 1267-1274. Stelinski, L.L., Gut, L.J. & Miller, J.R. 2005c: Occurrence and duration of long-lasting peripheral adaptation among males of three species of economically important tortricid moths. – Annals Entomol. Soc. Am. 98: 580-586. 94

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 95-100

New insights into codling moth, Cydia pomonella (L.), distribution and implications for mating disruption

David L. Epstein, Larry J. Gut, James R. Miller, Lukasz L. Stelinski Department of Entomology, Michigan State University, B18 NFSTC, East Lansing, MI 48824, USA; [email protected]

Abstract: A study aimed at determining the location of searching codling moth (Cydia pomonella [L.]) (Lepidoptera, Tortricidae) males and calling females in mating disrupted and non-disrupted plots was conducted in Michigan, USA. A leaf blower, converted into a vacuum for sampling codling moth adults on branches and in the tree canopy, had a 20-24% success in recovering released moths in orchard conditions. A series of four collections were made during the hours of 09:00-18:00 from May 25 through June 15 and a second series of four collections were completed during the hours of 18:00- 22:00 from July 20 to August 22. Only eight codling moth adults were collected during the four daylight samples; one female and two male moths were sampled from the top third of the tree canopy, four males were sampled from the middle third of the tree canopy, and one male was sampled from the lower third of the tree canopy. Distributions of adults were also assessed during daytime hours (09:00- 18:00) by fogging trees with various pyrethroid insecticides. No codling moth adults were collected in any of these samples. In contrast to the paucity of moths collected in the daytime samples, 94 moths were collected during four twilight samples, with equal numbers sampled in disrupted and non- disrupted plots. In mating disruption plots, 42% of females were found in the top third of the tree canopy, 46% were found in the middle third, and 12% were recovered in the lower third. The 22 females sampled from non-disrupted plots were more evenly distributed, with 36.4% in the top third, 36.4% in the middle third, and 27.2% in the lower third of the tree canopy. Releases of marked moths were conducted in 2006 in screened tents to identify the daytime (09:00-18:00) habitats for adult moths within the orchard. Of males released, 11.2% were recovered from the tree canopy and 6.2% were recovered from the ground (drive row grass and vegetation under the tree). Of females released, 18.6 % were recovered from the tree and 8.2% from the ground.

Key words: Codling moth, distribution, diel behavior

Introduction

Mating disruption of codling moth (Cydia pomonella [L].) using various hand-applied dispensers has become an accepted practice, with approximately 45,000 ha of apple in North America treated in 2002 (Gut et al., 2004). Research with pheromone baited traps showing male flight behavior to be concentrated in the top third of the tree canopy has resulted in the standard recommendation that dispensers be applied within the top meter of the tree canopy (Barret, 1995; Knight, 2000; McNally & Barnes, 1980). Witzgall et al. (1998) visually observed male moths flying and searching branches in the upper half of tree crowns, providing further support for deployment high in the canopy. However, the distribution of codling moth females within the tree canopy has yet to be determined. In addition, the location of both adult males and females during daylight hours, when they are thought to be inactive, has not been well documented. The studies reported here summarize a two-year investigation of the location of codling moth males and females in apple orchards during daylight and twilight hours. To directly measure moth spatial distribution within the tree canopy, a vacuum sampling technique was

95 96

developed. The long-term aim of this work is to improve the effectiveness of codling moth mating disruption based on a solid understanding of adult distribution within the canopy and diel patterns of activity.

Material and methods

Vacuum A 10 horsepower (Briggs & Stratton Intek engine) leaf blower (MacKissic Inc., PA, USA) with a 34 cm diameter impeller and 322 KPH air velocity, was converted into a vacuum powerful enough to remove codling moth adults from sampled surfaces. A 2 m length of 15 cm diameter reinforced rubber air hose (Cathey Co, Lansing, MI) was attached to the leaf blower air intake opening. The open end of the rubber hose was attached to a 2 m long wand constructed of 15 cm diameter heat duct sheet metal. A handle constructed of 1 m long, 2.5 cm diameter metal was attached to the wand with hose clamps. Four 3.5 cm long bolts were attached at 90-degree spacing 2.5 cm from the end of the sheet metal wand. Moths were collected from trees into a 19-liter nylon mesh paint strainer bag (Master Craft, El Monte, CA, USA) inserted into the wand, folded back over the bolts, and secured with rubber bands to prevent the bag from being suctioned into the impeller. A 90-degree sheet metal register box was attached to the wand end for vacuuming ground surfaces. Collection bags for ground collections were made from fiberglass window screening.

Recovery Efficacy of Vacuum Mark, release and recapture trials were used to test the efficiency of the vacuum. An initial 2005 test of whether the vacuum was capable of collecting moths from apple trees was done using 2 m tall potted Red Delicious trees in a glass greenhouse at Michigan State University. The procedure entailed three individual releases of twenty laboratory-reared moths onto four trees and immediately vacuuming the trees to recover moths. Two release/recapture trials were performed in the field in 2005 with the 10 hp leaf blower vacuum. Moths were marked by placing them in a 19 L plastic pail and spraying 0.5 g dye in 75ml acetone through cheesecloth covering the top opening of the pail. Forty laboratory-reared moths were marked and released on two occasions onto individual 16-year old trees, 3 x 6m Red Delicious, 4 – 5 m in height, at the Trevor Nichols Research Complex.

Orchard Vacuum Trials All orchard vacuum collections were done in six 0.4 ha apple plots at the Trevor Nichols Research Center, Fennville, MI. Four plots were 16 year old Red Delicious (3 x 6m planting), 4 – 5 m in height, and two plots were 23 year old Macspur, (5.5 x 6 m), 5 m in height. Two of the Red Delicious plots and one Macspur plot were treated with Isomate C+ (Shin-Etsu Chemical Co., Tokyo, Japan) at the full label rate of 1000 dispensers/ha. The remaining three plots were not treated with pheromone. Daytime samples were collected between the hours of 9:00 to 16:00, twilight collections occurred between 18:00 - 21:30 hours. Twelve trees from each of the six plots were sampled on four dates for both daytime and twilight samples in 2005, and on eight dates in 2006. The 2006 samples were collected from two of the Red Delicious plots described above; one treated with Isomate C+ at 1000 dispensers/ha, and the second with no mating disruption. In both years, six of the 12 sample trees were located on plot perimeters and six trees were located in plot middles. Thirty-second samples were collected from the top third, middle third, and lower third of each tree. The lower third of the tree sample included the trunk to the soil surface, and weeds growing within 0.5m of the tree trunk. Sample contents were transferred from the nylon mesh collection bag located in the vacuum wand into 4 L freezer bags, and identified in the laboratory. Only adult codling moths 97

were counted in these collections, even though the vacuum was powerful enough to remove cocooned larvae (5 individuals) and pupae (7 individuals) from the trunks and scaffold branches of the trees in 2005 field trials. One pheromone trap (LPD Scenturian Guardpost, Suterra, Bend, OR) baited with a 1 mg codlemone rubber septum lure was placed in the upper third of the tree canopy in each non-mating disrupted plot to monitor codling moth flight.

Tree Fogging Twelve trees were fogged with pyrethroid insecticides (tetramethrin and sumithrin) on two separate dates in 2005 to test this method for sampling codling moth adults in the tree canopy. Aerosol foggers were attached to the trunks of each tree. Trees were covered with 4ml plastic, and the foggers were activated until empty of contents. After a 10-minute interval, the plastic covers were removed, and trees shaken to dislodge codling moth adults, as well as other insects, onto tarps placed on the ground under each tree. All specimens were collected off of the tarps and taken to the laboratory for later identification.

Screen Tents Four tents measuring 3m high, 5.5m wide, and 6.5m long were constructed of 2.5cm PVC pipe and mosquito netting at the Trevor Nichols Research Center, Fennville, MI. The tents were erected over single apple trees, extending out over the drive row grass. Trees were 2.5m high Red Delicious, spaced 3X6m. Fourteen release/recaptures were made within screen tents over 7 dates in 2006 to test the efficacy of the vacuum for sampling moths from the tree and from ground surfaces, and to determine daytime moth habitat. Moths were marked by placing 20 individuals in a 13 cm Petri dish coated with 0.4 g luminescent powder (Bioquip Inc., CA, USA). The moths accumulated powder after several minutes of movement in the dish. Moths were released between 18:00 - 19:00 hours and recaptured the following day between 10:00 and 14:00 hours. Trees and drive-row grass were individually covered with 4 ml plastic for the recovery efficacy trials. Open release cups containing 20 moths each were placed under each cover to allow moths to freely exit on their own. Moths in the habitat determination trials were released by placing 2 open release containers, one with 20 males and one with 20 females, within the tree canopy. Male and female moths were released in equal numbers and were segregated in separate release containers prior to all releases.

Results and discussion

Recovery Efficacy of Vacuum The leaf blower vacuum recovered 70%-80% of moths released onto potted trees, and 24% of moths released in trees at the Trevor Nichols Research Center orchards in 2005. Recovery of released moths (14 releases) from covered trees within screened tents in 2006 was 20.6%. Recovery of moths from drive-row grass initially proved more challenging, and required the development of a collection bag that allowed fine particulate matter to pass through to prevent instantaneous blockage of air suction through the collection wand. Long, tapered bags made from fiberglass window screening were constructed for this purpose. The tapered design prevented the bag from clogging the collection wand passageway as it filled with dry grass and other plant matter. Recovery of released moths (14 releases) from covered grass within screened tents in 2006 was 21.7%.

Orchard Vacuum Trials In 2005, a total of 8 moths were collected during 576 individual daylight samples, 6 males and 2 females. The 2 females were captured from the top third of tree canopies (Table 1).

Table 1. Total number of moths collected with the leaf blower vacuum in orchard sampling during daylight hours in 2005

Top 1/3 Middle 1/3 Low 1/3 Canopy Canopy Canopy Male CM 1 4 1 Female CM 2 0 0

Of the total males captured, one was located in the top third, four in the middle third, and one in the bottom third of the tree canopies (Table 1). Twilight samples (576 individual vacuum samples) in 2005 resulted in the capture of 94 moths distributed throughout the tree canopies; 15 males and 18 females in the top third of tree canopies, 23 males and 19 females from the middle third, and 10 males and 9 females from the bottom third of the tree canopies (Table 2).

Table 2. Total number of moths collected with leaf blower vacuum in orchard sampling during twilight hours in 2005

Top 1/3 Middle 1/3 Low 1/3 Canopy Canopy Canopy Male CM 15 23 10 Female CM 18 19 9

25 Male 20 Female

15 # CM Adults 10

0 Edge Middle Edge Middle Figure 1. Vacuum Samples showed that males and females were present in both orchard edges and middles

There were only minor differences in moth distribution when the data were analyzed by treatment. Moths were distributed evenly between plot perimeters and plot centers in both mating disrupted and no-disrupted plots (Figure 1) in the 2005 vacuum samples. The majority of moths in disrupted and non-disrupted plots were in the upper and middle portions of the canopy. In disrupted plots, 46% of female moths and 43% of male moths were collected from the middle third of the tree canopies, while 42% of females and 22% of males were in the top third of the tree canopies. An equal percentage of females in non-disrupted plots were collected from the top (36%) and middle (36%) thirds of the tree canopies. Male distribution in no-disruption plots was especially skewed toward the top two thirds of the tree canopies, 99

with 52% collected from the middle and 40% from the top. Thus, there appeared to be a slight effect of the pheromone treatment on male distribution. Specifically, 35% of males were collected from the bottom third of the tree canopies in disrupted plots, while only 8% were vacuumed in the lower canopy in non-disrupted plots, Orchard vacuum samples (288 individual samples) taken in 2006, again collected moths from all areas in the tree canopies, with 25 moths captured from the top third (10 males 15 females), 13 from the middle third (7 males, 6 females), and 10 moths (1 male, 9 females) from the bottom third of the tree. Distribution of moths within varying tree strata according to treatment is presented in tables 3 and 4. All male moths collected in non-disrupted plots in 2006 were collected from the top third of the tree canopies, compared to 50% of males collected from the same strata in mating disrupted plots (Tables 3 and 4).

Table 3. Moths collected from varying tree strata using a leaf blower vacuum in mating disrupted blocks in 2005

Top 1/3 Middle 1/3 Low 1/3 Canopy Canopy Canopy Male CM 50% 44% 6% Female CM 57% 17% 26%

Table 4. Moths collected from varying tree strata using a leaf blower vacuum in non-mating disrupted blocks in 2005

Top 1/3 Middle 1/3 Low 1/3 Canopy Canopy Canopy Male CM 100% 0% 0% Female CM 28.5% 28.5% 43%

The paucity of moths collected during daylight hours in 2005 raised the possibility of moths moving to other habitats during this time. Do moths reside strictly in the tree canopy or in bark crevices, or do they move to alternative habitats after twilight flight activity ceases? The ability of the vacuum to collect cocooned larvae and pupae combined with high recapture rates provided evidence that the vacuum would have been effective in collecting adults from the canopy and bark crevices if they had been present during the daylight samples. Tree fogging with pyrethroid insecticides applied on 2 occasions to 12 covered trees during daylight hours did not result in the capture of any codling moth in 2005. It is possible that the failure to collect moths using this technique resulted from a lack of moths in the tree at the time of sampling, rather than just its inadequacy as an adult sampling method. Mark recapture studies were designed to ascertain whether codling moths potentially inhabited orchard locations other than the tree, such as grasses and weed growing on the orchard floor. Fourteen release/recapture trials in screened tents in 2006 resulted in 14.9% of moths being recovered from the trees, and 7.2% from drive-row grass and weeds under the trees. Moths were also recovered off of the tent screen (16.5%) and from the release cups (12.4%). The remaining 48.9% of moths were not recaptured. Of moths recaptured, 6.2% of males and 8.2% of females were recovered from drive-row grass and weeds growing under the trees. Data loggers (Hobo Pro series, Onset Computer Corporation, Bourne, MA, USA) 100

placed in the tree and in the grass at soil level showed that the mean temperature in the 2 microclimates did not vary over a 3-month period (tree 22.1, grass 22.1), but that mean relative humidity varied by 13.3% between the tree (78.7%) and the grass (92.0%). The higher availability of moisture found in the grass could provide an explanation for why moths may seek shelter in this habitat during hot, dry summer days. Orchard vacuum samples in 2005 and 2006 show codling moth adults to be distributed throughout all parts of the tree canopies during the time periods when female calling and male searching activity is occurring. Current protocols for the deployment of hand applied mating disruption dispensers call for dispensers to be placed exclusively in the top meter of tree canopies. Future research should address whether placement of dispensers at a range of heights within the tree may improve the overall performance of mating disruption targeting codling moth. The orchard vacuum samples, the vacuum samples in screened tents, and the fogging of trees with pyrethroid insecticides provided three lines of evidence that moths leave the trees for alternative habitats after twilight flight activity has ended. Potential opportunities to exploit this behavior, and whether it is a contributing factor in the difficulty of disrupting codling moth in apple will be explored in future research.

Acknowledgements

We thank the Michigan Apple Research Committee for funds supporting this research.

References

Barret, B.A. 1995. Effect of synthetic pheromone permeation on captures of male codling moth (Lepidoptera: Tortricidae) in pheromone and virgin female moth-baited traps at different tree heights in small orchard blocks. – Environ. Entomol. 24(5): 1201-1206. Gut, L.J., Stelinski, L., Thomson, D.R. & Miller, J.R. 2004. Behaviour-modifying chemicals: prospects and constraints in IPM. – In: Koul, O, Dhaliwal, G.S., and Cuperus, G.W. (eds.). Integrated pest management: potential, constraints and challenges. CAB Inter- national, Oxfordshire, UK: 73-121. Knight, A. 2000. Monitoring codling moth (Lepidoptera: Tortricidae) with passive inter- ception traps in sex pheromone-treated apple orchards. – J. Econ. Entomol. 93(6): 1744- 1751. McNally, P.S. & Barnes, M.M. 1980. Inherent characteristics of codling moth pheromone traps. – Environ. Entomol. 9: 538-541. Witzgall, P., Backman, A., Svensson, M., Koch, U. Rama, F., El-Sayed, A., Brauchli, J., Arn, H., Bengtsson, M., & Lofqvist, J. 1999. Behavioral observations of codling moth, Cydia pomonella, in orchards permeated with synthetic pheromone. – BioControl 44:211-237. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 101-105

Mating disruption of codling moth, Cydia pomonella (L.), using Puffer® CM, on apple orchards

Santiago Martí1, Abel Zaragoza2, Tom Larsen3 1 Departamento de Desarrollo, Suterra España Biocontrol S.L., Cervantes 22A, 25243 El Palau d´Anglesola, Lleida, Spain, [email protected] 2 Departamento de Ventas y Marketing, Suterra España Biocontrol S.L., Tenor Massini 93 3º; 6ª, 08028 Barcelona, Spain, [email protected] 3 Product Development, Suterra LLC, 213 S.W Columbia Street, Bend, OR 97702, USA, [email protected]

Abstract: The performance of Puffer® CM, aerosol formulation of the codling moth pheromone that is uniformly applied by mechanical means at pre-programmed and periodical intervals irrespective of weather conditions, was investigated in 2005 in European trials. Trap catches and fruit damage were compared in ca. 10 ha plots with different mating disruption products, dispenser [CheckMate CM, applied twice at 300 dispensers/ha, or Isomate CTT, applied once at 500 dispensers/ha] vs. Puffer® CM (applied once at 2 puffers/ha), during the 2005 season in two trials conducted in Northern Spain: Monzón (Huesca) and Ager (Lleida). Puffer® CM performed extremely well, at least comparable to mating disruption with dispensers (either CheckMate CM or Isomate CTT), leading to an almost complete codling moth catch inhibition and to extremely low fruit damage situation. Moreover, Puffer® CM pheromone release was constant through the season not being affected by weather conditions and persistence of Puffer® CM (average 185 days) was longer than both reference dispensers.

Key words: codling moth, mating disruption, Puffer® CM, apple

Introduction

Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is an important pest of pome fruit and walnut orchards throughout the world. Codling moth resistance to many insecticides has been observed in several areas, including Northern Spain and mating disruption as a control tactic is increasing in use against this pest. Currently, mating disruption is commonly implemented using hand applied dispensers. The application of dispensers is time-consuming and may be expensive. Cheaper and faster means of mating disruption are being developed and commercially used in some areas of the world, as puffers (Welter et al., 2005) and micro-encapsulated sprayable pheromone (Welter et al. 2005). Puffer® CM is an aerosol formulation of the codling moth pheromone that is uniformly applied by mechanical means at pre-programmed and periodical intervals, irrespective of weather conditions. The product consists of an aerosol can which is housed in a weatherproof cabinet. The objective of this study was to determine the efficacy and persistence of Puffer® CM in pome fruit orchards.

101 102

Material and methods

Trial sites Two trials were conducted during the 2005 season in Northern Spain, where there are normally two full codling moth generations and a partial third codling moth generation. Trial 1 was located in Monzón (Huesca) in a Golden Delicious and Royal Gala mixed apple orchard. Trial 2 was located in Ager (Lleida) in a Golden Delicious and Granny Smith mixed apple orchard. Trials were located in orchards with moderate codling moth pressure. Puffer® CM and dispensers were applied in 10 ha plots in both trial sites.

Mating disruption products Puffer® CM was compared vs. CheckMate CM (90-day dispenser applied twice at 300 dispensers/ha) in Trial 1 and vs. Isomate CTT (150-day dispenser applied once at 500 dispensers/ha). Puffer® CM was applied in both trial sites once at the beginning of first codling moth generation flight at a rate of 2 puffers/ha. Dominant wind direction was taken into account for puffer distribution inside the plot. Approximately half of the puffer cabinets were located in the upwind perimeter of the Puffer® CM plot and the rest of puffer cabinets were regularly distributed inside the plot. Both CheckMate CM and Isomate CTT dispensers were uniformly distributed in the mating disruption plot. The puffer was set to release codling moth pheromone at 15 minutes intervals from 15:00 until 03:00 hours during the day.

Trap catches and fruit damage Four Delta traps containing Wageningen lures and four traps having Biolure 1X lures were placed in each mating disruption plot. Wageningen lures were replaced at ca. 6 weeks intervals. Biolure 1X lures were replaced at ca. 8 weeks intervals. Sticky panes inside traps were replaced at ca. 6 weeks intervals. Traps were checked and catches recorded at ca. weekly intervals. Fruit damage assessment was conducted three times during the season (at least 1000 fruit per plot): at the end of the first codling moth generation, at the end of the second codling moth generation, and at harvest.

Puffer® CM pheromone emission rate Puffer cans were weighed periodically (ca. every 3 weeks) in order to determine the amount of product remaining in the aerosol cans and to predict/determine the persistence of the product in the field.

Results and discussion

Trap catches and fruit damage Codling moth adult males catches were almost completely inhibited both in dispenser and puffer mating disruption plots [≤1 moth/trap/week)] in both trial sites during the whole season (Figure 1 and 2)]. No fruit damage was observed in any mating disruption plot in Trial 1 (Monzón, Huesca). Fruit damage was extremely low in Trial 2 (Ager, Lleida), and it tended to be slightly lower in the Puffer® CM plot (≤0,1%) than in the Isomate CTT dispenser plot (≤0,2%) (Figure 3). However, some insecticide sprays that could have some effect on the codling moth, although not especially applied against it, were conducted in both trial sites. Trial 1 site received one chlorpyrifos spray at the end of June 2005 and one lambda cyhalothrin spray before Royal Gala harvest, for Medfly control. Trial 2 site received one carbaryl at mid-May 2005, for thinning purposes, one flufenoxuron spray for Lepidopteran eggs control at the second half of May 2005, and one deltametrin spray only in the Granny Smith cultivar at the end of the season for Medfly control. At least three to four insecticide 103

sprays per CM generation are usual in non-mating disruption plots in the area where trials were conducted in order to control CM. Up to 12-15 insecticide applications every 7-10 days may be required in heavily infested plots to try to control the codling moth (García de Otazo et al., 2004).

Codling moth Catches in Mating Disruption Plots - Spain 2005 - Trial 1(Monzón, Huesca)

3,0

2,5

2,0 Puffer CM (Biolure 1X Lure) 1,5 Puffer CM (Pherobank Lure) CheckMate CM Disp. (Pherobank Lure) 1,0

0,5

0,0 Codling moth catches/Trap/Week 5 5 5 5 5 5 5 0 -0 0 -05 0 0 -0 -0 y-05 y l l- p pr- ju jul- ju go-05 a -jun-05 - - - e ep-05 - ma -ma 4 9 a -s -s 7 6 09-jun- 24 0 11-jul-0518 2 6 2 06- 13-may-052 13- 29-ago 12 2 21-oct-05 Date

Figure 1. Codling moth catches in mating disruption plots (Trial 1: Monzón, Huesca)

Codling moth Catches in Mating Disruption Plots - Spain 2005 - Trial 2 (Ager, Lleida)

3,0

2,5

2,0 Puffer CM (Biolure 1X Lure) 1,5 Puffer CM (Pherobank Lure) Isomate CTT Disp. (Pherobank Lure) 1,0

0,5

Codling moth catches/Trap/Week 0,0

5 5 5 5 5 5 5 -0 -0 -05 -0 0 0 -0 -0 5 y n n-0 l- l- o p p-05 ay u u jul u ju g -j - - a se -m -ma 9 4 2-j 0 - - 6 3 0 25-j 0 1 19-jul-05 3 3 7 28-apr-060 1 26-may-05 1 26-ago-0510-se 2 25-oct-05 Date

Figure 2. Codling moth catches in mating disruption plots (Trial 2: Ager, Lleida)

Results indicate that Puffer® CM, applied just once at the beginning of season, performs extremely well, at least comparable to dispensers (either CheckMate CM or Isomate CTT), during the whole season, leading to an almost complete codling moth catch inhibition and to an extremely low or non-damage situation. 104

Codling Moth Fruit Damage - Puffer Trial - Spain 2005 - Trial 2 (Ager)

1,00 0,90 0,80 0,70 0,60 0,50 % Damaged Fruit 0,40 0,30 %Damaged Fruit 0,20 0,10 0,00 Puffer MD Isomate CTT Puffer MD Isomate CTT Puffer MD Isomate CTT Dispenser Dispenser Dispenser

15/06/05 15/06/05 19/08/2005 19/08/2005 10/09/2005 10/09/2005 (48DAA) (48DAA) (113DAA) (113DAA) (135DAA) (135DAA) (Golden Delicious (Golden Delicious Harv est) Harv es t) Treatment/Date

Figure 3. Fruit damage due to codling moth attack in mating disruption plots (Trial 2: Ager, Lleida)

Puffer Average Daily Weight Loss by Interval After Placement in the Field - Spain 2005 - Trial 1 (Monzón-Huesca)

2,50

2,00

1,50

Weight Loss (g)

1,00

0,50

PufferAverage Weight Loss/Day (g) 0,00

A A A A DA DA A DA DA 4 2 77 94 DAA 1-50 -101 DA -12 15 1 1 51-68DAA 69-87 DAA 8 5- 3- 8 02 2 5 1 1 1 178- Application Interval

Figure 4. Puffer Average Daily Weight Loss (Trial 1: Monzón, Huesca)

Puffer® CM pheromone emission rate Weight loss of puffer cans was very constant (ca. 2 g/day) basically throughout the whole season, up to 180 days after application (DAA) indicating that the Puffer® CM was not 105

affected by weather conditions and could provide a constant emission of the pheromone product (Figure 4 and 5). Average weight loss after 180 DAA decreased mostly due to battery failure observed in some of the Puffer units. This problem has been addressed and solved in the new units being used since the 2006 season. The average persistence of Puffer® CM was ca. 185 days, longer than both reference dispensers.

Puffer Average Daily Weight Loss by Interval After Placement in the Field - Spain 2005 - Trial 2 (Ager-Lleida)

2,50

2,00

1,50

Weight Loss (g)

1,00

0,50

Puffer AverageWeight Loss/Day(g) 0,00

A A A A A A AA AA D D D D D 6 DAA 7 8 4 8 2 5 1-48 -10 -1 49-67 DAA 68- 7 9-1 5-180 8 08 2 5 1 1 1 181-193 DAA Application Interval

Figure 5. Puffer Average Daily Weight Loss (Trial 2: Ager, Lleida)

References

García de Otazo, J.; Torà, R. & Almacellas, J. 2004: La carpocapsa de peras y manzanas en las plantaciones frutícolas de Lleida. Resistencia y control. – Terralia 42: 62-69. Welter, S.; Pickel, C.; Millar, J.; Cave, F.; Van Steenwyk, R. & Dunley, J. 2005: Pheromone mating disruption offers selective management options for key pests. – California Agricultura 59 (1): 16-22. 106

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 107-114

Field assays of new biodegradable controlled-release pheromone dispensers for mating disruption of Cydia pomonella (L.)

Beatriu Femenia-Ferrer1, Dolors Bosch2, Pilar Moya1, Jesús Avilla2,3, Jaime Primo1 1 Centro de Ecología Química Agrícola, Universidad Politécnica de Valencia, Camino de Vera s/n, Edif. 9B - Lab. 111, 46022 – Valencia, Spain. [email protected] 2 IRTA. Centre Udl-IRTA, Rovira Roure 191, 25198 – Lleida, Spain. [email protected] 3 Universitat de Lleida, Department of Crop and Forest Science, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]

Abstract: A new type of eco-friendly pheromone dispenser for mating disruption of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), based on sepiolite, has been developed. In order to evaluate its efficacy under field conditions, mating disruption trials were conducted during 2005 and 2006 in a 9.84 - ha orchard (Plot A) in Lleida, Spain. Pheromone dispensers were set up shortly before the beginning of the first flight of the pest in plots where apples and pears were cultivated. Each year, a reference orchard next to the trial (Plot C in 2005: 1.24 ha and Plot D in 2006: 1.29 ha) where insecticides were only applied when justified by the level of the pest, was considered as chemical standard. Results were compared with those obtained in a close 4.11 ha field (Plot B) with Isomate C- Plus dispensers (Shin-Etsu, Japan). The efficacy of the mating disruption technique was periodically evaluated with assessments of fruit damage and adult catches in monitoring traps, both in the mating disruption areas and in the reference plots. In addition, a comparative study of pheromone release patterns from new dispensers and a reference one (Isomate C-Plus), under field conditions, was conducted. The new dispensers were, at least, as highly efficient as Isomate C-Plus. In plots A and B, adult catches were highly inhibited respect to those recorded in the reference plot. Moreover, damage levels were below the treatment thresholds, so less supplemental chemical application were required. In addition, new dispensers showed more adequate first-order kinetics than Isomate C-Plus, under field conditions. Thus, new dispensers could be an eco-friendly alternative for the control of C. pomonella, as they show good release patterns and they are biodegradable.

Key words: Cydia pomonella, field assay, mating disruption, controlled-release, pheromone dispenser

Introduction

Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the most important pest of pear and apple worldwide (Witzgall et al., 2001). Chemical control programmes in the orchard growing area of Lleida, Spain, comprises continuous applications of insecticides along the activity period of adults and larvae. This heavy pesticide programme is expensive and disruptive to beneficial orchard fauna, requiring, in addition, the supplemental application of spraying against secondary pests. Resistance to these insecticides, the possibility of cross- resistance to others and the limited number of products registered against codling moth, are motivating the implementation of the pheromone-based IPM systems. In these programmes, the mating disruption technique (MD) is the most promising tactic used to control this key pest in pome fruit production areas around the world (Barnes and Bloemfield, 1997). One of the main factors contributing to the success of the MD is the right diffusion of the pheromone in the treated area; delivery of effective amounts of pheromone as well as a good distribution is essential. Although several types of dispensers are commercially available,

107 108

many of them suffer from a variety of problems. In some dispensers, pheromone load falls off very rapidly and, as a consequence, dispenser field-life is not long enough to cover the flight periods of the pest. In addition, most devices are not very environmentally benign (plastic discs, polyethylene tubes and rubber septa, etc.) not being biodegradable, which causes an accumulation of polymeric materials in the treated environment. These limitations inspired the search of environmentally safe materials which could be used as controlled-release delivery systems for pheromones. Among the numerous materials being studied, sepiolite, a natural mineral type-clay, seems to have the potential to be an appropriate one, due to its physical and chemical properties. Thus, these new pheromone dispensers consist on tablets of compressed sepiolite, containing the sex pheromone, several stabilizers and compacting additives. In order to adjust pheromone emission to field requirements, release studies of tablets with different formulations were previously conducted under controlled laboratory conditions, comparing them with commercial dispensers Isomate C-Plus (Shin-Etsu). Here, we report on the MD assays performed in 13.95 ha of commercial orchards in Lleida, Spain, during years 2005 and 2006, using these new dispensers and the Isomate C-Plus ones. Release patterns of both types of pheromone dispensers were studied under our field assay conditions, and the efficacy of the MD, using both dispenser types, was evaluated.

Material and methods

Materials Natural sepiolite with size particles from 240 to 420 µm (Tolsa S.A., Madrid, Spain) was used as support for the three main components of the sexual pheromone of codling moth; codlemone, dodecanol and tetradecanol. Codlemone (E8,E10-dodecadienol) was provided by Bedoukian Reserach Inc. (Danbury, CT., USA), with 91.7% purity; dodecanol (12:OH) and tetradecanol (14:OH) were provided by Fluka Chemical Corp. (Milwaukee, USA), with 95% and 97% purities, respectively. In order to avoid the degradation of the pheromone, several stabilizers were incorporated to the dispenser’s formulation; compacting additives were also included to prevent the disintegration of tablets when exposed to field conditions. Commercial pheromone dispensers Isomate C-Plus were supplied by Shin-Etsu (Tokyo, Japan).

Elaboration of the new dispensers The new dispensers were industrially manufactured (Ecología y Protección Agrícola, Carlet, Spain), by impregnating the sepiolite with a solution of the three main pheromone components (codlemone, 12:OH and 14:OH) and several additives, in dichloromethane (DCM). After evaporating the exceeding solvent, equivalent amounts of impregnated sepiolite were compacted in a hydraulic press in order to obtain 18 mm-diameter tablets, with the same pheromone load as Isomate C-Plus, previously determined by chemical analysis (160, 78 and 17 mg of codlemone, dodecanol and tetradecanol, respectively). Individual dispensers were hanged to trees in an inverted plastic cup to avoid direct exposure to sunlight and rain.

Distribution of pheromone dispensers The new dispensers were placed in a 9.84-ha orchard (5.9-ha apples and 3.94-ha pears); Isomate C-Plus dispensers were placed in a close 4.11-ha apple orchard. In both plots, previous MD assays had been performed with Isomate C-Plus dispensers since 2004 and 2002, respectively. A reference orchard adjacent to the MD areas (Plot C in 2005, and Plot D, in 2006), where chemical control was applied against codling moth only when justified by the 109

level of the pest, was considered. Field test was carried out from April 21st until September 13th, in 2005, and from April 20th until September 12th, in 2006. All pheromone dispensers were placed in MD plots at a density of 1000 per ha. In border areas, an higher number of dispensers were hanged up in order to create a pheromone barrier. All dispensers were hanged on the upper part of the canopy before the beginning of the first flight of the pest (April 18th in 2005, and April 20th in 2006).

Flights of C. pomonella Male flights of codling moth in reference plots were recorded in delta traps with sticky bases and L2 lures (1 mg) (Trécé Inc., Salinas, USA). In MD orchards (plots A and B) high dose (10 mg) lures BioLure (Suterra Inc., Bend, USA) were used. Pheromone traps were weekly checked both in the reference Plot and in the MD areas. Reduction of catches in plots A and B was expressed as an Inhibition of Catches Index (ICI), following the formula ICIMD=100-[(CMD/CSD)x100], where ICIMD is the reduction of catches in pheromone areas (%), and CMD and CSD are the catches registered per trap and day in MD and reference plots, respectively.

Damage levels In order to establish damage levels in standard orchards (Plots C and D) and to evaluate the efficacy of the MD in plots A and B, fruits were periodically checked. Fruits from the perimeter rows were checked fortnightly and fruits from the centre areas were only checked when damage levels in the borders were detected. One thousand fruits per ha and 15 fruits per tree were observed each time.

Patterns of release of dispensers Release studies of new dispensers and Isomate C-Plus were performed by aging them under field conditions. Periodically, 4 samples of each dispenser type were randomly taken for analysis. Duration of the assay was previously fixed in 5 months. For each kinetic determination, 4 samples per dispenser type were analysed, as fixed in previous assays. In all cases, dispensers were kept in a labelled stock pack bag and then placed in the freezer until extraction was performed. Residual pheromone from the new tablets and Isomate C-Plus was obtained by individual solid-liquid extraction, using 50 ml of DCM. The new tablets were previously disaggregated in order to facilitate the extraction of the pheromone and, for Isomate C-Plus, some cuttings along the rope were made. Then, the new dispensers were individually wrapped in a cellulose bag and placed into cellulose thimbles. Extractions were performed in a Soxtec 2043 Unit System (Foss, Hillerød, Denmark), during 5 hours. Isomate C-Plus dispensers were extracted in soxhlets (25 ml) during 7 hours (60 cycles, ca.). These extraction times were previously determined as the shortest times to avoid pheromone detection when the emitter was subjected to a second extraction. The amount of pheromone remaining in the dispenser was quantified by gas-liquid chromatography with 200 µl hexadecane (150 mg/ml) as internal standard. Hexadecane with a purity of 99% was provided by the Aldrich Chemical Company Inc., (Milwaukee, WI., USA). Analysis were performed in a Perkin Elmer Clarus 500-GC with FID detector (Perkin Elmer, Wellesley, USA), in a Zebron ZB-5 column (30 m; 0.25 mm i.d.; 0.25 µm film thickness) with a crosslinked 5% phenylmethylsiloxane phase from Phenomenex (Torrance, USA). Operating parameters were: injection port, 250ºC; detection port, 300ºC; gas, He (at 1ml/min); oven program, 120ºC (5 min), 120-180ºC (3ºC/min), 180ºC-230ºC (30ºC/min). Four analyses were performed per sample, with 1µl injections volume and 50 ml/min split. 110

Values corresponding to residual codlemone from dispensers were graphically represented versus time and then adjusted to first order kinetic rate equations. At the end of the field trial, dispensers´ half-lives, total amount of pheromone emitted and release rates were compared.

Statistical analysis Differences in inhibition of catches and damage levels observed in Plots A and B, were determined by the Kruskal-Wallis tests, and followed by a multiple comparison test. Analyses were performed with the STATGRAPHICS PLUS 5.1 statistical package (CPD, UPV Valencia).

Results and discussion

Flights of C. pomonella Pheromone trap catches in the reference orchards (Plots C and D) from April to September 2005 and 2006 are shown in Figure 1. In both years, the first adult males of C. pomonella were caught at the beginning of May, but the seasonal flight patterns of moths were not well defined, differing considerably between the two years of assay.

6,0

5,0 Plot C (2005)

4,0 Plot D (2006) 3,0

2,0 Catches/trap·day

1,0

0,0 18-Apr 28-May 7-Jul 16-Aug 25-Sep

Figure 1. Seasonal flights of adult males of C. pomonella in reference plots, in 2005 and 2006.

Table 1. Mean and standard error of the inhibition of catches (%) in the MD plots (Plot A provided with new dispensers and Plot B with Isomate C-Plus) in 2005 and 2006.

Year Plot A Plot B 2005 96.2 ± 2.1 a 92.4 ± 3.3 a 2006 79.4 ± 10.9 b 55.3 ± 12.6 b

n=34 (2005) and n=23 (2006). Values in the same row followed by the same letter do not show statistical differences (P<0.05).

In the MD areas, the total number of adults caught in high-lure traps was much lower compared to those registered in the reference plots, especially in 2005 (trap catches ranged from 0 to 0.04 moths · trap-1 · day-1 in Plot A, and from 0 to 0.11 moths · trap-1 · day-1, in Plot 111

B). In 2006, however, males showed less difficulties to find high dose pheromone lures in monitoring traps, ranging from 0 to 0.14 moths/trap·day in Plot A, and from 0 to 0.5 moths/trap·day in Plot B. There were not significant differences in the inhibition values registered in both MD plots, both in 2005 and in 2006 (Table 1).

Damage levels In reference orchards, 8 and 7 chemical applications against codling moth were required along the growing season in 2005 and 2006, respectively, to ensure damage levels at harvest below the economic threshold (2%). With these sprayings, the average levels of infested fruits at harvest in apples, the most susceptible crop, were 0.09% and 0.39%, in 2005 and 2006, respectively (Figure 2).

2 Border 1,6 Center 1,2

0,8

Infested(%) fruits 0,4

B C D t C ot o ot ot Plot A Pl Pl Plot A Pl Plot A Plot B Pl Plot A Apple Pear Apple Pear

2005 2006

Figure 2. Damage levels at harvest in MD areas (Plots A and B) and in reference plots (Plots C and D).

In Plots A and B, the MD alone against the 1st and the 2nd generation of the codling moth was not efficient enough to ensure a good control of the pest until the end of the growing season, especially in the border areas. As a consequence, several supplemental chemical sprayings were required in both MD plots. In Plot A, using the new dispensers, most of the damage caused by the larvae was done on apples; consequently, specific treatments (1 and 2 chemical sprayings in 2005 and 2006, respectively) were only applied on apple trees. Then, only 0.17% of apples in the centre areas were damaged in 2005, and around 1% in 2006, at harvest. In pear trees, the 4 and 8 chemical sprayings applied to control other pests, were enough to ensure a total control of the codling moth. In plot B, with Isomate C-Plus dispensers, 3 chemical sprayings against the codling moth were necessary to achieve the control of the pest in 2005. In 2006, as MD alone was not able to control efficiently the pest, 6 additional chemical sprayings were needed to keep the fruit damage at harvest at 0.02% . Chemical sprayings were based on azinphos methyl, chlorpyriphos methyl, phosmet, phosalone, lambda cyalothrin and flufenoxuron. 112

Release patterns of dispensers under field conditions In 2005, the release patterns of both dispenser types where similar, with adequate first-order kinetics when exposed to field conditions (Figure 3). In 2006, however, the new dispensers maintained almost the same release characteristics while Isomate C-Plus showed higher emission rates. As a consequence, during the second year of assay, half-life of Isomate C-Plus was only 53.32 days (Table 2). These differences in the performance of Isomate C-Plus dispensers could be presumably attributed to the slight increase (around 1.5°C) of the average temperatures (medium and maximum) recorded in 2006.

120 New dispensers (2005) 100 New dispensers (2006) 80 Isomate C-Plus (2005) Isomate C-Plus (2006) 60

20 Residual codlemone (%) Residual codlemone 0 0 25 50 75 100 125 150 Time (days)

Figure 3. Codlemone release curves of the new dispensers and Isomate C-Plus under field conditions.

During 2005, the new dispensers and Isomate C-Plus released similar amounts of codlemone/ha, as they showed similar release rate and patterns (110.73 and 98.58 mg/ dispenser, respectively) and were placed at the same density. As the new dispensers released only 12.5 mg more than Isomate C-Plus, along the 143 days of exposure, differences in the performance of the MD in plots A and B, in 2005, should not be attributed to differences in pheromone emission. In fact, similar levels of activity were achieved. In 2006, and despite the higher release of codlemone of Isomate C-Plus dispensers in Plot B (123.17 mg), especially during the period corresponding to the first flight of the pest (Figure 4), the inhibition of catches was lower than in Plot A, where each new dispenser released an average of 81.6 mg of codlemone. In the field trials, a direct relationship between the degree of inhibition of catches in the MD areas and the efficacy of the MD was observed. In fact, the best efficacy of the technique was registered in Plot A during 2005, when catches where mostly inhibited (96.2%). It agrees with Jones et al. (1998) in the way that inhibiting catches less than 95%, the efficacy of the technique decreased, as the concentration of pheromone in the air could let males detect females allowing the copula. On the other hand, the inhibition of catches was not in agreement to the total amount of pheromone released per hectare by the release rates of the two types of dispensers and so, by the total amount of pheromone released. In fact, in Plot B, during 2006, when higher amounts of codlemone were released (more than 123 mg per dispenser) males showed less difficulties to find females (only 55.3% of inhibition of catches). In 2005, however, the best inhibition 113

was observed in Plot A (more than 96%) despite releasing less pheromone than Isomate C- Plus in plot B (Table 2). This apparent contradiction could be explained taking into account that in Plot B, possessing a lesser size than Plot A, adverse effects due to other environmental factors such as wind speed can be promoted lowering the efficacy of the MD.

Table 2. Release parameters of dispensers under field conditions.

2005* 2006* Release parameter New Isomate New Isomate dispensers C-Plus dispensers C-Plus K (days -1) 0.0074 0.0092 0.0067 0.013 R2 0.9905 0.9918 0.972 0.9803 Half-life (days) 93.67 75.34 103.45 53.32 Codlemone released (mg/dispenser) 110.73 98.58 81.60 123.07

Parameters were obtained from the first order-rate equations, after 143 days of exposure of dispensers under field trial conditions.

1,6 e 1,2 New dispensers Isomate C-Plus 0,8

0,4

(mg/dispenser·day) 0,0

Release rates of codlemon of rates Release 5 6 005 006 0 0 005 006 2 2 20 20 2 2 1st Flight 2nd Flight 3rd Flight

Figure 4. Release rates of codlemone released by new dispensers and Isomate C-Plus.

In summary, the MD with the new dispensers and the commercial Isomate C-Plus, supplemented with chemical spraying, was effective for the control of the codling moth in the orchards of the growing area of Lleida, Spain. The highest efficacy was achieved using new pheromone dispensers, which released the main pheromone component, codlemone, at the average rate of 0.73 mg/day. Moreover, in the same plot the chemical treatments were reduced (87.5% and 71.4%, less, in 2005 and 2006 respectively). In Plot B, with Isomate C- Plus dispensers releasing codlemone to average rates of 0.77 mg/day, reduction of pesticides was around 62.5%, in 2005, and it was unusually low (14.3%) in the second year. Nowadays, two important factors that affect the implementation of the MD are the insufficient control in plots with high population densities and the high price of pheromones (Gordon et al. 1995). In this study it was showed that, under our growing conditions, with 114

pest levels traditionally high (requiring up to 8 chemical applications to keep damage levels below 2% of infested fruits), the MD was not efficient enough, requiring supplemental insecticide spraying. However, insecticide applications were saved in comparison with reference plots in 2005 and 2006. In addition, as population levels in MD programs regularly decrease, a progressive improvement in codling moth control in successive seasons under a reduced insecticide programme is expected.

References

Barnes, B.N. & Blomefield, T.L. 1997: Goading growers towards mating disruption: the South African experience with Grapholita molesta and Cydia pomonella (Lepidoptera, Tortricidae). – IOBC/wprs Bulletin 20 (1): 45-56. Gordon, D.; Zahavi, T.; Anshelevich, L.; Harel, M.; Ovadia, S.; Dunkelblum, E. & Harari, A.R. 1995: Mating disruption of Lobesia botrana (Lepidoptera: Tortricidae): effect of pheromone formulations and concentrations. – Journal of Economic Entomology 98: 135-142. Jones, O.T. 1998. Practical applications of pheromones and other semiochemicals. – In: Insect pheromones and their use in pest management. Howse, P.E., Jones, O.T. & Stevens, I. (eds.), Chapman and Hall, London, UK. Witzgall, P.; Bengtsson, M.; Rauscher, S.; Liblikas, I.; Bäckman, A-C.; Coracini, M.; Ander- son, P. & Löfqvist, J. 2001: Indentification of further sex pheromones in the codling moth, Cydia pomonella. – Entomologia Experimentalis et Applicata 101: 131-141.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 115-122

Towards high performance mating disruption of codling moth, Cydia pomonella (L.)

Lukasz L. Stelinski, Larry J. Gut, Peter McGhee, James R. Miller Department of Entomology, Michigan State University, East Lansing, MI 48824, USA; [email protected]

Abstract: Recently, we have explored novel approaches of achieving “high-performance” mating disruption, defined as exceeding 98 % efficacy even under high population densities and without the need for companion insecticides. This work has been grounded in earlier investigations of disruption mechanisms, which suggested that competitive attraction between calling females and synthetic pheromone sources is an important component of effective and economical disruption. For Oriental fruit moth (OFM), Grapholita molesta (Busck), we have developed a mechanized applicator for high- speed deployment of pheromone dispensers made of paraffin or microcrystalline wax to tree fruit. One ha of crop can be treated in ca. 20 min. By modifying our applicator to dispense larger 0.3 g wax drops at an intended rate of 20 / tree and increasing the pheromone concentration to 10 % (by weight) in an improved wax formulation manufactured by ISCA Technologies, we achieved high-performance disruption of OFM for more than 100 days with a single application of wax in early spring. For codling moth (CM), Cydia pomonella (L.), we are investigating experimental “Cidetrack” dispensers manufactured by Trécé Co. intended to better protect the active ingredient, codlemone, and to co- release both codlemone and the pear ester (ethyl (E,Z)-2,4-decadienoate). In 2005, Trécé Cidetrack dispensers loaded with codlemone (40%) and pear ester (60 %) disrupted CM better than standard Isomate-C Plus dispensers. In 2006, disruption of male CM with Trécé Cidetrack dispensers loaded with pear ester and codlemone (60:40 ratio) was not superior to Cidetrack dispensers loaded with 100 % codlemone; however, Cidetrack treatments loaded with codlemone were superior or equivalent to Isomate treatments. Deploying high densities (3,600 - 18,500 pieces / ha) of Cidetrack CM pieces containing ca. 1/7th of the standard codlemone loading each resulted in 92–99 % disruption of male CM. These results suggest that this new dispenser technology may enhance disruption of CM by better protecting codlemone from degradation, compared with Isomate dispensers, rather than because of an additive effect of the plant kairomone. Furthermore, high densities of Trécé Cidetrack pieces that are 1/7th the size of a standard dispenser provided "high performance" disruption, and thus superior efficacy compared to that achieved using a standard application of dispensers 1000 / ha.

Key words: Grapholita molesta, Cydia pomonella, mating disruption, false-plume-following, competitive attraction, kairomone, pear ester, wax

Introduction

Over the past five years we have been investigating the physiological and behavioral mechanisms underling pheromone-based mating disruption in tortricid moth pests of fruit (eg., Stelinski et al., 2004; 2005a; 2006a). Collectively, this series of studies has led us to conclude that false-plume following to synthetic pheromone dispensers in the field is a critical mechanism mediating disruption of tortricid moths. In cases where high release dispensers are deployed, habituation rather than adaptation may play an additional role in mediating disruption; however, recovery of normal sensitivity occurs in pheromone-free air. Thus habituation is contingent upon an initial bout of false-plume following and its relative contribution to disruption depends on the level of pheromone exposure.

115 116

We have also dedicated considerable energy into refinement of a paraffin-wax formulation for dispensing pheromones (Atterholt et al., 1998) with the aim of deploying it as drops at high densities to maximally exploit competitive attraction. Paraffin wax drops (0.1 ml) containing 5 % pheromone and deployed at 30 and 100 per tree (8,200 and 27,300 / ha) completely disrupted mating of Oriental fruit moth (OFM), Grapholita molesta, under heavy population densities as measured by tethered female moths and disrupted orientation of feral males to optimally-baited traps above 99 % relative to control plots (Stelinski et al., 2005b). In addition, the level of mating disruption using high-application densities of wax drops was superior to that with label-recommended applications of Isomate M-Rosso dispensers, which resulted in ca. 17 % mating of tethered virgin females. Extensive field observations revealed that male OFM briefly (< 30 s) approached within 130 cm of wax drops. In 2005, we developed a tractor-mounted mechanized applicator capable of precisely deploying up to 100 wax drops per tree and covering one ha in ca 20 - 25 min (Stelinski et al., 2005b). A hydraulic piston precisely meters the emulsified wax formulation through a high- pressure hose running up a manoeuvrable boom positioning a spinning-cup dispenser spun by a hydraulic motor. Droplet size and number delivered per time is governed by the number and size of holes at the bottom of the side-walls of the spinning cup, in conjunction with its rpm’s. A 2005 test with very high populations of OFM yielded 100 % disruption of tethered female mating and 99 % disruption of pheromone traps with machine-applied wax drops during the spring generation of moth flight (Stelinski et al., 2005b). But during hot summer months, excellent disruption lasted not quite ten days, because of insufficient droplet size (0.05 ml) to prolong pheromone release at the hot temperatures. A modified version of this applicator that deposits ca. twenty 0.3 mg drops of a newly-formulated wax formulation containing 10 % pheromone by weight and manufactured by ISCA Technologies provided season-long high performance disruption (>99%) of OFM with a single application of wax in early spring (Stelinski et al., unpublished). A formulation similar to that described for OFM above is in development for CM; however, currently this formulation is effective for only a limited 2 week period in the field likely due to degradation of codlemone. Substantial improvements in CM mating disruption will likely require major improvements in codlemone protection offered by commercially-manufactured dispensers. This will be of particular importance for CM disruption given the chemical nature of codlemone and its propensity for degradation in commercial disruption formulations (eg., Millar, 1995). Our goal was to improve disruption of CM and reach the high performance standard that has been achieved for OFM as described above. We define high performance disruption as: exceeding 98 % efficacy even under high population densities and without the input of companion insecticides. To do so, we have been investigating a new dispenser technology under development by Trécé Corporation, which has been designed to 1) protect codlemone from degradation, and 2) co-release an attractive plant volatile (pear ester, (ethyl (E,Z)-2,4- decadienoate) with codlemone.

Material and methods

Initial comparison of Cidetrack CM-PE dispensers with Isomate C Plus (Experiment 1) This experiment directly compared disruption efficacy of a newly developed dispenser for semiochemicals manufactured by Trécé with the industry standard in North America, Isomate-C Plus. The Cidetrack dispenser is made from rubbery PVC polymer and can be formulated to release codlemone alone, pear ester alone, or both compounds simultaneously. Cidetrack CM-PE dispensers were loaded with codlemone and pear ester and their efficacy was directly compared to that of Isomate-C Plus dispensers. The two dispenser treatments 117

were applied at an equivalent rate of 1000 units / ha. Treatments were applied to 16 tree plots (0.07 ha) in experimental orchards described in Stelinski et al. (2004). Five replicates were conducted in a randomized complete block design. Disruption of CM was measured using pheromone-baited traps and tethered virgin females. Two delta traps, baited with 0.1 mg codlemone lures, were placed within each plot. Traps were hung ca. 2 - 3 m above ground level in the upper third of the tree canopy. Moths captured in traps were counted and removed twice weekly. Tethered virgin females were deployed in each plot according to the protocol described in Stelinski et al. (2005b). Females were deployed for 24 h intervals in treatment plots and mating status was determined by dissections examining the bursa copulatrix for presence or absence of a spermatophore.

Small-plot evaluation of Cidetrack dispensers (Experiment 2) This experiment tested the hypothesis that mating disruption of CM can be improved under high moth population densities using a newly developed dispenser by Trécé Incorporated. The impact of the following treatments on CM disruption was compared in a randomized complete block design with five replicates: Trécé Cidetrack dispensers releasing 1) codlemone alone, 2) releasing pear ester alone, 3) releasing the same combination of codlemone (40%) and pear ester (60%) as tested in Experiment 1, 4) releasing a combination of codlemone and pear ester in a 40: 60 ratio but at half of the loading rate of treatment 3, 5) a no pheromone control, and 5) Isomate-C Plus dispensers as a positive control. Treatments were applied to 25 tree plots (0.11 ha) in experimental orchards described above. Disruption was measured using pheromone baited traps and virgin females as described for Experiment 1 above.

Large-plot evaluation of Cidetrack dispensers (Experiment 3) This experiment was conducted as a companion to experiment 2 (above), but in large (2.0 ha) grower-maintained plots. The same treatments were evaluated except for pear ester alone, treatment number 2, which was not included in the large plot trial. Dispenser treatments were applied at a rate of 1000 units / ha. Four replicates were conducted in a randomized complete block design. Disruption of CM was measured using a trapping grid consisting of 9 LPD (Large Plastic Delta) traps baited with Trécé L2 lures per replicate plot. The trapping grid was deployed in the center of each block so that each trap covered 0.2 ha.

High-density deployment of Cidetrack pieces (Experiment 4) This experiment tested the hypothesis that disruption of CM above 98 % could be achieved by deploying higher densities of smaller Trécé Cidetrack pieces loaded with 1/7th of the total codlemone of the standard Cidetrack CM dispenser deployed at 1000 / ha. Trécé Cidtrack dispensers were chopped into 7 smaller pieces of approximately equal mass (ca. 30 mg codlemone / piece). These were stapled to bread clips and deployed at various densities in 0.15 ha plots in a non-commercially maintained apple orchard. The densities directly compared were 0, 250, 1,250, 6,250, and 18,750 / ha. Disruption of CM was measured with pheromone traps baited with 0.1 mg of pheromone as described above.

Results and discussion

Initial comparison of Cidetrack CM-PE dispensers with Isomate-C Plus Our data from the initial evaluation of Trécé Cidetrack dispensers releasing both codlemone and pear ester were exciting; disruption was substantially better than that achieved using Isomate-C Plus dispensers, the industry standard for the past 15 years (Figure 1 A, B). Mean captures of moths and mating of tethered virgin females (Figure 1 A, B) were statistically compared by analysis of variance and separation of pairs of means for each sampling date to show significant (P < 0.05) efficacy changes over time. For the first 4 wks of this test, 118

inhibition of moth catch in pheromone traps was substantially better for Trécé dispensers releasing both codlemone and pear ester than for Isomate-C Plus dispensers releasing just pheromone. Moreover, mating of tethered virgin females under high moth densities was completely impeded over this period in plots treated with Trécé dispensers (Figure 1 A). In contrast, mating in Isomate-C Plus treated plots averaged ca. 15% during the initial four weeks of the study (Figure 1 A). This is the first time we have been able to achieve complete disruption of CM mating under high pest pressure. However, the high degree of efficacy achieved with Trécé dispensers lasted only four weeks. For the remainder of the season, effectiveness of Trécé dispensers was similar to that of Isomate-C Plus. The Trécé Cidetrack dispenser contains an equivalent amount of codling moth pheromone to that in Isomate-C Plus dispensers. The content of PE was somewhat higher.

1 Control 0.8 Cidetrack CM:PE (40:60) Isomate-C 0.6 Plus

0.4 CM females 0.2 Proportion of mated of mated Proportion 0 7/11/05 7/25/05 8/8/05 8/22/05 B. 40 Control Cidetrack CM:PE (40:60) 30 Isomate-C Plus 20 Figure 1. A. Proportion of tethered virgin CM females mated after 24 10 hr deployment in plots treated with various pheromone treatments in Mean no. trap no. Mean per CM 0 small-plot trials. B. Mean weekly 7/8/05 7/22/05 8/5/05 8/19/05 9/2/05 9/16/05 captures of male CM in pheromone Date traps.

However, based on our release-rate studies of both compounds from paraffin wax, the PE volatilizes ca. 3 times faster than codlemone, which suggested that depletion of PE after 4 wks of deployment may have been responsible for the abrupt decrease in efficacy for Trécé dispensers after 4 wks in the field. Other hypotheses that still needed to be ruled out by direct evidence were that: 1) the release rate and active ingredients may not have fallen below threshold, but something else changed in the orchard environment, 2) a breakdown of codlemone or PE occurred with concurrent build up of antagonists such as codlemone acetate that rendered dispensers unattractive after 4 wks, or 3) superior efficacy for Cidetrack was due to better protection of codlemone than Isomate-C Plus rather than due to the effect of the kairomone degradation. Unfortunately, during this first test, Cidetrack dispensers releasing

119

250 a

200 36 % b 150

100

50 92 % 92 % 91 % cc95 % c /CM Treatment Mean number of c 0 Control PE (100) CM:DA CM:DA CM (100) Isomate (full (half B. rate) rate) 0.3

28 % 0.2

0.1 87 % 93 % 90 %

/ TreatmentCM 97 % 0 matedProportion of Control PE (100) CM:DA CM:DA CM (100) Isomate (full (half rate) rate) C. 15 a

5 81 % 90 % b 90 % 92 % b b CM /CM Treatment b Mean number of 0 Control CM:DA CM:DA CM (100) Isomate (full (half rate) rate)

Treatment

Figure 2. A. Mean number of male CM captured in pheromone traps per treatment in small- plot (0.11 ha replicates) experiment. B. Proportion of tethered virgin females mated after 24 hr deployment in plots treated with various pheromone treatments in small- plot trials. C. Mean number of male CM captured in pheromone traps per treatment in large plot (2 ha replicates) trial. Percentage above means indicates percentage disruption calculated as 1 - (mean moth catch per trap in the pheromone-treated plot / mean moth catch per trap in the control plot) x 100. Means followed by the same letter are not significantly different (P > 0.05). 120

codlemone only were not available for direct comparison. Thus, based on this initial test of efficacy relative to that of Isomate-C Plus, it was unknown whether this higher disruption efficacy was due to the co-release of the pear-ester plant volatile or do to other properties of the dispenser such as protection of codlemone.

Small-plot evaluation of Cidetrack dispensers. Significantly (P < 0.01) fewer male CM were captured in traps in plots treated with PE only (100) compared with the control (Figure 2 A). Significantly fewer male CM were captured in plots treated with each Cidetrack treatment containing pheromone compared with Cidetrack PE and the control (Figure 2 A). Although there were no significant differences between Cidetrack treatments containing pheromone and Isomate-treated plots, % disruption was greatest in plots treated with Cidetrack CM (codlemone only) (Figure 2 A). Disruption of virgin female CM mating was consistent with trap disruption data (Figure 2 B). Disruption of virgin female mating was 10 % greater with Cidetrack CM (codlemone only) than with Isomate C Plus; however, the difference in disruption between Cidetrack with and without PE was only 5-7 %.

Large-plot evaluation of Cidetrack dispensers Data from the large-plot trials were highly consistent with the small-plot results. Significantly (P < 0.01) fewer male CM were captured in each pheromone treatment compared with the control; however, there were no significant (P > 0.05) differences between the pheromone treatments. The highest level of disruption was achieved with dispensers containing codlemone only (Cidetrack CM); disruption was 10 % greater with this treatment compared with Isomate (Figure 2 C).

High-density deployment of Cidetrack pieces Disruption of CM males increased proportionally with an increase in density of Cidetrack CM (100% codlemone) pieces per hectare. High performance disruption (> 98 %) was achieved with 18,500 dispensers per ha (ca. 75 / tree) (Figure 3). However, CM male orientation to traps was not completely disrupted even with 18,500 / ha (Figure 3).

200

160

120

80 56 %

Number of CM / Trap 90 % 92 % 99 % 0 0 250 1,200 3,600 18,500 Number of Cidetrack codlemone only pieces per ha

Figure 3. Disruption of male codling moth orientation to pheromone traps as a function of Cidetrack CM piece density. Each Cidetrack piece comprises 1/7th of a total mass of a standard Cidetrack dispenser (ca. 30 mg of codlemone per piece) 121

Cidetrack dispensers containing codlemone with and without the addition of pear ester performed equally or better than Isomate-C Plus dispensers in disrupting CM male orientation to pheromone-baited traps and mating of tethered virgin female CM. Addition of PE to Cidetrack dispensers did not increase the level of CM disruption; however, a half rate PE:CM combination treatment was equivalent to the 100% CM and full rate combination treatment. These data suggest that other factors, such as optimized release or protection of codlemone, may be responsible for the improved CM disruption achieved using Cidetrack dispensers as compared with Isomate dispensers. Also, this demonstrates that the level of codlemone active ingredient per dispenser can be reduced by half and still provide comparable efficacy as compared with Isomate-C Plus. Future research should focus on the impact of the pear ester active ingredient in Cidetrack combination dispensers on the behavior of female moths. Deployment of high densities (> 18,000 / ha) of Cidetrack CM dispenser pieces per unit area of crop resulted in a very high level of CM disruption and was superior to that achieved by 1000 standard Cidetrack CM dispensers / ha. This improved disruption was likely due to a greater level of competitive attraction at the higher deployment densities. However, 18,500 pieces per ha (75 per tree) of Cidetrack CM plastic pheromone-impregnated material were necessary to achieve 99 % disruption of male CM orientation to traps. This suggests that “high-performance” disruption of CM at high population densities will require both excellent protection of codlemone from degradation as well as high deployment densities of synthetic point sources. It is intriguing that Cidetrack dispensers loaded with pear ester only disrupted male CM orientation to pheromone traps by 36 % and disrupted mating of tethered virgin female CM by 28 %. This suggests that deploying a kairomone impacts pheromonal communication of CM. In companion flight-tunnel studies, we have found that Cidetrack dispensers loaded with pear ester alone (ca. 200 mg) were as attractive as optimal 0.1 mg codlemone lures to males CM, when those Cidetrack pear ester dispensers were fume-hood aged for 16 – 100 days (Stelinski et al., unpublished). Although flight-tunnel data indicate that Cidetrack PE dispensers are highly attractive to male CM, disruption of CM male orientation using this treatment in the field was poor. This suggests that desensitization of male CM response to codlemone following an attraction event to a synthetic codlemone dispenser may play a role in effective disruption of male CM. Our recent flight-tunnel investigations have shown that male CM are highly susceptible to desensitization following brief orientations and exposures in plumes from Isomate-C Plus dispensers (Stelinski et al., 2006a). Alternatively, it is also possible that we observed more total CM approaching PE-containing dispensers given that female CM (especially mated females) are attracted to this kairomone (Knight, 2006). Finally, our deployment density experiment (number 4) suggests that a high density of point sources is also a critically important factor in disrupting CM, likely maximizing the role of competitive attraction.

References

Atterholt, C.A., Delwiche, M.J., Rice, R.E. & Krochta, J.M. 1998: Study of biopolymers and paraffin as potential controlled-release carriers for insect pheromones. – J. Agric. Food Chem. 46: 4429-4434. Knight, A.L. 2006. Assessing the mating status of female codling moth (Lepidoptera: Tortricidae) in orchards treated with sex pheromone with traps with ethyl (E/Z)-2,4- decadienoate. – Environ. Entomol. 35: 894-900. 122

Millar, J.G. 1995: Degradation and stabilization of (E,E)-8,10-dodecadien-1-ol, the major component of the sex pheromone of the codling moth (Lepidoptera: Tortricidae). – J. Econ. Entomol. 88: 1425-1432. Stelinski, L.L., Gut, L.J., Pierzchala, A.V. & Miller, J.R. 2004: Field observations quantifying attraction of four tortricid moth species to high-dosage, polyethylene-tube pheromone dispensers in untreated and pheromone-treated orchards. – Ent. Exp. Appl. 113: 187-196. Stelinski, L.L., Miller, J.R. & Gut, L.J. 2005a: Occurrence and duration of long-lasting peripheral adaptation among male moths of three species of economically important tortricids. – Ann. Entomol. Soc. Am. 98: 580-586. Stelinski, L.L., Gut, L.J., Mallinger, R.E., Epstein, D., Reed, T.P. & Miller, J.R. 2005b: Small plot trials documenting effective mating disruption of Oriental fruit moth, Grapholita molesta (Busck), using high densities of wax-drop pheromone dispensers. – J. Econ. Entomol. 98: 1267-1274. Stelinski, L.L., Gut, L.J. & Miller, J.R. 2006a: Orientational behaviors and EAG responses of male codling moth after exposure to synthetic sex pheromone from various dispensers. – J. Chem. Ecol. 32: 1527-1538. Stelinski, L.L., Miller, J.R., Ledebuhr, R. & Gut, L.J. 2006b: Mechanized applicator for large- scale field deployment of paraffin-wax dispensers of pheromone for mating disruption in tree fruit. – J. Econ. Entomol. 99: 1705-1710.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 123

Pheromone mating disruption of Cydia pomonella (L.) in California pears: Balancing dispenser emission rates and program performance

S. Welter, F. Cave Department of Environmental Science, Policy, and Management, 201 Wellman Hall, University of California, Berkeley, CA, USA, [email protected]

Abstract: Alternative pheromone mating disruption programs were explored that mixed multiple types of emitting devices either to improve program performance for high densities of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) or to optimize application costs with program performance. Combinations programs using either high dose aerosol emitters (Suttera Corp) at low numbers per ha (ca. 1 emitter per 2 ha) or sprayable formulations of codlemone were used to supplement treatments using traditional hand-applied dispensers. No additional efficacy was observed with the inclusion of sprayable formulations of codlemone. Stability of sprayable formulations was significantly influenced by position in canopy and canopy structure with fully light exposed microcapsules no longer emitting detectable codlemone within several days of application using EAG or extraction protocols. The addition of high dose emitters at low numbers of dispensers per ha increased control in all plots with significant codling moth pressure. The combination program increased program performance with relatively low increase in program cost. Use of experimental emitters that released at intermediate levels (e.g. 20-30 mg per day) were deployed at ca. 38 dispensers per ha. A modified membrane dispenser and a new wax matrix dispenser were evaluated in 2006 within orchards with high and low populations of codling moth. Low pressure orchards were untreated with insecticides for the season, whereas high pressure programs received insecticidal supplements given the preliminary nature of the trial. Seasonal suppression of traps >90% were achieved at all levels of codling moth pressure and no significant damage has been observed to date.

Key words: Cydia pomonella, pheromone, mating disruption

123 124 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 125

Olfactory sensitivy of different populations of Cydia pomonella (L.) to sex pheromone E8E10-12:OH and kairomone ethyl (2E, 4Z)-2,4-decadienoate

A. De Cristofaro1, G. Anfora2, G.S. Germinara1, C. Ioriatti2, S. Vitagliano1, M. Guida1, G. Rotundo1 1 Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis, I-86100 Campobasso, Italy, [email protected] 2 IASMA Research Center - Plant Protection Department, Via E. Mach 1, 38010 – San Michele all’Adige (TN), Italy, [email protected]

Abstract: Codling moth (CM), Cydia pomonella (L.) (Lepidoptera: Tortricidae), has a close ecological association with its main host plants: apple, pear, and walnut. Ethyl (2E, 4Z)-2,4- decadienoate (Et-2E,4Z-DD), a kairomonal compound derived from ripening pears, has been found to be highly attractive to the CM larvae and both male and female (virgin and mated) adults in walnut and apple orchards. The main component of CM sex pheromone (E,E)-8,10-dodecadien-1-ol (E8E10- 12:OH) (Codlemone) is widely used in monitoring systems and direct control methods. The knowledge of the olfactory sensitivity to both compounds of the different populations coming from different host-plants may have a practical impact in the choice of the more appropriate quantities of attractants to be used in the diverse ecological contexts. In the present study electrophysiological responses (electroantennography, EAG) E8E10-12:OH and Et-2E,4Z-DD of several CM populations coming from different host plants (apple, pear, walnut) were recorded. The dose-response curves to the volatile compounds were calculated and differences in olfactory sensitivity of virgin and mated males and females of the different populations are discussed. Between physiological status (virgin and mated) of both sexes and among populations from different host-plants no significant differences were observed. As it was expected, females were highly less sensitive to codlemone than males, and males showed higher responses to the kairomone, too, thus confirming the results of our previous studies. It was also observed that adults of the walnut populations show a higher sensitivity (not significantly different) to both compounds. Instead, significant differences were sometimes recorded in populations coming from the same host plant. Setting up the more effective dosage of attractants to be used in CM monitoring systems or control methods, the variability in olfactory sensitivity of different populations living on the same plant may be at least as important as the variability showed by populations living on different hosts.

Key words: Cydia pomonella, pear ester, codlemone

125 126 Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 127-132

Use of ethyl (E, Z)-2,4-decadienoate for the control of Cydia pomonella (L.) on apple orchards

Silvia Schmidt1, Cristina Tomasi2, Massimiliano Melandri4, Gianfranco Pradolesi4, Edison Pasqualini3, Claudio Ioriatti2 1 SafeCrop Centre, IASMA , Via E. Mach, 1 - 38010, S. Michele a/A (TN), Italy, [email protected] 2 IASMA Research Center - Plant Protection Department, Via E. Mach, 1 - 38010 – S. Michele a/A (TN), Italy, [email protected] 3 DiSTA (Dipartimento di Scienze e Tecnologie Agroambientali), Facoltà di Agraria dell’Università di Bologna, Viale G. Fanin 42, I-40137 Bologna, Italy, [email protected] 4 Agronomica R&D - Coop. Terremerse, Via S .Alberto 325, I-48100 Ravenna, Italy

Abstract: The ethyl (E,Z)-2,4-decadienoate (pear ester) is an adult and larval kairomonal attractant for Cydia pomonella (L.) (Lepidoptera: Tortricidae). The possibility of using a microencapsulated formulation of pear ester (Da-Mec, a.i. 5%, Trécé inc.) to interfere with the host location behaviour has been evaluated. Field efficacy trials were carried out on apple orchards with a sprayable formulation to verify the potentiality of the kairomone for direct pest control. The tests performed revealed a significant effect of the Da-Mec formulation in reducing the fruit injury compared to an unsprayed control. These preliminary results support the use of the pear ester in direct control of codling moth for improving the efficacy of larval contact insecticides.

Key words: codling moth, pear ester, host location disruption, pest control

Introduction

A few plant and fruit volatiles are able to affect as single compounds the behaviour of adults and larvae of Cydia pomonella (L.) (Lepidoptera: Tortricidae). (E,E)-α-farnesene attracts adults and neonate larvae over a short range (Wearing and Hutchins, 1973; Hern and Dorn, 1999). In olfactometer the esters hexyl and butyl hexanoate attract females (Hern and Dorn, 2004). The pear ester, (E,Z)-2,4-ethyldecadienoate (Et-E,Z-DD), allows to capture males and females of codling moth in the field (Light et al., 2001; Ioriatti et al., 2003; De Cristofaro et al., 2004) and laboratory bioassays showed an attractant effect of that compound also on neonate larvae (Knight and Light, 2001). Knight and Light (2001) suggested that the application of pear ester in sprayable formulation could increase the time neonate larvae spend walking on foliage prior to entry into the fruit. As a consequence, the mortality of larvae could be enhanced because of the longer exposure time to biotic and abiotic factors. Hughes et al. (2003) evaluated the use of apple odour and (E,E)-α-farnesene on larvae and mated females to disrupt host location in laboratory trials and they obtained a certain effect with the apple odour but not with the (E,E)- α-farnesene. Pasqualini et al. (2005a; 2005b) assessed the effect of a microencapsulated formulation of the pear ester, Da-Mec (a.i. Et-E,Z-DD 5 %, Trécé Inc.), on the egg-laying behaviour of mated females and on the fruit searching behaviour of neonate larvae. They reported a disorienting effect of Da-Mec on egg laying females in semi-field trials.

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Preliminary laboratory studies on larvae indicated that pear ester may disrupt host location also of the neonates, both on apple and pear cultivars. The aim of the present study was to verify the potentiality of the kairomone in direct pest control strategies. Field efficacy trials in apple orchards with the formulation of pear ester Da- Mec (a.i. 5 %, Trécé inc.) were carried out. Improvement of insecticide performance by blending pear ester and insecticide was also investigated.

Material and methods

During the seasons 2004, 2005 and 2006 efficacy trials were carried out in a 10 years old experimental apple orchard at IASMA Research Center, in S. Michele all’Adige (Trento, Italy). Double rows of G. delicious variety on M9 rootstocks are interspersed with single rows of Florina which act as guard rows. The plant order is 3.5 m x 1.20 m, the high of the trees is approximately 3 m, the trailing system Spindel. The treatments were adjusted in a randomized complete block design with four blocks and performed in plots including 40 trees distributed on two rows. The trials were carried out against the first generation of codling moth. The efficacy was measured by the extend to which fruit damage attributable to C. pomonella was reduced by each treatment, compared with the unsprayed control. The applications were performed using an experimental sprayer (Dieter Waibl) and a water volume of 15 hl ha-1. The treatments tested are reported in Table 1.

Table 1. Treatments tested in the three year field trials in S. Michele all’Adige.

Treatment Dose Year Commercial product Active ingredient (ml/hl) 2004 Da-Mec 5 % Et-E,Z-DD 12 2005 2005 Da-Mec 5 % Et-E,Z-DD 1 2006 Madex 3 x 1013 granule/l CpGV 7 2004 Madex + Da-Mec 3 x 1013 granule/l CpGV +5 % Et-E,Z-DD 7 + 12 2004 Madex 1.5 x 1013 granule/l CpGV 16 2005 Madex + Da-Mec 1.5 x 1013 granule/l CpGV +5 % Et-E,Z-DD 16 + 12 2005 Madex + Da-Mec 1.5 x 1013 granule/l CpGV +5 % Et-E,Z-DD 16 + 1 2005 2004 Gusathion 20 SC 18.4 % Azinphos-methyl 250 2005 Alisé 75 WG 75 % Chlorphyriphos-ethyl 70 (g/hl) 2006 Alisé 75 WG 75 % Chlorphyriphos-ethyl 35 (g/hl) 2006 Alisé 75 WG 75 % Chlorphyriphos-ethyl 70 + 1 2006 + Da-Mec + 5 % Et-E,Z-DD Alisé 75 WG 75 % Chlorphyriphos-ethyl 35 + 1 2006 + Da-Mec + 5 % Et-E,Z-DD Untreated control 2004, 2005, 2006 The formulation of the commercial product Madex changed from 3 x 1013 granules/l into 1.5 x 1013 granules/l and subsequently the dosage applied in the field was adjusted to 16 ml of product hl -1 129

In 2004, the first spray was timed at the beginning of eggs hatching on May 25th. The following applications were performed at 8 days intervals (every other weeks for azinphos- methyl) with the last one carried out on July 2nd. Six applications were needed to cover the first generation flight. On July 8th, 1000 fruits/treatment were examined for codling moth damage. Damage caused by either active still living larvae (with presence of wet brown frass) or by larvae stopped in its activity (superficial stings) was distinguished. In 2005, two treatments were added to the experimental design in order to evaluate the activity of a lower dosage of the kairomone, alone and blended with CpGV (Table 1). The first spray was timed on May 10th at the beginning of oviposition, in order to completely exploit the disorienting effect of the pear ester on egg laying females. The following applications were made at intervals of approximately 8 days (every 14 days for azinphos- methyl) with the last one carried out on June 28th. Seven sprays with kairomone and CpGV were necessary to cover the first generation flight. Fruit damage was checked on July 6th by inspecting 1320 fruits/treatment. In 2006, the first spray was positioned at beginning of oviposition on May 19th. Successively the applications were carried out at about 14 days interval. Four sprays were executed; the final one was done on June 30th. Fruit damage was checked on July 6th by inspecting 1300 fruits/treatment. An additional efficacy field trial was carried out during the first generation period in 2005 in S. Antonio (Ravenna, Italy). The apple orchard consists of 12 years old plants variety Golden B, plant order 4 m x 2 m. The treatments were used in a randomized complete block design with four blocks and a single plant-plot. The treatments tested were an untreated control, azinphos-methyl (Gusathion 20 SC) at 250 ml of product hl -1, azinphos-methyl (Gusathion 20 SC) at 125 ml of product hl -1, azinphos-methyl (Gusathion 20 SC) at 125 ml of product hl -1 + Et-E,Z-DD (Da-Mec 5% a.i.) at respectively 1 ml, 6 ml or 12 ml of product hl - 1 and Et-E,Z-DD (Da-Mec 5% a.i.) at 12 ml of product hl -1. The applications were made with a backpack sprayer using a water volume of 10 hl ha-1. The first application was sprayed at beginning of eggs hatching on May 23th and the following two applications were performed at 8 days interval. Fruit damage was checked on June 20th by inspecting 100 fruits/treatment. Fruit damage was processed by analysis of variance. Data obtained were subjected to one-way ANOVA after Bliss angular transformation. Levene’s test was used to verify homogeneity of variances. Duncan test was used to compare unsprayed control vs. mean of all other treatments.

Results and discussion

The field efficacy trials carried out in 2004 and 2005 in S. Michele all’Adige showed a control activity of Da-Mec. In both seasons, the fruit damage occurred in the Da-Mec treated plots was significantly lower than the one recorded in the unsprayed control plots. In 2005, the lower dosage of kairomone (1 ml hl-1) gave the same result than the dosage of 12 ml hl-1. The addition of Da-Mec to the CpGV product, at the tested dosages, did not improve the control activity of the granulovirus on C. pomonella. No significant differences in the percentage of total damaged fruits were found between Da-Mec and CpGV formulation. A different type of damage was found in the kairomone treated apples compared to the untreated control: a significant lower percentages of injury caused by still living larvae was observed. (Table 2). In 2006, the lower dosage of the pear ester (1 ml hl-1) did not provide the same result as the previous year. In fact no control effect of the Da-Mec (1 ml hl -1) could be evidenced at the end of the codling moth first generation. This was probably due to the longer interval 130

between applications (14 instead of 8 days). Nevertheless a significant difference compared to the untreated control was assessed in the preliminary visual check performed ten days before. As the previous year, the trial confirmed an effect of Da-Mec on the type of damage caused to a certain extent by larvae successively stopped in their activity (Table 3). The results of the trial carried out in 2005 in S. Antonio are shown in table 4. The best efficacy was obtained with the tank mix Da-Mec (1 ml/hl) and Azinphos-methyl at half the recommended dose. With the highest dosages of the kairomone, the percentage of damaged apples unexpectedly increased even if not significantly. All the treatments where insecticide was blended with pear ester significantly differed to the untreated control.

Table 2. Percentages of damaged apple fruits obtained in the field trials carried out in 2004 and 2005 in S. Michele all’Adige (TN).

2004 2005 Treatment % damaged % active % damaged % active apples larvae apples larvae Untreated 51.4 a 41.9 a 24.3 a 19.5 a Da-Mec 12 ml/hl 18.2 b 6.5 b 12.0 b 6.5 b Da-Mec 12 ml/hl + Madex 16.0 b 1.9 b 10.4 b 2.7 b Madex 17.9 b 4.3 b 12.1 b 3.4 b Da-Mec 1 ml/hl 11.6 b 5.0 b Da-Mec 1 ml/hl + Madex 15.8 b 4.3 b Gusathion SC 0.8 c 0.0 c 0.8 c 0.2 c 2004 ANOVA on % damaged apples F=23.43, p=0.00; on % active larvae F=59.24, p=0.00 2005 ANOVA on % damaged apples F=5.53, p=0.003; on % active larvae F=37.85, p=0.00 Means within each column followed by the same letter are not significantly different by Duncan test (P=0.05)

Table 3. Percentages of damaged apple fruits obtained in the field trial carried out in 2006 in S. Michele all’Adige (TN), preliminary evaluation (27.06.2006) and final check (06.07.2006).

27 June 2006 6 July 2006 Treatment % damaged % active % damaged % active apples larvae apples larvae Untreated 13.2 a 11.7 a 17.0 a 15.0 a Da-Mec 1 ml/hl 9.0 b 5.5 b 12.6 a 7.0 b Alisè 70 g/hl 2.3 c 1.5 c 2.3 b 1.4 c Alisè 35 g/hl 3.5 c 2.8 c 3.8 b 2.5 c Alisè 70 g/hl+Da 1 ml/hl 2.5 c 2.2 c 3.9 b 2.3 c Alisè 35 g/hl+Da 1 ml/hl 4.5 c 3.3 c 5.8 b 4.3 bc 27 June ANOVA on % damaged apples F=20.48, p=0.00; on % active larvae F=20.86, p=0.00 6 July ANOVA on % damaged apples F=9.96, p=0.00; on % active larvae F=13.11, p=0.00 Means within each column followed by the same letter are not significantly different by Duncan test (p=0.05)

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Table 4. Percentages of injured fruits per plots observed in field trial carried out in S. Antonio (2005).

% damaged Treatment apples Untreated 8.00 b Gusathion SC 250 ml/hl 0.75 a Gusathion SC 125 ml/hl 3.25 ab Da-Mec 1 ml/hl + Gusathion SC 125 ml/hl 0.00 a Da-Mec 6 ml/hl + Gusathion SC 125 ml/hl 1.25 a Da-Mec 12 ml/hl + Gusathion SC 125 ml/hl 1.75 a Da-Mec 12 ml/hl 2.75 ab ANOVA on % of damaged apples F=3.12, p=0.0281. Means within each column followed by the same letter are not significantly different by SNK test (p=0.05).

It was not possible to improve the control efficacy of the insecticides Madex and Alisè 75 WG by blending them with the pear ester formulation at the tested dosages and in the experimental conditions applied. However, a trend in that sense could be evidenced by combining the kairomone with half the recommended dose of Gusathion SC. We hypothese that the different results could be due to: • dissimilar physical contact of the larvae with the insecticides because of the formulations; • different time period larvae are exposed to the toxic effect because of the different mode of action of the insecticides. In conclusion this study showed that the kairomone Da-Mec (a.i. 5%) formulation was able to reduce the codling moth fruit injury. Investigations are in progress to identify the insecticide that can take advantage of blending with pear ester in order to lower the recommended field dosages of the insecticide.

Acknowledgements

Research supported by the Autonomous Province of Trento (Research Projects Bioinnova and SafeCrop Centre). We thank Dr. Bill Lingren (Trécé Inc., Adair, OK, USA) for supplying Da- Mec (a.i. Et-E,Z-DD 5%).

References

De Cristofaro, A.; Ioriatti, C.; Pasqualini, E.; Anfora, G.; Germinara, G.S.; Villa, M. & Rotundo, G. 2004: Electrophysiological responses of codling moth, Cydia pomonella (L.) (Lepidoptera Tortricidae) to codlemone and pear ester ethyl (E,Z)-2,4-decadienoate: peripheral interactions in their perception. – Bull. Insect. 52: 137-144. Hern, A. & Dorn, S. 1999: Sexual dimorphism in the olfactory orientation of adult Cydia pomonella in response to α-farnesene. – Ent. Exp. Appl. 92: 63-72. Hern, A. & Dorn, S. 2004: A female-specific attractant for the codling moth, Cydia pomonella, from apple fruit volatiles. – Naturwiss. 26: 77-80. 132

Hughes, W.O.H.; Gailey, D. & Knapp, J. 2003: Host location by adult and larval codling moth and the potential for its disruption by the application of kairomones. – Ent. Exp. Appl. 106: 147-153. Ioriatti, C.; Molinari, F.; Pasqualini, E.; De Cristofaro, A.; Schmidt, S. & Espinha, I. 2003: The plant volatile attractant Ethyl (2E, 4Z)-2,4-decadienoate (DA 2313) for codling moth monitoring. – Boll. Zool. Agr. Bachic. 35 (2): 127-137. Knight, A. & Light, D.L. 2001: Attractants from Bartlett pear for codling moth, Cydia pomonella (L.) larvae. – Naturwiss. 88: 339-342. Light, D.; Knight, A.; Henrick Clive, A.; Rajapaska, D.; Lingren, B.; Dickens, J.C.; Reynolds, K.M.; Buttery, R.G.; Merill, G.; Roitman, J. & Campbell, B.C. 2001: A pear-derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.) – Naturwiss. 88: 333-338. Pasqualini, E.; Schmidt, S.; Espinha, I.; Civolani, E.; Ioriatti, C.; De Cristofaro, A.; Molinari, F.; Villa, M.; Ladurner, E. & Sauphanor, B. 2005a: Effects of the kairomone ethyl (2E, 4Z)-2,4-decadienoate (DA 2313) on the oviposition behaviour of Cydia pomonella (L.) (Lepidoptera Tortricidae). – Bull. Insect. 58: 119-126. Pasqualini, E.; Villa, M.; Civolani, S.; Espinha, I.; Ioriatti, C.; Schmidt, S.; Molinari, F.; De Cristofaro, A.; Sauphanor, B. & Ladurner, E. 2005b: The pear ester ethyl (E,Z)-2,4- decadienoate as a potential tool for the control of Cydia pomonella larvae: preliminary investigation. – Bull. Insect. 58: 65-69. Wearing, C.H. & Hutchins, R.F.N. 1973: α-farnesene, a naturally occurring oviposition stimulant for the codling moth, Laspeyresia pomonella. – J. Insect Physiol. 19: 1251- 1256.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 133-140

Experimental use of the micro-encapsulated pear ester kairomone for control of codling moth, Cydia pomonella (L.), in walnuts

Douglas Light Plant Mycotoxin Research Unit, Western Regional Research Center, Agricultural Research Service, USDA, Albany, CA 94710, USA, [email protected]

Abstract: The larval attractant kairomone, ethyl (2E, 4Z)-2,4-decadienoate or pear ester (PE), was tested in a Californian walnut orchard for its control efficacy against codling moth larvae, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in a microencapsulated bait spray formulation (PE-MEC) combined with various insecticides (choropyrifos, phosmet, methoxyfenozide and a granulosis virus, Cyd-X). PE-MEC was tank-mixed as an adjuvant with reduced rates of insecticides and applied by a handgun-sprayer to replicate walnut trees in an 8 ha orchard. Results were significant, with PE-MEC adjuvant + insecticide treatments reducing CM harvest damage by 77% (choropyrifos), 85% (phosmet), 54% (methoxyfenozide)and 47% (Cyd-X) below the damage incurred with insecticides alone. Also with the secondary pest, the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae), the PE-MEC adjuvant significantly reduced damage levels when combined with the two OP insecticides. The PE kairomone shows promise for improving insecticide efficacy and contributing to both resistance management and new IPM tactics for C. pomonella and the accompanying A. transitella.

Key words: codling moth, Cydia pomonella, kairomone, pear ester, attract – kill, insecticide adjuvant, navel orangeworm, Amyelois transitella

Introduction

Food Quality Protection Act 1996 enacted by the U.S. government is banning the use of most organophosphate (OP) insecticides. Alternative insecticides must be made more effective and affordable. Knight and Light (2001) reported that the pear ester (PE), ethyl (2E, 4Z)-2,4- decadienoate, kairomone was highly attractive to neonate codling moth larvae, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Since the PE was found to increase rates of both crawling and turning and to stimulate orientation to point sources by C. pomonella larvae (Knight and Light, 2001), then PE-MEC applications might promote “wandering” thereby increasing temporal and spatial exposure to insecticides (Pasquilini et al., 2005a; Schmidt et al., 2007, Vitagliano et al., 2007). Experimental objective was to field test the larval control efficacy of bait-sprays of PE kairomone co-sprayed with various insecticides. PE, micro-encapsulated into a sprayable formulation, “PE-MEC,” was tank-mixed as an adjuvant with reduced rates of insecticides and applied by a handgun-sprayer to individual replicate walnut trees in a 8 ha orchard. To challenge the control efficacy of the insecticides, the rate of application was reduced down to 50% below the insecticides’ label rates. Also, treatments were judged for impact on the level of infestation by the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae), which typically penetrates the nut through the entries by C. pomonella.

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Material and methods

Treatments Objective was to reveal potential synergism and/or improvement in control efficacy of insecticide sprays when using the PE-MEC spray adjuvant targeted against neonate C. pomonella and A. transitella larvae. Design was to challenge the insecticides’ effectiveness by using low label-rates or reduced rates of as low as ½ the standard label-rate of insecticide, thereby potentially revealing the contribution of the tank-mixed PE-MEC spray adjuvant. Treatments were reduced or ½ label-rates of insecticides-alone vs. insecticides tank-mixed with PE-MEC adjuvant (“DA-MEC,” experimental formulation, Trécé, Inc., Adair, OK, USA) (Table 1). Three rates (approx. 0.59, 1.75, & 4.35 grams PE A.I./ha) of the PE-MEC adjuvant were variously tested in combination with four insecticides. Three classes of insecticides were tested, including two organophosphates (choropyrifos, Lorsban, Dow CropScience and phosmet, Imidan, Gowan, Inc.), an insect growth regulator (methoxyfenozide, Intrepid, Dow CropScience), and a formulation of the CM granulosis virus (Cyd-X Virus, Certis, Inc.). Also tested were the PE-MEC alone and a “blank” or empty capsule MEC formulation (Trécé, Inc.), with all treatments compared with “control” trees that were not sprayed throughout the season. The phosmet treatments were buffered to 4.5 – 5.0 pH using a buffer (Trifol, Wilbur- Ellis Co.).

Design and application technique Study was conducted in a mature, 8 ha Chandler variety walnut orchard (Esparto, CA) that received no grower applied insecticide applications throughout the season. Test orchard was comprised of 31 rows with 30 trees/row, or 118 trees/ha. The 13 treatments were replicated eight times (total of 104 sprayed trees, each of a 3 row X 3 tree separation) in a randomized complete block design throughout the orchard. All other orchard trees (total of 826 trees) served as ‘buffers’ and were not sprayed. From these buffer trees, 20 random trees were picked as ‘control’ trees for damage assessment comparisons. An ATV (all terrain vehicle) mounted gasoline engine powered, diaphragm pump sprayer (Honda) (operating at 110 psi) was used to apply by handgun (D5 nozzle, Spray Systems, Inc.) foliar sprays at ca. 13.3 L/tree, or an equivalent spray volume of 15.6 hL/ha.

Treatment timing Application timing was based on female biofix monitoring using kairomone-baited traps (Pherocon DA lures, Trécé, Inc.) and the predicted onset or peak in egg hatch (Knight and Light, 2005a; b). Cool weather, rain delays, and the subsequent late emergence of female CM caused the first spray applications to be conducted after the peak egg hatch following the 1B flight. Due to persistent and increasing flight intensities (from 2 to 12 moths/trap/night), spray applications were conducted six times for all insecticide treatments, while the virus insecticide was applied an additional two times. Treatment applications were sprayed on 7th and 10th June for the post 1B flight, 6th – 8th July, 19th – 22nd July, and 2nd – 5th August for the 2nd flight, and 23rd – 26th August and 7th – 9th September for the 3rd flight (Figure 1).

Damage assessments A preharvest sampling of nut damage and worm infestation was conducted by ‘nut knock- down’ on the 21st of September 2005, about three weeks prior to the grower’s shake-down harvest of nuts on the 10 – 12th October 2005. This preharvest sampling was performed three weeks prior to harvest in order to assure that the knocked-down and collected nuts had intact and attached husks, so that assessments of husk confined damage and larval presence could be accomplished. Hired laborers used 3.7 m fiberglass poles to randomly knock-down 100+ nuts at canopy heights ranging from 1.8 - 6 m from each treatment tree and control tree. Random 135

samples of 100 nuts were collected then examined and cut open to assess husk stings and penetrations, shell staining and entries, and kernel damage or instar presence of C. pomonella and/or A. transitella (differentiated by a specific thoracic “u” shaped chevron black marking on worms, webbing, and presence of multiple worms of many age – classes). Collected nuts had their husks, shells and kernels examined for: 1) source of damage/ defect whether worm, mold or shrevel, or combinations, 2) location of damage in husk, shell, and/or kernel, 3) presence and number of larvae and species, 4) if A. transitella present then evidence of prior C. pomonella infestation, and 5) age-class or particular flight period in which damage occurred (fresh, new 3rd flight with moist frass, mid-season 2nd flight with dried frass and mold, or old 1st flight with dried, shriveled and powdery mold). The proportion of damaged nuts was subjected to angular transformation and ANOVA. Means were separated with Fisher protected LSD, and Duncan’s multiple range test, P < 0.05.

18 # 2: # 3: # 4: # 5: # 6: SPRAY DATES: # 1: 6/7, 6/10 7/6-8 7/19-22 8/2-5 8/23-26 9/7-9 16 IA IB II III 14 (7/14 (7/27 virus) virus) 12

DA MEAN 10

6 mean mothsmean caughtpernight 4

0 4/1 5/1 6/1 7/1 8/1 9/1 10/1

Figure 1. The dates of experimental spray applications (arrows) in relationship to female Cydia pomonella emergence, biofix, initiation of flights, flight peak, and flight duration and intensity as delineated by daily moth capture rates using PE kairomone or “DA” lure baited traps.

Results and discussion

Population monitoring and spray timing The 2005 Spring was characterized, as similarly for the last four years, by cool and wet weather, with sunset temperatures not exceeding 16° C until mid-April. These cool wet conditions are believed to retard the emergence of female C. pomonella, who require a sustained period of sunset temperatures exceeding 16° C. However, sunset temperatures during April did exceed the male 14° C temperature threshold for flight activity, contributing to the occurrence of a distinct period of ‘protandry,’ male emergence preceding females’. In this orchard, male biofix was observed on 11th April, based on the strong male 1A flight 136

initiation detected by both pheromone-baited traps and traps baited with pheromone + kairomone or ‘combo’ lures (Pherocon CM-DA Combo lures, Trécé, Inc.). The initial female biofix was not registered until on 4th May, followed by a concerted sustained flight on 11 th May, the biofix date for the 1B flight. Only the PE-based ‘DA’ lure- baited traps detected the 1A and 1B biofixes and the actual emergences of the over wintered female moths. The six spray applications were timed for the periods of hatch and emergence of neonate larvae (Knight and Light 2005a; b). Because of 1) the prolonged high trap capture levels or population pressure of C. pomonella females and 2) the shortened residual concentration of the lower rate insecticide applications, it was judged appropriate/necessary to perform five repeated spray applications during the 2nd and through the 3rd flights.

Damage assessment of ‘controls’ and MEC – alone ‘standards’ 2005 was a high pressure year for C. pomonella in this orchard, with ‘control’ trees averaging 29.4% (+ 1.7% SEM) damage by C. pomonella (Table 1). Damage attributable to A. transitella averaged 8.4% (+ 1.1%). Of total ‘worm’ damage found, C. pomonella damage represented 77.8%, while A. transitella damage represented 22.2%. Virtually all A. transitella damage and infestation presence was attributable to them following a prior shell penetration and kernel infestation by C. pomonella larvae. Therefore, all C. pomonella caused damage reported represents both C. pomonella alone caused damage and that of dual species presence. The C. pomonella damage rates for the tested ‘standards,’ PE-MEC and ‘Blank-MEC,’ were the same, averaging 23.9% (+ 2.1%) and 26.4% (+ 2.3%) respectively (F = 0.65, P = 0.44), and were not significantly different from the infestation level of the unsprayed ‘control’ trees (P = 0.08 and 0.33, respectively) (Table 1). Similarly, no differences were found in A. transitella infestations between the PE-MEC and ‘Blank-MEC’ treated trees and ‘control’ trees. Thus, it’s concluded that neither the PE-MEC nor the MEC formulation itself (‘Blank MEC’) had any obvious, overt effect on either species’ infestation level or ability to attack walnuts. The spraying at the neonate hatch periods with PE-MEC adjuvant alone did not appear to directly effect larval survival in comparison to the nearby unsprayed ‘control’ trees. However, we could not assess effects of the PE-MEC adjuvant on female attraction to treated trees, or perhaps stimulation of egg laying frequency (Knight & Light, 2004) and egg placement (Pasqualini et al., 2005b; Schmidt et al., 2007) on those kairomone treated trees.

Damage assessment of insecticides augmented with PE-MEC adjuvant First, damage surveys showed the expected, that these registered insecticides alone, when applied at these reduced or low rates, did significantly decrease damage by C. pomonella (68 to 77% reduction) (F = 61, P < 0.0001) in comparison to non-sprayed control trees (Table 1). Moreover, the tank-mixing of the PE-MEC adjuvant with these four different insecticides at reduced or low rates caused highly significant reductions in C. pomonella damage below the damage found with the paired applications of the insecticide-alone (Table 1). Various tank-mixed rates of the PE-MEC adjuvant were tested in combination with the two OP insecticides, with choropyrifos paired with 0.59, 1.75 and 4.35 grams PE A.I./ha and phosmet paired with 1.75 and 4.35 grams PE/ha. No significant differences between the rates of PE-MEC tested were found for either OP, except for the 0.59 vs. 4.35 g PE/ha rates in combination with choropyrifos ) (F = 4.9, P = 0.044) (Table 1). An apparent improvement or decrease in C. pomonella damage percentage was observed for both of the OP treatments, with the higher rate(s) of the PE-MEC reducing damage over that of the lower experimental rate (Table 1).

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Table 1. Insecticide treatments tested with and without the pear ester microencapsulated adjuvant (PE-MEC), their equivalent rates of application per hectare, and the incurred percentage of walnut damage, with husk and kernel damage combined, attributed to Cydia pomonella larvae observed in nut knock-down assessments; Esparto, California, 2005.

Statistical Comparison Percentage Percentage Insecticide alone vs. Reduction Equivalent Rate per Harvest Insecticide + by Treatments: Hectare: Damage: PE-MEC: Adjuvant:

PE- Mean + ANOVA LSD Insecticide: MEC: SEM* F value P value

Choropyrifos 2.76 l/ha - 7.6 + 1.3bc Choropyrifos + 2.76 l/ha 0.59 g/ha 3.1 + 0.8de 9.1 0.009 59% PE-MEC low rate Choropyrifos + 2.76 l/ha 1.75 g/ha 1.5 + 0.4e 21.0 0.0004 80% PE-MEC medium rate Choropyrifos + 2.76 l/ha 4.35 g/ha 1.3 + 0.1e 22.9 0.0003 83% PE-MEC high rate

Phosmet 3.29 kg/ha - 9.4 + 0.9b Phosmet + 3.29 kg/ha 1.75 g/ha 2.0 + 0.4de 50.9 <0.0001 79% PE-MEC medium rate Phosmet + 3.29 kg/ha 4.35 g/ha 0.9 + 0.4e 71.3 <0.0001 90% PE-MEC high rate

Methoxyfenozide 0.86 l/ha - 6.8 + 0.8c Methoxyfenozide + 0.86 l/ha 1.75 g/ha 3.1 + 0.8de 9.6 0.008 54% PE-MEC medium rate

Granulosis Virus 260 ml/ha - 8.3 + 1.0b Granulosis Virus + 260 ml/ha 1.75 g/ha 4.4 + 0.8cd 9.2 0.009 47% PE-MEC medium rate

PE-MEC alone - 1.75 g/ha 23.9 + 2.1a 3.4 0.08 19% Medium rate NS Blank-MEC alonec - - 26.4 + 2.3a 1.0 0.33 10% (medium rate) NS

Controls (no sprays) - - 29.4 + 1.7a (n = 20) * Means followed by a different letter significantly different, Duncan’s multiple range test, P < 0.05.

Analysis of the relative proportion of the total C. pomonella caused damage that occurred during the three specific flight periods is portrayed in Figure 2. For all insecticide treatments, MEC standards, and unsprayed controls a higher proportion of the seasonal damage detected is attributed to having occurred during the 2nd flight, followed by the 1B flight damage and then the damage during the 3rd flight (Figure 2). One interesting observation is the lack of detected 3rd flight damage for phosmet + PE-MEC and methoxyfenozide + PE-MEC, relative 138

to the 19% damage with these insecticides alone and an average of 19.7 (+ 6.5%) for the other treatments.

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0% Lorsban Lorsban + Lorsban + Lorsban + Imidan Imidan + Imidan + Intrepid Intrepid + Virus Virus + PE-MEC Blank- Controls PE 0.2 PE 0.6 PE 1.7 PE 0.6 PE 1.7 PE 0.6 PE 0.6 MEC FLIGHT IB DAMAGE FLIGHT II DAMAGE FLIGHT III DAMAGE

Methoxy Choropyrifos - Phosmet - Virus – -fenozide Blank-MEC PE-MEC Lorsban Imidan Cyd-X Controls -Intrepid % of total + PE 0.2 + PE 0.6 + PE 1.7 + PE 0.6 + PE 1.7 + PE 0.6 + PE 0.6 Alone damage alone alone alone

FLIGHT IB 38 28 33 40 45 63 14 28 48 14 23 29 36 27

FLIGHT II 41 52 33 50 36 38 71 54 52 53 57 51 40 49

FLIGHT III 21 20 33 10 19 0 14 19 0 33 20 20 24 23

Figure 2: Relative proportions – percentages of the Cydia pomonella caused total damage incurred during the three flight periods, 1B flight, 2nd flight and 3rd flight, 2005 trial.

Treatment effects on secondary pest, Amyelois transitella All four insecticides applied alone significantly reduced A. transitella caused damage below the levels found for the ‘control’ trees (F = 63, P < 0.0001), with reductions in infestation of 71 to 98%. The tank-mixing of the PE-MEC adjuvant with either of the OP insecticides at ca. ½ label-rate caused significant reductions in A. transitella damage (PE-MEC + choropyrifos: 0.1 and 0%, or phosmet: 0.5 and 0%) below the damage found with these insecticides alone (choropyrifos: 1.1% and phosmet: 2.0%). The PE has not been found to attract A. transitella 139

adults in our nine years of field PE trap studies (Light et al., 2001, Light & Knight 2005) and there is no host plant basis to suspect that PE would attract or affect the behavior and host selection of A. transitella larvae. Thus, the observed improvement in A. transitella control by the addition of the PE-MEC adjuvant to the two OP insecticides is presumed to be an accompanying attribute of the direct control improvement effects on C. pomonella larvae (adults also?) and/or their behaviors. Thus, the efficacy of the OP contact poisons was improved for both C. pomonella and A. transitella by the use of PE-MEC adjuvant.

Summary and impact The PE adjuvant’s synergistic enhancement of the efficacy of the OP, IGR and microbial insecticides is portrayed in Table 1 as the percentage reduction in damage or improvement in the insecticides’ efficacy. The combination of the PE-MEC adjuvant with insecticide reduced C. pomonella damage over the reduced-rate insecticide alone by 59 - 83% for choropyrifos, 79 - 90% for phosmet, 54% for methoxyfenozide, and 47% for Cyd-X virus. Navel orangeworm, A. transitella, attributed damage was also significantly reduced by 75 – 100% for the rates of PE-MEC with the two OP insecticides. Combining the activity for all four insecticides, the mean (+ SEM) reduction in damage provided by the PE-MEC adjuvant in these particular experiments averaged 66.6% (+ 9.4%) for reduction in C. pomonella damage and 63.9% (+ 23.7%) for reduction in A. transitella damage. These studies show promise that the kairomone can improve insecticide efficacy and contribute to new IPM tactics for C. pomonella and A. transitella and the integrated reduction of Aspergillus mold infection and incidence of aflatoxin contamination of walnuts. The pear-ester compound has the potential to be used to manage C. pomonella in an ‘attract and kill’ (AK) bait-spray by allowing for the death and removal of neonate larvae by promoting a suggested increase in the temporal and spatial exposure to insecticides. The ability to selectively attract, prolong exposure and kill crawling C. pomonella larvae, could provide a critical and environmentally rationale means of direct and selective control using insecticides that will improve efficacy and resistance management (Chicon et al., unpublished; Sauphanor et al. submitted, Schmidt et al., 2007). C. pomonella neonates are known to be more attracted to, prefer, and feed/penetrate more readily into pome-fruits over walnuts (Mills, Walnut Research Reports 2003, unpublished). C. pomonella larvae were found to be relatively ‘hesitant’ to ‘accept’ and feed/penetrate into walnut husks. It has been demonstrated in laboratory studies of various designs that the PE kairomone is the most effective, genetically-based attractant know for neonate C. pomonella (Knight and Light 2001). The current intent is to exploit this C. pomonella larval preference by presenting pear ester in AK bait-spray MEC formulation droplets to larvae to increase larval temporal and spatial exposure to, and thereby lethality of, insecticides in walnut, apple and perhaps pear orchard environments. Many concurrent studies investigating the efficacy of the PE-MEC adjuvant in pome fruits have been underway for the last two-plus years internationally, in Europe (e.g., studies by Ioriatti, Pasqualini, Schmidt and Vitagliano), in South America (e.g., Chicon) and North America (e.g., Dunley, Gut, Hilton Knight, Reidl, Stelinski, etc.). This report conveys that the slow-release MEC formulation of the kairomone, PE-MEC, can effectively enhance and synergize the control efficacy of various insecticides, ranging from contact-poison OPs, to an ingestion-based IGR, and a microbial virus. These bait-spray adjuvant enhancement effects were attained using modest amounts of A.I. material, being effective at 1.75 grams/ha. These results were revealed, and allowed to reach statistically significant proportions, through our experimental design to test reduced or lower than label rates of these established insecticides. However, the practical commercial use of this kairomone would be as an tank-mixed spray adjuvant to insecticides at their registered ‘label rates.’ The goal and benefit of the adjuvant being, to provide a degree of increased efficacy, 140

but of a more subtle contribution nature, and moreover adding perhaps a higher dimension of assurance of control for the practitioner and grower.

Acknowledgements

The development and supply of the PE-MEC experimental formulation and technical assistance by Bill Lingren, Clive Henrick, Jeff Downs and Janet Haworth of Trécé, Inc. (Adair, OK, USA) is appreciated. Also, appreciated is the supply of insecticide materials by Certis, Corp., Dow Cropscience and Gowan, Inc.

References

Knight, A.L. & Light, D.M. 2001: Attractants from ‘Bartlett’ pear for codling moth, Cydia pomonella (L.), larvae. – Naturwissenschaften 88: 339-342. Knight, A.L. & Light, D.M. 2004. Use of ethyl (E,Z)-2,4-decadienoate in codling moth management: stimulation of oviposition. – J. Entomol. Soc. British Col. 101:53-60. Knight, A.L. & Light, D.M. 2005a: Developing action thresholds for codling moth (Lepidoptera: Tortricidae) with pear ester and codlemone-baited traps in apple orchards treated with sex pheromone mating disruption. – Can. Entomol. 137: 739-747. Knight, A.L. & Light, D.M. 2005b: Timing of egg hatch by early-season codling moth (Lepidoptera: Tortricidae) predicted by moth catch in pear ester- and codlemone-baited traps. – Can. Entomol. 137: 728-738. Light, D.M.; Knight A.L.; Henrick, C.; Rajapaska, D.; Lingren, B.; Dickens, J.C.; Reynolds, K.M.; Buttery, R.G.; Merrill, G.B.; Roitman, J.N. & Campbell, B.C. 2001: A pear- derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.). – Naturwissenschaften 88: 333-338. Light, D. M. & Knight, A. L. 2005: Specificity of codling moth (Lepidoptera: Tortricidae) for the host plant kairomone, ethyl (2E, 4Z)-2,4-decadienoate: field bioassays with pome fruit volatiles, analogue, and isomeric compounds. – J. Agric. Food Chem. 53: 4046- 4053. Pasqualini, E.; Villa, M.; Civolani, S.; Espinha, I.; Ioriatti, C.; Schmidt, S.; Molinari, F.; De Cristofaro, A.; Sauphanor, B. & Ladurner, E. 2005a: The pear ester ethyl (E,Z)-2,4- decadienoate as a potential tool for control of Cydia pomonella larvae: preliminary investigation. – Bull. Insectology. 58: 65-69. Pasqualini, E.; Schmidt, S.; Espinha, I.; Civolani, S.; Ioriatti, C.; De Cristofaro, A.; Molinari, F.; Villa, M.; Ladurner, E. & Sauphanor, B. 2005b: Effects of the kairomone ethyl (2E, 4Z)-2,4-decadienoate (DA 2313) on the oviposition behaviour of Cydia pomonella L. (Lepidoptera: Tortricidae). – Bull. Insectology. 58: 119-124. Schmidt, S.; Tomasi, C.; Melandri, M.; Pradolesi, G.; Pasquilni, E. & Ioriatti, C. 2007: Use of ethyl (E,Z)-2,4-decadienoate in the control of Cydia pomonella (L.) on apple orchards. – IOBC wprs Bulletin 30(4): 127-132. Vitagliano, S.; Germinara, G.S.; Lingren, B.; Ioriatti, C.; Pasqualini, E.; Rotundo, G. & De Cristofaro, A. Behavioral responses of Cydia pomonella (L.) neonate larvae to a microencapsulated formulation of ethyl (E,Z)-2,4-decadienoate. – IOBC wprs Bulletin 30(4): 141. Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 141

Behavioural responses of Cydia pomonella (L.) neonate larvae to a microencapsulated formulation of ethyl (2E, 4Z)-2,4-decadienoate

S. Vitagliano1, G.S. Germinara1, B. Lingren2, C. Ioriatti3, E. Pasqualini4, G. Rotundo1, A. De Cristofaro1 1 Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via De Sanctis, I-86100 Campobasso, Italy, [email protected] 2 Trécé Incorporated, Insect Monitoring Systems & Pheromones, 7569 Highway 28 West, PO Box 129, Adair, OK 74330, USA, [email protected] 3 IASMA Research Center - Plant Protection Department, Via E. Mach 1, 38010 – San Michele all’Adige (TN), Italy. [email protected] 4 Dipartimento di Scienze e Tecnologie Agroambientali, Università di Bologna, V. le G. Fanin 42, I-40127 Bologna, Italy, [email protected]

Abstract: Codling moth, Cydia pomonella (L.) (Lepidoptera Tortricidae) is known as a key pest of apples, pears and walnuts wordlwide. Ethyl (2E, 4Z)-2,4-decadienoate (Et-2E, 4Z–DD) has been found to be an effective attractant for both male and female of this pest, as well as for its larvae. The use of this attractant for direct control methods which target both male and female moths or larvae is important for Codling moth management. Field studies, carried out using a microencapsulated formulation of Et-2E, 4Z–DD (5% A.I. DA MEC, Trécé Inc.), showed results not always coherent to exclude a biological role played on Codling moth neonate larvae by the coformulants in the microencapsulated formulation. Therefore, the aim of this study was to evaluate the biological activity of this formulation compared to the active ingredient alone and the coformulants present in the commercial product on the behaviour of codling moth larvae. We show preliminary olfactory responses of C. pomonella neonate larvae to different doses (distilled water soluted) of the complete microencapsulated formulation (5% A.I. DA MEC, Trécé Inc.), its pure active compound (DA 2313 Neat, Trécé Inc.) and the coformulants (Blank MEC, Trécé Inc.) in separate behavioural bioassays (Petri dish multi-choice arena). As control stimulus only distilled water was tested. Preliminary results showed that at lower doses of each tested compound, neonate larvae did not show any preference in comparison with control. As expected, only responses to the microencapsulated formulation and to the active ingredient were dose-related. In response to the pure complete microencapsulated formulation, codling moth larvae showed the stronger preference (64%) toward the tested compounds. Whereas the pure coformulant was tested, neonate larvae showed a similar response to the control. So far, codling moth larvae behavioural responses lead on considering that the tested coformulants don’t have a biological role on neonate larvae, if considered separated from the active compound.

Key words: Cydia pomonella, pear ester, attraction, larva

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 143-148

Effect of Madex® (codling moth granulovirus) on Cydia pomonella (L.) egg laying on two apple varieties: Relationships with plant surface metabolites

Nadia Lombarkia1, Claudio Ioriatti1, Edouard Bourguet2, Sylvie Derridj2 1 IASMA Research Center - Plant Protection Department, Via E. Mach 1, 38010 – San Michele all’Adige (TN), Italy, [email protected] 2 INRA, UMR 1272 Insect physiology: Signalisation and Communication, Route de Saint-Cyr, 78026 Versailles Cedex, France, [email protected]

Abstract: Variations of Madex® (codling moth granulovirus) treatment efficiencies against Cydia pomonella larvae were observed in San Michele orchard according to apple tree varieties. Our hypothesis was that it could be linked to a different population pressure due to an insect egg laying preference among varieties. We verified in two years the differential efficacy of Madex® on larval damage on Golden Delicious vs. Red Chief and observed an egg laying preference for Golden Delicious. But surprisingly we observed also i) a reduction of the numbers of eggs after Madex® treatment ii) and that the reduction was greater on Golden Delicious than on Red Chief. Water-soluble carbohydrates and sugar-alcohols have been already identified from surfaces of apple tree organs as signals for C. pomonella egg laying. The blend of glucose, fructose, sucrose, sorbitol, quebrachitol and myo-inositol particularly stimulates insect egg laying. The Madex® treatment modified the blend (quantities and ratios) differently according to the apple variety. Here we verified on artificial substrates impregnated with blends found on the corymb leaves of each variety, that Madex® modified blends reduce the number of eggs. The reduction could be explained on Red Chief by a more drastic effect on egg-laying stimulation than on Golden Delicious on which the reduction was partly due to less acceptance of the substrate by females for egg laying and fewer eggs laid per female.

Key words: Cydia pomonella, egg-laying, Madex®, primary metabolites, leaf surface.

Introduction

The use of Madex® (codling moth granulovirus) as a biological control against C. pomonella larvae shows a differential efficacy on apple damage in IASMA San Michele (Trento, Italy) orchards, on two apple tree varieties: Golden Delicious and Red Chief. Our hypothesis is that this can be linked to different population pressures due to the insect egg-laying preference among varieties. Water soluble metabolites: sugar and sugar-alcohols (ng per cm²) have been already identified from surfaces of apple tree organs. They discriminate apple tree sites such as leaves according to their function within the tree, leaf side, organ as fruit, varieties and also plant growth stages and stage of fruit maturity (Derridj and Borges, 2006). They influence plant site acceptance after alighting and stimulate C. pomonella egg laying. Fructose, sorbitol and myo- inositol are then specially concerned within a blend of six metabolites (Lombarkia and Derridj, 2002). They also influence examination step behaviour of the substrate by the neonate larvae, before penetrating into the fruit (Derridj et al., 1999). The purpose of the study was to analyse the effect of Madex® on codling moth apple damage on the two varieties by looking at: i) egg numbers, ii) fruit damage and iii) primary

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metabolites on leaf surface already known as signals influencing egg laying (Lombarkia and Derridj, 2002). The first and second points were already presented and discussed by Lombarkia et al. (2005). Here we focus on the third point to verify the relationship between leaf surface chemical composition modifications due to Madex® spraying on plant and C. pomonella egg-laying.

Material and methods

Bio-assays on artificial substrate Metabolite blends: Usually, treatments with Madex® were carried out every 9 days and collections of leaf surface washings were done 6 days after treatment. Washings were collected during the second flight egg-laying period and the blends of metabolites were then analysed. The corymb leaf surface metabolite compositions were chosen to be tested. Six metabolites: glucose, fructose, sucrose, sorbitol, quebrachitol and myo-inositol, mixed as blends representing leaf surface compositions of Golden Delicious and Red Chief, non-treated and treated with Madex® (Table 1), were respectively tested for C. pomonella egg-laying.

Table 1. Blends representing leaf surface compositions of Golden Delicious and Red Chief non-treated and treated with Madex®

Golden Control Golden Treated Relative Relative (pg/cm²) % (pg/cm²) % Fructose D 30,4 28,4 4,6 15,6 Glucose D 9,4 8,8 5,0 16,9 Saccharose 5,9 5,5 6,4 21,7 Quebrachitol 0,9 0,8 1,3 4,4 Sorbitol 58 54,2 11,4 38,6 Myo-inositol 2,5 2,3 0,8 2,7 107,1 100,0 29,5 100,0

Red Chief Red Chief Control Treated Relative Relative (pg/cm²) % (pg/cm²) % wFructose D 9,2 16,6 7,9 24,2 Glucose D 6,5 11,8 5,4 16,6 Saccharose 8,1 14,6 2,4 7,4 Quebrachitol 1,4 2,5 1,3 4,0 Sorbitol 24,9 45,0 13 39,9 Myo-inositol 5,2 9,4 2,6 8,0 55,3 100,0 32,6 100,0

Insects The codling moths used in the bioassays came from an INRA mass rearing in Le Magneraud (France) where they had been kept on artificial diet for 10 years and were considered to be susceptible to insecticides and granulovirus.

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Bio-assays Bio-assays were done in a climatic chamber under a photoperiod of L 16: D 8, at 80 ± 10% and 23 ± 2°C. One day after emergence, two males and a single female were transferred to a cylindrical plastic box (10 cm in diameter and 8 cm high) for mating. Females, which had been laying eggs, during two days, in theses boxes, were tested. Each isolated gravid female was confined in a small cylindrical cage of 11 cm in diameter and height, lined at the top, bottom and wall with dried nylon cloth (200 cm² in area and 0,5 µm mesh) impregnated with metabolite blends representing non-treated and treated varieties in no-choice conditions. Four replicates of 11 insects were tested on four different days. Artificial substrates were given to the females one hour before the start of scotophase. Egg-laying was observed after 63 min (60 min of light and 3 min of darkness) of contact with the substrate. In these conditions in the control (nylon cloth impregnated with ultra-pure water) about 50 % of females lay eggs. The six metabolites were obtained from commercial sources with synthetic SIGMA products (Ultra): sucrose (S 7903), D-sorbitol (S7547), myo-inositol (I 5125), L-Quebrachitol (Q 3629), anhydrous cell culture test: D (+) glucose G 7021, D (-) fructose (F 0127), diluted in ultra-pure water (resistivity 18 M/Ω/cm or 0.15 µs/cm and with a filtration thresholds < 0.05 µm). Concentrations of the solutions in which the substrates were soaked were calculated to obtain on the nylon surface quantities in the same ratios as those collected on leaves:

Statistical analysis Results on eggs were analysed by non-parametric Wilcoxon and Man Whitney tests. The proportions of females laying eggs is expressed in percentage related to the total of females tested and were analysed by the χ2 test. The level of significance taken was equal to 0.05.

Results

Incidence of modified apple leaf surface blends by Madex® on egg laying The quantities and ratios of the leaf surface metabolite known as egg-laying signals were modified by Madex® treatment. The modifications induced and their consequences were different according to the original composition of each variety. The 6 metabolite blends found on the corymb leaf surface treated by Madex® vs control reduced significantly the egg laying on both varieties (-44% on Golden Delicious and –57% on Red Chief). The Red Chief modified blend composition reduced mainly the number of eggs laid per female (lower egg laying stimulation/and or egg laying inhibition?). The Golden Delicious modified blend reduced less egg number but it was compensated by a reduction of the number of females laying eggs (acceptance) (Figures 1 and 2). In our tests in which we considered the surface of the corymb leaves we observed no differences of egg-laying between the two varieties tested on a total of 53 females (water control : 6.96 ± 1.39 eggs, Red Chief : 4.32± 1.03, Golden : 5.28± 1.21).

Direct effect of Madex® product and its formulation on egg laying: We considered it would be also interesting to know in a first step if the Madex® could have by itself a direct activity on egg-laying by contact with C. pomonella. We limited the tests to immediate effect just after the Madex® spraying, on leaf surface composition already modified by the treatment. Madex® deposited 1h before the egg laying test on nylon, stimulated egg-laying only with Red Chief surface composition. It increased by 36,6% the egg-laying and was mostly due to an increase of the acceptance of the substrate by females (75 % laying eggs with Madex® vs 49% on control or formulation alone).

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aa 60 60 a a 50 50 a b 40 40

30 30 eggs 20 20 laying eggs 10 10 Percentage femalesPercentage

Percentage females laying 0 0 Control (U.P. G.NT. G.T Control (U.P. R.NT. R.T Water) Water) A Type of substrate B Type of substrate

Figure 1: Proportions (%) of females laying eggs on artificial substrates impregnated with: ultra-pure water (U.P. water), blend of six metabolites found on the corymb leaves of non-treated and treated Golden Delicious and Red Chief. (Different letters indicate significantly different percentages at P = 0,05, χ² test). G. NT.: Golden Non-Treated, G.T.: Golden Treated, R. NT.: Red Non-treated, R.T.: Red treated.

18 18 16 16 aa 14 14 12 12 10 a 10 b a 8 8 6 6 b 4 4 2 2 0 0 Number of eggs (mean +/- s.e.) Number

of eggs (mean +/- s.e.) Number Control (U.P. R.NT. R.T Control (U.P. G.NT. G.T Water) Water) A Type of subsrate B Type of substrate

Figure 2: Number of eggs laid per female (mean +/- s.e.) on artificial substrates impregnated with: ultra-pure water (U.P. water), blend of six metabolites found on the corymb leaves of non-treated and treated Golden Delicious and Red Chief. (Different letters indicate significantly different numbers of eggs at P = 0,05, Wilcoxon and Man Whitney tests).

The formulation (blank) used in Madex® had no effect on egg-laying whatever the leaf surface variety composition tested.

Conclusion

Previous observations carried on in the IASMA orchard throughout two consecutive years showed there were more eggs and more fruits damaged by larvae on Golden than on Red Chief. In addition, the treatment with Madex® also differentially reduces egg numbers and larval damage on the two varieties.

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According to the variety, Madex® treatment modifies the quantities and ratios of leaf and fruit surface metabolite blends. Consequences were a reduction of egg-laying by different behavioural ways according to the variety of apple tree. The leaf surface composition interacts with a direct stimulant effect of freshly deposited Madex® and can influence also egg laying for a short time only on Red Chief. Consequently the variety is at the origin of differences of pressure due to egg laying preference before and after Madex® treatment.

Discussion and perspectives

The effect of Madex® on leaf surface composition does not exclude an eventual effect of Madex® on volatiles which could be emitted by treated plants and which could play a role on alighting of females. The effect of contact studied here should occur only on females which can alight on the plant surface. Egg number reduction by Madex® treatment on artificial substrate is nevertheless in the same order as it was observed in orchard. In addition to egg-laying, we cannot leave out also an effect of the leaf surface composition on larval behaviour. Among the different types of fruit damage the proportion of stopped damage due to neonate larvae is much higher on Golden Delicious (26% ± 4) than on Red Chief (5% ± 1). Derridj et al. (1999) showed that apple surface metabolites especially fructose, sorbitol and myo-inositol influence larval behaviour and biting. It would be interesting to look at the different commercial products of granulovirus. It has not appeared that formulation is influencing directly egg laying and we do not know still which part of the Madex® product is inducing leaf surface chemical modifications. Knowledge of the components of Madex® which are at the origin of modifications of leaf surface of apple trees and modifications of insect behaviour could open new directions of improvement of this product and orchard protection against C. pomonella. Concerning the direct effect of Madex®, we studied what would happen in a short time just after spraying. It would be also interesting to study as a function of time the granulovirus persistence on leaves of each variety and its effect on egg laying (Ballard et al., 2000) within the season with the evolution of composition of the leaf surfaces on each variety. Because of the effect on egg-laying of Madex®, pest management against C. pomonella should take into consideration the egg laying period and not only egg hatching. This study shows also that apple tree susceptibility to C. pomonella egg laying and Madex® effect on larval damages could be considered associated in a protection method of apple trees. Only a small part (40 to 30%) of females tested lay eggs on modified plant surface blends by Madex® treatment. This should be already a selective pressure based on the response of some insects to these modifications of signals. The eventual selective pressure by the granulovirus would happen on the larval progeny of these females. It would be interesting to test the egg laying response to the modified blends of females born from resistant populations to the granulovirus and to look at any relationships between egg laying stimulation response and resistance to the virus.

Acknowledgements

Research supported by Safecrop Center - Autonomous Province of Trento and INRA.

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References

Ballard, J.; Ellis, D.J. & Payne, C.C. 2000: The role of formulation additives in increasing the potency of Cydia pomonella granulovirus for codling moth larvae, in laboratory and field experiments. – Biocontrol Science and Technology 10: 627-640. Derridj, S. & Borges, A. 2006: Apple tree resistance against an insect pest by an elicitor (ASM). Investigations by analyses of the leaf surface metabolites of the tree sites. Induced resistance in plants against insects and diseases. – IOBC/WPRS Bull. 29(8): 9- 13. Derridj, S.; Cabanat, I.; Cochet, E.; Couzi, P.; Lombarkia, N. & Wu, B.R. 1999: Incidence des métabolites présents à la surface des organes du pommier sur le comportement de Cydia pomonella (Lepidoptera, Tortricidae). – A.N.P.P. 5ème conférence internationale sur les ravageurs en agriculture. Montpellier, 7-8-9 décembre, II, 279-286. Lombarkia, N. & Derridj, S. 2002: Incidence of apple and leaf surface metabolites on Cydia pomonella oviposition. – Entomologia Experimentalis et Applicata 104: 79-87. Lombarkia, N.; Ioriatti, C. & Derridj, S. 2005: Relationships between granulovirus Madex® efficacy on Cydia pomonella fruit damages and apple tree surface metabolites. – IOBC/WPRS Bull. 28 (7): 419-424.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 149-152

Effect of a potential biocontrol agent of apple diseases on the egg laying of Cydia pomonella (L.)

Aude Alaphilippe1, Yigal Elad2, Sylvie Derridj3, Cesare Gessler1 1 SafeCrop Centre. Istituto Agrario San Michele all'Adige. Via Mach, 1. 38010 - San Michele TN, ITALY. [email protected] 2 Department of Plant Pathology and Weed Research, ARO, The Volcani Center, Bet Dagan. 50250 – Israel, [email protected] 3 INRA. Unité de Phytopharmacie et médiateurs chimiques. Route de St Cyr, 78 026 - Versailles cedex, France

Abstract: With the development of pest resistance towards pesticides and the decrease of chemical input, the research on alternative methods for plant protection, such as the use of microorganisms as biocontrol agents (BCA), is developing fast. But the side effects of BCA on non-targeted organisms and on the host plant are poorly known. Mass introduction of an organism into a system can change it substantially. Similarly, the introduction of an epiphytic microorganism may change the phylloplane chemical composition. Furthermore, sugar quantities and ratios in the phylloplane can influence the egg laying behaviour of Cydia pomonella (L.) (Lepidoptera Tortricidae). In this work, we studied the effect on the egg quantity laid by C. pomonella of an epiphytic yeast, as BC-model organism, introduced to apple canopy surfaces. Two seasons of trial, during the second flight of C. pomonella, showed an effect of the yeast spray on the quantity of eggs laid on apple trees. In the preliminary greenhouse experiment, the number of eggs laid on the yeast treated trees was lower as compared with those laid on the untreated trees. But for the second year of experiment, performed in semi-artificial conditions, the yeast treatment increased significantly the quantity of eggs laid, especially on the sites close to the fruits. These conflicting results could be explained by the different environmental and experimental conditions, which may affect the yeast activity and as consequences the phylloplane chemical composition. We analysed the chemical composition of leaf water washings using gas chromatography. On the first year of analysis, we observed modifications of quantities and ratios within the sugar blend on the upper side surfaces compared to untreated ones. We are currently analysing the samples from the second season using liquid chromatography. Finally, the correlation between yeast treatment, sugar quantities and qualities and egg laying behaviour will be analysed.

Key words: codling moth, epiphytic yeast, phylloplane, primary metabolites

Introduction

Biocontrol agents (BCA) are used as control method against different pathogens, such as the ® bacterium Bacillus subtilis, active ingredient in Serenade , against Podosphaera leucotricha, responsible of the powdery mildew on apple trees. The mode of action of a BCA is not always known or it is only partially known. As a consequence, a BCA spray could affect non-targeted organisms and the host-plant itself. After screening epiphytic yeasts and bacteria for their ability to decrease the severity of apple powdery mildew, we checked the side effects of the introduction of an isolate of epiphytic yeast isolate on the egg quantities laid by the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) on trees and on the chemical composition of the phylloplane, as it is known that primary metabolites (sugars) of the phylloplane stimulate the egg laying of the codling moth.

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Material and methods

All the experiments were conducted on 3-4 years old apple ‘Golden Delicious’ trees, cultivated in containers.

Yeast cultures and suspensions The yeast has been isolated in Israel by the team of Yigal Elad, Volcani Center, ARO. It has been selected for its effect on the powdery mildew severity and its capacity to grow up on the apple tree canopy. The yeast cultures were maintained on Petri dishes and then transferred to cell suspension cultures. The media used in Petri dishes was Potato Dextrose Agar (Oxoid), and in the cell suspension cultures, the Potato Dextrose Broth (Sigma) was used. For the yeast treatment, the yeast was cleaned from the growth media by a double centrifugation of 20 minutes at 4°C. The cells were then suspended in water supplemented with Tween 80 (0.01%), at a concentration of about 107 cells/ml. The cell concentration was estimated with a spectrophotometer.

Yeast effect on the codling moth oviposition in semi-natural condition trials The experiment was conducted in the Trentino, Italy, during the second flight of the codling moth corresponding to the month of August in 2005 and it was repeated in 2006. This experiment was carried under a roof in order to have a light intensity close to the one in the tree canopy of a commercial orchard. The yeast suspension was sprayed on the trees 24 hours before the insect release. Untreated trees were used as control. An experiment consisted of eight treated and eight untreated trees, a tree corresponding to a replicate. Each replicate was characterized by different tree morphology. For the year 2005, the 8 replicates were separated in two groups of four replicates separated by a 2 day interval, with similar climatic conditions. At 5:00 pm local time around 20 gravid females (Andermatt mass-rearing, Switzerland) were released under a white net cage, each of them containing one apple tree. Counts of the number of eggs on trees were made after the end of the release periods on different tree sites: fruits, leaves from the corymb, leaves from the bourse shoot, and “distal” leaves (“distal” leaves = all the other type of leaves). The “distal” leaves were separated in two groups: leaves at less than 20 cm from the fruits, and leaves at a distance over 20 cm from the fruits, since around 95% of the eggs are laid on fruits and leaves at less than 20 cm from fruits (Mattedi and Zelger, 2006).

Yeast effect on the plant surface metabolites Samples of 6 leaves were collected in the 2005 experiment: 3 leaves from the corymb and 3 from the bourse shoot, corresponding to the preferred site of the codling moth for egg laying. Four treated trees and four untreated trees were collected and two samples of six leaves were taken from each tree at 8:00 pm solar time, during the insect release. Leaves were washed using the method described by Fiala et al. (1990). The petioles were covered with paraffin. For one sample, each side was sprayed separately with c. 10 ml of ultra-pure water per 100 cm2. Sugars and sugar alcohols as well as free amino acids in the other hand were quantified by liquid chromatography.

Results and discussion

Only part of the results is presented in this manuscript, as they are still been analysed.

Yeast effect on the codling moth oviposition in semi-natural condition trials Egg quantities laid on the control trees for the year 2005 is similar to 2006 (Figure 1). But for the yeast treated trees they are both different from the control and from the control tree. In

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2005 we observed an increase of the number of eggs laid on the treated trees compared to the control, although in year 2006 the yeast treatment decreased the number of eggs laid. The yeast had an effect on C. pomonella egg laying, however it can be seen that a contrasting effect was observed in the two years.

Average of laid eggs 5

corymbe distal <20 fruits Bourse sh.

control 2005 control 2006 Y+ T80 2005 Y+ T80 2006

Figure 1. Raw results of the two years 2005 and 2006 of experiment. Average of egg number laid per tree and within the tree.

Yeast effect on plant surface metabolites The data are currently analysed and the results will not be presented here. The first analyses of the data of year 2005 show a tendency to a general decrease of almost all substances analysed: sugar, sugar, alcohols and free amino acid in the yeast treated plant.

Discussion

The yeast suspension affected the egg laying stimulation of the codling moth. Moreover, it modified the chemical composition of the phylloplane. The modification of leaf surface signals could partly explain the effect of the yeast suspension on the egg laying stimulation of the insect as already shown in other researches (Lombarkia, 2002; Derridj and Borges, 2004). But the effect of the yeast population is linked to the year. We may hypothesize that the plant–yeast interaction is linked to environmental factors, such as climatic factor. This yeast is a potential BCA of apple powdery mildew. Before developing this yeast as a BCA, it is needed to establish the effect of this BCA on this insect. Indeed, the codling moth is economically one of the most important pest of apple trees production in Europe. Although the effect of the yeast treatment is year linked, an increase of the insect damages on the production due to the BCA spray can nulify its beneficial effect gained by the control of powdery mildew.

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Acknowledgements

This research was supported by SafeCrop Centre, funded by Fondo per la Ricerca, Autonomous Province of Trento.

References

Derridj, S. & Borges, A. 2004: Apple tree resistance against an insect pest induced by an elicitor (ASM). Investigations by the analyses of the leaf surface metabolite on tree sites. – IOBC workshop on methods in research on induced resistance Nov. 2004, Delémont. Suisse. Fiala, V.; Glad, C.; Martin, M.; Jolivet, E. & Derridj, S. 1990: Occurrence of soluble carbohydrates on the phylloplane of maize (Zea mays L.): variations in relation to leaf heterogeneity and position on the plant. – New Phytol. 115: 609-615. Lombarkia, N. & Derridj, S. 2002: Incidence of apple fruit and leaf surface metabolites on Cydia pomonella oviposition. – Entomol. Exp. App. 104: 79-87. Mattedi, L. & Zelger, R. 2006: Untersuchungen zur Eiablage des Apfelwicklers. – Obstbau. 5: 267-271.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 153-156

Field assessment of behaviorially-based management tactics for Conotrachelus nenuphar (Herbst), and Rhagoletis pomonella (Walsh) in the northeastern US

Arthur Agnello1, Jaime Piñero2, Ronald Prokopy3 1 Department of Entomology, New York State Agricultural Experiment Station, PO Box 462, Geneva, NY, 14456, USA, [email protected]. 2 Institute of Plant Sciences, Applied Entomology ETH, Schmelzbergstrasse 9 LFO, 8092, Zürich, Switzerland, [email protected]. 3 Department of Entomology, Univ. of Massachusetts, Amherst, MA, 01003, USA, (deceased)

Abstract: Field trials were conducted for 2 years to validate and demonstrate the effectiveness of advanced-level IPM tactics. For plum curculio (PC), Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), trap trees using benzaldehyde and grandisoic acid lures determined need and timing of insecticides compared with calendar-driven sprays or heat-unit models. For apple maggot (AM), Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), odor-baited pesticide-treated spheres (PTS) were deployed for direct control of AM compared with calendar-driven sprays or monitoring trap capture-driven sprays. Trials were conducted in 24 commercial orchards in New York and 6 New England states: Connecticut, Massachusetts, Maine, New Hampshire, Rhode Island and Vermont. These "Advanced IPM" approaches proved effective in the majority of the sites over 2 years. In those few cases where fruit injury was above 1–2%, the cause could be attributed to a missed insecticide spray, the occurrence of intense pest pressure from within the orchard block, or to a planting arrangement consisting of very small apple trees. In both years there was a significant reduction in pesticide use in the "Advanced IPM" treatment.

Key words: apple maggot, plum curculio, trap trees, pesticide-treated spheres, behavioral control

Introduction

Apples are grown in all states of the northeastern US. Annual production in the region (including MA, NY, CT, NH, ME, NJ, DE, MD, PA, WV) is about 46 million bushels with a value exceeding $225 million. Apple maggot (AM, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae)), and plum curculio (PC, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae)) are the two key insect pests in the region. Unsprayed trees typically have fruit with 90% injury by both pests. Orchards using conventional spray programs target 6 out of their 7 annual insecticide sprays against these two pests (May-June for PC and July-August for AM). By far the most commonly used sprays are the broad-spectrum organophosphates, phosmet and azinphosmethyl. These materials are high-risk for worker exposure, toxicity to non-targets and fruit residues. Azinphosmethyl has a new 14-day re-entry interval and may be restricted further under FQPA. For these reasons and for concerns over pesticide drift beyond orchard boudaries, growers are asking for reduced-pesticide programs and biologically-based alternatives to pesticides. This study represents the culmination of over 20 years of research by Ronald Prokopy and his associates to develop advanced IPM reduced-risk approaches to manage AM and PC. 2005 was the 2nd year of the 2-year demonstration phase of the study, in which the odor- baited trap-tree approach for monitoring and managing PC damage and odor-baited spheres for directly controlling AM were tested in more than 20 blocks of apple trees in the Northeast.

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Material and methods

Plum curculio The objective was a validation and demonstration in 24 (2004) and 21 (2005) commercial orchards of the effectiveness of an optimal trap-tree approach to determine need for and timing of insecticide use against PC in comparison with existing approaches that are based on calendar-driven sprays or heat-unit-accumulation models. The 0.4-ha plots were set up in commercial blocks of apple trees adjacent to woods. There were 11 in MA, 2 each in NH, VT, NY, and RI, and 1 each in in CT and ME. Each orchard block was divided into 3 plots of about 1 acre each. The "Conventional" plot received 3 whole-plot applications of phosmet (at petal fall + 2 covers). The "1st-Level IPM" plot received a whole-plot spray at petal fall, followed by a whole- plot cover spray which was dependent upon a degree-day model (Reissig et al. 1998), in which the last spray has residual activity until the accumulation of 171 degree days (base 10°C) since petal fall. In the "Advanced IPM" plot, a perimeter-row trap-tree was baited with 1 dispenser of grandisoic acid (pheromone) plus 4 dispensers of benzaldehyde (host-plant attractant) at the time of petal fall, and a whole-plot spray was applied to kill any PC that had overwintered in the plot or had immigrated earlier from the woods. A week later, 25 fruit were tagged and numbered and for 6 weeks fruit were examined every 3–4 days for fresh PC egg-laying scars. PC immigrating from woods into the orchard were lured into the trap-tree and arrested there and in neighboring trees. Occurrence of a new scar indicated the need for a perimeter spray (to the outer 2 rows). The combined bait resulted in 20 times more damage by PC to fruit on a trap-tree than on unbaited trees, thereby greatly reducing the time needed to sample for this key pest. The effectiveness of the trap-tree approach was compared with the two other approaches. Efficacy of each method was assessed by sampling 10 fruit at random from each of 10 trees in each of 9 rows in each plot. Assessments were made both during early July and also 1 week before harvest.

Apple maggot An improved pesticide-treated sphere and a new method for calculating the number of spheres to place on the perimeter of a block of trees were tested. The placement method used an index developed from four variables: orchard tree size, quality of pruning, susceptibility of cultivar composition, and nature of bordering habitat. In previous trials in 2003, this approach reduced the number of spheres needed by 40% from previous methods. The same plots as described above for PC were used for AM. All plots received 4 unbaited sticky sphere traps to estimate penetration of AM adults into the block. These spheres were inspected weekly. Management of AM in the "Conventional" plot consisted of 3 calendar-driven applications of phosmet to the entire plot (mid-July, early August, mid-August). Phosmet application in the "1st-Level IPM" plot to the entire plot was driven by accumulation of AM on the 4 unbaited sticky red monitoring traps (threshold: avg of 2 AM/trap). For direct trap-out control of AM in the "Advanced IPM" plot, odor-baited pesticide- treated spheres were deployed on perimeter trees of all four sides of the plot. The Pesticide- Treated Sphere (PTS) was composed of a contoured compressed top cap bearing sugar (as a feeding stimulant), spinosad (Entrust), and paraffin wax coupled to a hollow plastic sphere. Using the new placement system, an average of 22 PTS, each baited with attractive odor (a 5- component apple volatile blend), were deployed per plot. There were no insecticide sprays in

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this plot after the plum curculio season was over (early July). At harvest, 900 fruit per plot were sampled for AM injury.

Results and discussion

Plum curculio In 2004, plum curculio injury was not significantly higher in trap-tree (Advanced IPM) plots compared with the other two management tactics (0.8-1.5% injury). A 35% reduction in insecticide use was achieved in these plots for PC compared with the Conventional plots in the 14 plots in Massachusetts. In 2005, the average percentage of fruit injured was <1% for all three types of plots in both the early July count and in the harvest count. In 2005, we decided to analyse the pesticide data more thoroughly so that we could report the costs of pesticides for the different management systems. Spray events were converted to dosage equivalents (DE) (dividing the actual rate used by the manufacturer’s recommended field rate) (MRFR) to adjust for the wide range of field rates used in the different commercial operations. Of the 14 (Mass.) plots analyzed so far, the total PC insecticide DEs were 44.5 for the Conventional plots (or an average of 3.2 DEs per orchard site), 40.45 for the 1st-Level IPM plots (avg of 2.9 DEs per site), and 30.97 for the Advanced IPM plots (avg. of 2.2 DE per site). These results are preliminary, but appear to support the use of Advanced IPM methods to significanly reduce the amount of insecticide used in the trap-tree plots.

Apple maggot In 2004, apple maggot injury was again low in all 3 types of plots (0.2 to 0.9% fruit injured). The injury in the Advanced IPM plots was higher (0.9%) than in the Conventional plots (0.23%), but at a level <1%, which is not a serious concern. We have not yet calculated the 2004 reductions in insecticide sprays in the Basic and Advanced IPM plots compared with the Conventional plots, but we expect it will be significant. In 2005, the AM injury was even lower (0.09-0.23% fruit injured) and again the Advanced IPM plots had more injury than the Conventional plots, but the level is economically insignificant. Among the 14 orchard sites that we have analyzed so far for 2005 insecticidal inputs, the total dosage equivalents are: 21.4 for the Conventional plots (avg. of 1.5 per site), 11.2 for the 1st-Level IPM plots (avg. of 0.8 per site), and 1.4 for the Advanced IPM plots (avg. of 0.1 per site); this last plot should have had 0, but there were 2 sprays applied in error. In the NY sites, insect damage during both years was very low in all plots. Apple maggot injury was only nominal overall (0 to 0.3%), and there were no significant differences across any of the treatments. In the New England locations, population pressure for both AM and PC was substantially greater than in NY, although there was high variability among the sites. Some trends noted in these locations were: • The Advanced-Level IPM plots usually exhibited the highest fruit damage readings at harvest. • A definite edge effect was noted in the incidence of pest injury, with worse damage occurring generally nearer to non-orchard habitats adjacent to the plots. • Because it was not always possible to set up all plots in the same varieties, a varietal effect was also apparent; greater pest susceptibility was seen in: McIntosh, Red Mac, Cortland, Jonamac, Mutsu, Liberty, to Redfree. In at least two cases, the Advanced-Level IPM plot was set up in plantings having an indigenous AM population in the orchard. This led to poor performance of the pesticide

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treated spheres in controlling this pest, as a behavioral tactic of this nature would not be expected to be effective in such a case.

Table 1. Mean percent fruit damage caused by plum curculio and apple maggot in Calender, 1st-Level IPM and Advanced IPM plots across all sites during 2004 and 2005.

Treatment Plum curculio Apple maggot 2004 2005 2004 2005 Conventional 1.19 a 0.7 a 0.35 a 0.1 a (Calendar-driven) 1st-Level IPM (PC, Oviposition model 1.55 a 0.6 a 0.32 a 0.2 a AM, Trap threshold) Advanced IPM (PC, Trap tree 3.21 a 1.6 a 4.37 b 0.3 a AM, Pesticide-treated spheres)

Acknowledgements

The following participants' efforts are acknowledged for overseeing this research in their respective states: Heather Faubert, Research Assistant, Univ. of Rhode Island; Lorraine Los, Fruit Crops IPM Coordinator, Univ. of Connecticut; Glen Koehler, Associate Scientist, Univ. of Maine Cooperative Extension; Kathleen Leahy, Independent Consultant, Polaris Orchard Management (sites in NH and VT); Glenn Morin, Independent Consultant, New England Fruit Consultants (sites in NH and VT). Also, this work could not have been accomplished without the efforts of the following people: Arthur Tuttle, Extension Educator, Univ. of Massachusetts; Suzanne O’Connell, Research Assistant, Univ. of Massachusetts; Starker Wright, President, Pest Management Innovations, LLC; David Kain, Research Support Specialist, Cornell Univ.

References

Piñero, J.C. & Prokopy, R.J. 2005: Spatial and temporal within-canopy distribution of egglaying by plum curculios (Coleoptera: Curculionidae) on apples in relation to tree size. – J. Entomol. Sci. 40: 1-9. Reissig, W.H., Nyrop, J.P. & Straub, R. 1998: Oviposition model for timing insecticide sprays against plum curculio (Coleoptera: Curculionidae) in New York State. – Environ. Entomol. 27: 1053-1061.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 157

Four years of mediterranean fruit fly (Ceratitis capitata Wied.) control in fruit orchards of Girona (NE of Spain) by using the mass trapping method

Josep Lluis Batllori1, Adriana Escudero2, Mariano Vilajeliu2 1 Servei de Sanitat Vegetal, DARP, Aiguamolls de l’Empordà, 17486 - Castelló d’Empúries, Girona, Spain, [email protected] 2 IRTA-Estació Experimental Agrícola MAS BADIA, Canet de la Tallada, 17134 - La Tallada d’Empordà, Girona, Spain, [email protected]; mariano.vilajeliu @irta.es

Abstract: Mediterranean fruit fly (MFF) (Ceratitis capitata Wiedemann) (Diptera: Tephritidae) is a polyphagous pest that can be potentially very harmful in fruit orchards, particularly in temperate and hot growing areas. Common control strategies to avoid fruit damage are based on chemical insecticide sprayings just before harvest. In 2001 there was serious fruit damage in the growing areas of Girona (NE of Spain), especially in mid and late season stone fruit cultivars. The production losses encouraged the use of the mass trapping control method, as an alternative to chemicals, in the context of Integrated Fruit Production methodology to which most of the fruit growers fit. From 2002 to 2005 several mass trapping trials were carried out on apple and stone fruit orchards with different attractants, number of fly-traps per hectare (from 50 to 75) and layout trap systems (homogeneous versus perimeter) in the field. In the last four years, pressure of the MFF fluctuated between middle and low level in the Girona fruit area; in these conditions, the amount of 50 to 75 fly-traps per hectare with any of the used attractants and methods of the traps layout in the orchards, reached satisfactory control of this pest. The higher damage level estimated was 1.3 % in only one peach fruit orchard. The conditions of these trials and their results will be discussed.

Key words: Mediterranean fruit fly, Ceratitis capitata, mass trapping, perimeter trapping, attractant, fly-traps

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 159-167

Exploiting the sex pheromone of the apple leaf midge, Dasineura mali, for pest monitoring and control

Jerry Cross1, David Hall2 and Peter Shaw1 1 East Malling Research, Kent, ME19 6BJ, UK, [email protected] 2 Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK, [email protected]

Abstract: Work by East Malling Research (EMR) and Natural Resources Institute (NRI) to identify the female sex pheromone of the apple leaf curling midge, Dasineura mali Kieffer, is overviewed. The pheromone was collected by the trapping of volatiles which proved far more effective than gland extraction methods used previously. Even so the single component of D. mali was present at only 2 pg per female per hour. This was detected by electroantennography (EAG) linked to gas chromatographic (GC) analysis, and identified as (Z)-13-acetoxy-8-heptadecen-2-one by interpretation of the mass spectra and comparison with synthetic standards. A rubber septum impregnated with 1 µg of the synthetic, racemic pheromone proved highly attractive to male D. mali, and release rate measurements indicate this lure will remain active for several years in the field. Only the S enantiomer of the chiral component is produced by the insect and field experiments showed that only this enantiomer is attractive and that the R enantiomer has no activity. The D. mali sex pheromone is a keto-ester, a variation on a new class of midge pheromone structures that is currently the subject of a patent application. Other midge pheromones previously identified are mono or diesters. The D. mali pheromone has proved very useful for monitoring pest populations in the field. Results from preliminary trials in the UK in 2004 are reported. White delta traps (20 x 20 cm), with rubber septum dispensers containing 3µg of the pheromone racemate and deployed at a height of 0.5 m are recommended as a standard. They were made available to growers in the UK for Beta testing in 2006 as part of a coordinated programme of experiments to establish treatment thresholds. An experiment in 2006 showed that the traps can be used to time sprays of synthetic pyrethroids for control of the pest but that multiple applications were required to give a high degree of control. Large scale field experiments started in the UK in 2004 to test the use of the pheromone for control by mating disruption and attract and kill approaches. Early season results were promising but control failed later in the season because the pheromone was degraded by UV light more rapidly than expected. Results so far have indicated the need to deploy the treatments on a large scale and in newly planted orchards where populations are initially low and to protect the pheromone from degradation by UV light.

Key words: sex pheromone, apple leaf midge, apple leaf curling midge, pest monitoring, mating disruption, attract and kill

Introduction

The apple leaf curling midge, Dasineura mali Kieffer, is a pest of apples in Europe, North America and New Zealand and in the UK it is widespread and abundant. It is particularly damaging in nurseries and newly planted or re-grafted orchards. Natural enemies are important in regulating leaf midge numbers, especially the egg parasitoid Platygaster demades and anthocorid predatory bugs which feed in galls on larvae. However, these natural enemies, especially adult P. demades, are sensitive to broad-spectrum insecticides which are commonly used to control other pests in apple orchards. There is currently no satisfactory

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method for controlling outbreaks of the pest. Synthetic pyrethroids are considered to be partially effective. All apple varieties are susceptible and it is unlikely that resistant varieties can be developed. Prior to our work, it was shown that virgin female adults of the apple leaf midge have been shown to produce a powerful sex pheromone that attracts conspecific males (Harris et al., 1996). The compounds constituting the sex pheromone of the apple leaf midge had not been identified. However, the components of the female sex pheromones of several other species of midge had been identified and synthesised including the Hessian fly, Mayetiola destructor (Foster et al., 1991), pea midge, Contarinia pisi (Hibur et al., 1999; 2000; 2001), orange blossom wheat midge, Sitodiplosis mosellana (Gries et al., 2000) and the Douglas-fir cone gall midge, C. oregonensis (Gries et al., 2002). In this paper, we overview the recent research work we have done to identify the female sex pheromone of Dasineura mali Kieffer and some of the preliminary work we have carried out to investigate its use for monitoring and control of the pest.

Identification and synthesis of female sex pheromone of apple leaf midge

For identification of the female sex pheromone of apple leaf midge, volatiles were collected from over 2,000 virgin female midges which proved more effective than gland extraction methods used previously. Analysis of collections by gas chromatography (GC) coupled to electroantennographic (EAG) recording from the antenna of a male midge antenna showed a single active component which was not present in similar collections from virgin male midges and was assumed to be the sex pheromone (Hall et al., 2005). Although this was present at less than 20 pg per female, mass spectra were obtained and the compound was identified as (Z)-13-acetoxy-8-heptadecen-2-one by comparison of GC retention times and mass spectra with those of synthetic standards and microanalytical reactions. The synthetic compound had GC and MS data identical with those of the natural compound and elicited a strong EAG response from a male D. mali midge. A convenient route was developed for synthesis of the compound from giving 63% yield in eight steps. The two enantiomers of the compound were separated and isolated by high performance liquid chromatography on a chiral column. The first-eluting enantiomer was predicted to be the S enantiomer by nuclear magnetic resonance spectroscopy of the (R)-2-methoxy-2-trifluoromethyl-2-phenylacetyl esters.

Optimisation of pheromone dispenser and trap for apple leaf midge

The pheromone racemate was found to be highly attractive to male apple leaf midge in the field with rubber septa lures containing 1 µg being significantly attractive. Rubber septa dispensers were shown to give a relatively constant release over at least 374 days under laboratory conditions while sealed polyethylene vial dispensers showed a lag period before release began followed by relatively rapid release which then declined. Only one enantiomer of the pheromone, probably that with S configuration, was attractive, but the racemic mixture was equally attractive, the latter being much more economic and easier to synthesise. A range of traps was evaluated and white sticky delta traps found to be most effective. Trap colour did not affect catches of midges in delta traps, but there were indications that red traps were less contaminated by non-target insects and this needs to be further investigated. The height of deployment of traps was shown to have a large effect on midge catches with highest catches in traps at ground level. Traps were shown to attract midges over a range of at least 50 m. White sticky delta traps baited with a rubber septum containing 3 µg of the racemic pheromone positioned at a height of 0.5 m above ground are recommended for monitoring of apple leaf midge by growers.

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Use of pheromone-baited traps for monitoring apple leaf midge

In 2004, two standard white delta traps baited with standard 3 µg rubber septa lures were deployed in each of 6 commercial apple orchards at Broadwater Farm, West Malling, Kent (England), and monitored at 3-4 day intervals from 28 May – 1 September. The orchards were of different varieties, rootstocks and ages and thus had widely varying populations of apple leaf midge: 1) a newly planted intensive M9 Braeburn orchard which had very low midge populations; 2) a 1 year old intensive M9 Braeburn orchard which had slightly higher populations of apple leaf midge; 3) an established M26 Bramley orchard with moderate populations of leaf midge; 4) a young established Gala/Meridian orchard with moderate populations of midge; 5) an established Gala orchard with moderate midge populations and 6) a vigorous old established Bramley orchard with MM106 rootstocks and high populations of apple leaf midge. On each monitoring date, 100 shoots in the centre of each orchard (5 selected at random on each of 20 trees) were examined for the presence of ovipositing females and the numbers of leaves with apple leaf midge galls were counted. Then the distribution of galls in an additional 25 new shoots (5 from 5 trees) was scored. The pheromone trap catches showed two clear generations of apple midge after 28 May 2004 (the first generation was missed because the synthetic pheromone was not available earlier). For clarity, the results from 4 of these orchards, representing the range, are shown in Figure 1. The second generation occurred in June and the third in July and August. There were clear, large differences in total numbers of males caught, which reflected the population density in the particular orchard. A mean total of 67 males were captured in the newly planted Braeburn orchard, mostly in July and August. A mean total of 718 males were caught in the lightly infested 1 year old Braeburn orchard. Mean totals of 3090 – 3377 males were captured in the three moderately infested orchards and a mean total of 20,400 males were caught in the heavily infested old vigorous Bramley orchard. Numbers of ovipositing females in shoots followed similar trends, peak numbers broadly coinciding with the latter part of the peaks of the male flights and total numbers correlating with the total numbers of males. Total numbers of galls per 100 shoots showed an upward trend throughout the monitoring period in the moderately and heavily infested orchards. Numbers of galls broadly correlated with the numbers of midges caught. Only in the lightly infested 1 year old Braeburn orchard was a second generation peak in galling damage clearly apparent. These results, taken together, were the first indication that the pheromone traps could be used for pest monitoring purposes and that that catches reflected the degree and timing of damage.

Use of pheromone traps to time insecticide sprays

In 2006, a replicated field experiment was done to determine whether apple leaf midge sex pheromone trap catches can be used to time sprays of the insecticide cypermethrin to control the first generation of apple leaf midge and to examine the numbers and timings of sprays required to achieve good control. The experiment was done in a young apple plantation at EMR with alternating rows of Bramleys Seedling and Jonagold on the M9 rootstock. A standard white delta pheromone trap with 3 µg rubber septum lure was deployed in the centre of the plantation at a height of 0.5 m on 18 April and catches monitored every 3-4 days. Treatments were 1, 2, 3 or 6 applications of the synthetic pyrethroid insecticide cypermethrin (Toppel 10, United Phosphorous) applied at a rate of 350 ml product in 500 l water per ha per spray with a motorised air-assisted knapsack sprayer, and an untreated control. A 5 x 5 Latin square experimental design was used for the experiment. Each plot consisted of 4 adjacent

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Bramley trees which received the spray treatments, but only the central two trees were used for assessment. Plots were separated by 4 unsprayed guard trees and a guard row on each side on the variety Jonagold. The first spray applications were made on 4 May, at the beginning of the flight of apple leaf midge males, as indicated by catches in the sex pheromone trap (Figure 2).

Males in pheromone traps

800

600

400

200

Mean no. males / trap / day 0

Ovipositing females

0 Mean no. females / 100 shoots

Galls in shoots

800

600

400

200

Mean galls / 100 shoots galls / 100 Mean 0 28 04 11 18 25 02 09 16 23 30 06 13 20 May Jun Jun Jun Jun Jul Jul Jul Jul Jul Aug Aug Aug

1 year Braeburn New Braeburn Old MM106 Bramley Establsihed Gala

Figure 1. Mean numbers of apple leaf midge males captured per day (top), numbers of ovipositing females per 100 shoots (middle) and numbers of galls per 100 shoots (bottom) in four orchards at Broadwater Farm, West Malling, Kent (England) in 2004.

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14 12 10 8 6 4

Catch / trap / day 2 0 21 28 05 12 19 26 02 Apr Apr May May May May Jun

Figure 2. Catches of first generation male apple leaf midge in the sex pheromone trap in the insecticide trial in 2006

Counts of eggs and oviposting females were made on 5 shoots on each of the two central trees in each plot on 8 May (50% bloom), 16 May (50% petal fall), 25 May (end of petal fall) and 7 June (early fruitlet). A full assessment of galling damage on 20 shoots per plot was done on 7 June 2006. Weather conditions were not favourable for midge development and the first generation flight of D. mali was small. On the untreated control, only 3.4 % of shoots were damaged by apple leaf midge galls on 7 June with a mean of 12.8 galls per tree (Table 1). The treatment with 6 sprays of cypermethrin virtually eliminated the gall infestation, but the treatments with 1, 2 or 3 sprays performed similarly, reducing gall numbers by 75% on average.

Table 1. Shoot and gall damage by apple leaf midge in insecticide trial (measured on 7 June 2006)

No. shoots per tree No. galls per tree No. sprays (dates 2005) No. % n damaged damaged n log10(n+1) 0 54.2 5.0 3.40 12.8 1.059 1 (4 May) 47.1 1.9 4.45 5.1 0.629 2 (4,15 May) 49.2 0.9 2.32 1.9 0.308 3 (4,11,18 May) 46.4 1.6 3.72 3.5 0.403 6 (4,8,11,15,18,23 May) 48.6 0.1 0.14 0.1 0.030 Fprob 0.002 SED (12 df) 0.1882 LSD (P=0.05) 0.410

Use of pheromone for control of apple leaf midge by attract-and-kill (A&K) and mating disruption (MD)

In July 2004, a preliminary field experiment was done at EMR to investigate the use of the apple leaf midge sex pheromone for control of apple leaf midge using an attract-and-kill (A&K) strategy. A&K devices were 20 x 20 cm squares of plastic laminated cardboard surface coated on both sides with a microencapsulated formulation of the SP insecticide lambda cyhalothrin developed for control of olive fly (Agrisense). These were positioned

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5 cm above ground level and baited with a rubber septum lure impregnated with 100 µg of apple leaf midge pheromone fixed centrally. Four small heavily infested apple orchards at EMR were selected for the experiment: On 5 July, a single standard white delta trap baited with a standard 3 µg rubber septum lure was deployed in the centre of each plot. Catches of males were counted on six occasions between 7-27 July 2004. On 7 July, 28 and 12 A&K devices respectively were deployed in lattices in two of the plots to give a density of approximately 100/ha. On 27 July, 100 shoot terminals in the centre of each plot were examined for ovipositing females and presence of apple leaf midge galls.

Table 2. Pheromone trap catches of male D. mali and damage assessments in preliminary attract and kill experiment at EMR (pheromone trapping 7-27 July; damage assessments on 27 July 2004)

Plot DM153 DM167 DM169 WM135 Variety Bramley Cox Fiesta Mixed dessert Area 0.28 ha 0.08 ha 0.26 ha 0.09 ha No. A&K devices 28 12 0 0 Trap catch/day 40.5 7.6 122.2 409.1 % shoots galled 100 84 100 100 No. females/100 shoots 3 0 2 5

The two orchards where the A&K treatment was deployed had considerably lower trap catches than the untreated plots, but the treatments failed to shut down catches completely (Table 2). The shoots assessment on 27 July revealed that 100% of shoots were galled on 3 of the plots with 84% galled on the other. Small numbers of ovipositing females were also recorded. The results showed the treatment was not able to adequately suppress mating and that a much higher dosage of pheromone would be required with many more devices per ha and/or a higher pheromone dose. Bioassays of the effect of contact of apple leaf midge adult males with the lambda cyhalothrin target devices were conducted on 1 and 2 September 2004. A&K devices were observed in the field and at intervals 10 attracted male midges were pootered from the surface of the device or shortly after they had made contact, and held in tubes. Midges were pootered in a similar way from the surface of a comparable device not treated with lambda cyhalothrin. After 1 hr, all the midges that had been exposed to the lambda cyhalothrin card, even for 5 min, were severely affected by the insecticide. They were unable to fly and lay trembling in the bottom of the tube. After 2 hr, all were trembling or moribund. After 3 hr, all were dead. Similar results were obtained with A&K devices that had previously been exposed for two months in the field. A very large scale field trial was carried out in commercial apple orchards in Kent during 2005 to evaluate the use of the apple leaf midge sex pheromone for control of apple leaf midge by mating disruption (MD) or attract and kill (A&K) approaches. MD devices were polythene caps each initially loaded with 500 µg of the apple leaf midge sex pheromone. These caps each released the pheromone at approximately 10 ng/hr at 27 ºC in the laboratory. The attract and kill target devices were 10 cm x 6.7 cm oblongs of the microencapsulated lambda cyhalothrin surface treated cardboard with a polythene cap lure containing 100 µg of the apple leaf midge sex pheromone fixed to the centre with a drawing pin. These caps each released the pheromone at approximately 2 ng/hr at 27 ºC in the laboratory. Both MD and

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A&K devices were deployed at 500 devices/ha or 2000 devices/ha, fixed to tree stakes so that the lure was at a height of approximately 15 cm above the ground in a regularly spaced lattice. Thus for the MD treatments, pheromone application rates were 0.25 g/ha or 1 g/ha respectively and release rates were approximately 5 µg/ha/hr and 20 µg/ha/hr. For the A&K treatments, pheromone application rates were 0.05 g/ha or 0.2 g/ha respectively and release rates were approximately 1 µg/ha/hr and 4 µg/ha/hr. A fully randomised experimental design was used with six replicate 1 ha plots of each treatment, requiring 30 plots of 1 ha in 11 orchards on six different fruit farms in Kent. Three of the plots for each treatment were in newly planted orchards where leaf midge populations were low and three in established orchards where leaf midge populations were high. Untreated plots were well separated from those which had MD or A&K treatments which themselves were adjacent. Experimental Approvals allowing this work to proceed without crop destruction were applied for and granted by the Pesticides Safety Directorate. The effectiveness of the treatments was assessed by weekly monitoring of catches of adult male midges in a delta trap baited with a 3 µg rubber septum lure in the centre of each plot and by counting the number of galls present in 200 shoots in the centre of each plot for each of the three main generations, at the peak of damage expression on 17-23 May, 20-25 June, 4-6 July and 30-31 August 2005.

Table 3. Pheromone trap catches of male D. mali and damage assessments in mating disruption (MD) and attract-and-kill (A&K) trials (April-September 2005; 1 ha plots, 3 replicates per treatment)

Mean pheromone trap catch Mean % shoot damage Apr- Jun- Aug- 17-23 20-25 4-6 30-31 Treatment May July Sept Total May Jun Jul Aug Heavily-infested orchards Heavily-infested orchards MD 500/ha 3 5 43 52 50 1 73 99 MD 2000/ha 6 1 30 36 28 2 62 99 A&K 500/ha 7 29 73 109 43 37 58 95 A&K 2000/ha 7 1 9 16 21 13 83 93 Untreated 429 4094 817 5340 29 5 29 94 Newly-planted orchards Newly-planted orchards MD 500 /ha 0 0 3 3 0 0 3 87 MD 2000/ha 0 1 2 2 0 0 3 87 A&K 500/ha 0 0 4 4 0 0 5 88 A&K 2000/ha 0 0 0 0 1 0 4 85 Untreated 0 11 7 18 0 0 14 90

In the analyses of variance of the log transformed total counts, the only differences of significance were whether or not any 'treatment' had been applied, with no differences between type and number of lures. All the MD and A&K treatments suppressed the catches of males in the traps in the centres of the plots compared to the untreated control (Table 3). In the established orchards with higher populations, catches were decreased by > 98% in April- May, by > 99% in June-July, but by only >91% in August-September (Table 3). In the newly planted orchards with very low populations of the midge, trap catches were zero in April-May in all plots, were very low but suppressed by 90% by the MD and A&K treatments in June- July, but rose somewhat in August September being suppressed by about 80% in the treated plots (Table 3).

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Regrettably, there was no evidence that either the MD or A&K treatments were suppressing numbers of galls in shoots in the established orchards (Table 3) and newly planted orchards. On 9 September 2005, bioassays of the effectiveness of target devices which had been deployed in the field since the start of the A&K trials were conducted in a similar way to that described above for the preliminary experiment. The results were less clear cut than in the previous bioassays with substantial mortality in the untreated controls. However, the experiments did show that the devices maintained their activity. Measurement of release of pheromone from the open polyethylene caps used in these trials for both MD and A&K treatments showed relatively uniform release for at least 270 days under laboratory conditions. However, lures recovered from the field at the end of the above experiments were found to contain no detectable pheromone. This has subsequently been shown to be due to degradation of the pheromone, the lures being unprotected from direct sunlight in both MD and A&K treatments. It is possible that the photo-degradation of the pheromone was the cause of the failure of the MD and A&K treatments to control the midge but further work is needed to determine whether or not the midge can be controlled even if uniform release rates are maintained through the season. Other factors such as pheromone dose, density of devices and scale of deployment also require further investigation. MD and A&K treatments generally need to be deployed on a large scale and where populations are initially low (i.e. for apple leaf midge in newly planted orchards) for success. Control of apple leaf midge by mass trapping is under investigation in New Zealand using pheromone supplied by NRI (Suckling et al., in press).

Conclusions

• The female sex pheromone of the apple leaf curling midge, Dasineura mali, has been shown to be (Z)-13-acetoxy-8-heptadecen-2-one, a keto-ester, a variation on a new class of midge pheromone structures that is currently the subject of a patent application. • Only the S enantiomer is attractive; the R enantiomer has no activity. The racemate is highly attractive. • The D. mali pheromone has proved very useful for monitoring pest populations in the field. A standard 20 x 20 cm delta trap baited with a rubber septum lure loaded with 3µg of the pheromone racemate and deployed at a height of 0.5 m in apple orchards is recommended. • An experiment in 2006 showed that the traps can be used to time sprays of synthetic pyrethroids for control of the pest, but that multiple applications were required to give a high degree of control. • Large scale field experiments started in the UK in 2005 to test the use of the pheromone for control by mating disruption and attract and kill approaches. Results so far have indicated the need to protect the pheromone from UV degradation and to deploy the treatments on a large scale and in newly planted orchards where populations are initially low.

References

Foster, S.P., Harris, M.O. & Millar, J.G. 1991: Identification of the sex pheromone of the Hessian fly, Mayetiola destructor (Say.). – Naturwissenschaften 78: 130-131.

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Gries, R., Gries, G., Khaskin, G., King, S., Olfert, O., Kaminski, L-A., Lamb, R. & Bennett, R. 2000: Sex pheromone of orange wheat blossom midge, Sitodiplosis mosellana. – Naturwissenschaften 87: 450-454. Gries, R., Khaskin, G., Gries, G., Bennett, R.G., Skip King, G.G., Morewood, P., Slessor, K.N. and Morewood, D. 2002: (Z,Z)-4,7-Tridecadien-(S)-2-yl acetate: sex pheromone of Douglas fir cone gall midge. – Journal of Chemical Ecology 28: 2283-2297. Hall, D. R., Cross, J. V., Farman, D., Innocenzi, P. J., Ando, T. and Yamamoto, M. 2005. Sex pheromones of apple leaf curling midge, Dasineura mali, and raspberry cane midge, Resseliella theobaldi: a new class of pheromone structures. – Abstracts of the 21st meeting of International Society of Chemical Ecology, Washington July 2005. p 46. Harris, M.O., Foster, S.P., Agee, K., and Dhana, S. 1996: Sex pheromone communication in the apple leafcurling midge (Dasineura mali). – Proceedings of New Zealand Plant Protection Conference 49: 52-58. Hillbur, Y., Anderson, P., Arn, H., Bengtsson, M. Löfqvist, J., Biddle, A.J., Smitt, O., Högberg, Plass, E., Franke, S. & Francke, W. 1999: Identification of sex pheromone components of the pea midge, Contarinia pisi (Diptera: Cecidomyiidae). – Naturwissenschaften 86: 292-294. Hillbur, Y., El-Sayed, A., Bengtsson, M. Löfqvist, J., Biddle, A.J., Plass, E., and Francke, W. 2000: Laboratory and field study of the attraction of male pea midges, Contarinia pisi, to synthetic sex pheromone components. – Journal of Chemical Ecology 26: 1941-1952. Hillbur, Y., Bengtsson, M. Löfqvist, J., Biddle, A.J., Pillon, O., Plass, E., Franke, W. & Hallberg, E. 2001: A chiral sex pheromone system in the pea midge, Contarinia pisi. – Journal of Chemical Ecology 27: 1391-1407. Suckling, D.M., Walker, J.T.S., Shaw, P.W., Manning, L. Peter Lo, P., Wallis, R., Bell, V., W.R., Sandanayaka, W.R.M., Hall, D.R.,Cross, J.V. & El-Sayed., A.M. Trapping Dasineura mali (Diptera: Cecidomyiidae) in Apples. – Journal of Economic Entomology. In press.

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 169-173

Investigations on the sex pheromone of pear leaf midge, Dasineura pyri (Bouché), and other gall midge pests of fruit crops

Lakmali Amarawardana1, David Hall1, Jerry Cross2 1 Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, U.K. [email protected], [email protected] 2 East Malling Research, New Road, East Malling, Kent ME19 6BJ, UK. [email protected]

Abstract: The pear leaf midge, Dasineura pyri (Bouché), is a pest of pear orchards, with nursery stocks and new shoots on young and fruiting trees being particularly vulnerable. Although a sex pheromone has not been demonstrated in this species, females of related midge species produce a sex pheromone to attract males for mating. Late larvae of D. pyri were collected from pear orchards and reared to adulthood individually. After sexing, volatiles were collected from both males and females by air entrainment. Collections were analysed by gas chromatography (GC) coupled to electroantennographic (EAG) recording from a male antenna, and by GC coupled to mass spectrometry (MS). Male midges responded to two components in the female volatile collections. The mass spectrum of the major compound indicated it was a 17-carbon diacetate with one double bond, and both mass spectrum and GC retention times were found to be identical with those of (Z)-2, 13-diacetoxy-8-heptadecene prepared previously. However, no male midges were caught in traps baited with the synthetic compound in pear orchards. Further work is required to confirm this identification, to determine which of the four possible isomers is produced by the female and to identify the minor component.

Key words: Dasineura pyri, pear leaf midge, pheromones, electroantennography,

Introduction

Leaf curling and gall midges (Diptera, Cecidomyiidae) on fruit crops affect the economic viability of host plants. They are currently controlled using broad-spectrum insecticides, but concerns have grown over the usage of insecticides due to adverse effect on environment, beneficial organisms and human health. Use of female sex pheromones provides more environmentally-safe ways of controlling pests through manipulating the behaviour of male insects. The scope of this project is to identify the female sex pheromones of several midge species for monitoring and mass trapping purposes. The pear leaf midge, Dasineura pyri (Bouché), is a serious pest in pear orchards in the UK and many other European countries. Nursery stocks and new shoots on young and fruiting trees are most vulnerable to this pest which retards the growth leading to yield loss. The adult lays eggs on rolled margins of the young leaves. Eggs hatch with in a few days and larvae feed on the upper epidermis, causing leaves to be tightly rolled. Fully grown larvae drop to the ground and pupate in the soil in a silken cocoon. After two weeks adults emerge, mate and lay eggs (Barnes, 1948). Although production of a sex pheromone by this species has not been demonstrated, a powerful pheromone is produced by the closely related apple leaf midge, D. mali, and this has been identified and synthesised (Cross et al., 2007). We here report preliminary work on identification of the female sex pheromone of the pear leaf midge.

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Material and methods

Insects Late larvae of pear leaf midge were collected in May 2006 from heavily infested pear shoots in pear orchards of Broadwater Farm, West Malling, UK. Shoots were stored in plastic boxes (19 cm x 10.6 cm x 7.5 cm). Mature larvae crawled out from the leaves for pupation, and these were collected and reared individually in clear plastic tubes (1.5 cm i.d. x 2.3 cm) containing a piece of wet filter paper which acted as the substratum for pupation. Tubes were closed with plastic caps and stored under controlled conditions, at 23°-18°C and 16L:8D light cycle. Adults emerged after 7-12 days and males and females were separated on the basis of antennal morphology. Approximately 6300 larvae were reared out of which 70% adults emerged as adults.

Pheromone collection Volatiles from male and female midges were collected by air entrainment under the same conditions as used for insect rearing. Midges were placed in a glass vessel (5.3 cm i.d. x 13 cm) with a glass frit at one end. Air was drawn into the vessel (0.5 l/min) with a vacuum pump (M 361C, Charles Austen Pump Ltd, UK) through an activated charcoal filter (20 cm x 2 cm; 10-18 mesh, Fisher Chemicals, UK) and out through a collection filter consisting of a Pasture pipette (4 mm i.d.) containing Porapak Q (200 mg; 80-100 µm, Waters Associates Inc., USA) held between two glass wool plugs. The Porapak was purified by Soxhlet extraction with chloroform and prior to use filters were cleaned with dichlomethane (1.5ml, pesticide grade) and dried by a stream of nitrogen. Midges were introduced at 24 hr intervals for three days. Three sets of volatiles from both males and females were collected with batches of between 300 to 1160 midges.

Electrophysiological recording Male antennal responses to female volatiles were analysed by gas chromatography (GC) linked to electroantennography (EAG) (Cork et al., 1990). The GC used was a HP6890 instrument (Aglilent Technologies) with a flame ionisation detector and fused silica capillary columns (30m x 0.32mm x 0.25µm film thickness) coated with polar (Supelcowax-10, Supelco, USA) and non polar (SPB-1, Supelco, USA) phases. The column ends were connected to a push-fit Y-connector, the outlet of which was connected to a second Y- connector. This was connected with identical pieces of deactivated silica capillary column, one going to FID and the other to a glass T piece. A stream of nitrogen (200 ml/min) blew the contents of the T piece directly over the antennal preparation every 3 sec. The oven temperature was maintained at 50°C for 2 min, then programmed at 10°C /min to 250°C and held for 5 min. Injection was splitless at 220°C and helium was used as carrier gas (2.4 ml/min). EAG responses were recorded using a portable recoding unit (INR-2, Synthech, The Netherlands) comprising integrated electrode holders and amplifier. Glass electrodes were pulled and were filled with electrolyte (0.1 M solution of KCl with 1% polyvinylpyrrolidine). The tips of the electrodes were broken off so that both antennae could be inserted into the recording electrode and the abdomen inserted into the reference electrode. Signals were amplified and analysed with EZChrom software (Elite v3.0).

Gas chromatograph linked to mass spectrometry The identification of compounds giving EAG responses from male midges was carried out by GC (HP6890 Agilent Technologies) coupled to mass spectrometry (MS; HP 5973, Agilent Technologies). Injection was splitless (220°C) with fused capillary columns (30m x 0.25 mm i.d.) coated with polar and non-polar phases as above. The temperature of the oven was held

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at 60°C for 2 min then programmed at 10°C/min to 250°C and held for 5 min. Helium was used as the carrier gas (1.0 ml/min). GC retention times were converted to Retention Indices (RI) relative to those for normal hydrocarbons.

Field assessment Preliminary field tests were carried using white delta traps (28 cm long × 20 cm sides; Agrisense, Treforest, UK) with sticky bases baited with rubber septa containing 10 µg of the synthetic pheromone. These were deployed in four separate plots of pears in East Malling Research Station and Broadwater farm, West Malling, UK. In each plot an unbaited control and a pheromone-baited trap were hung on the 10th and the 20th pear trees on the centre row. The sizes of the plots varied from 7 ha to 0.25 ha. Insect counts were taken once a week for a period of one month during August, 2006.

Results and discussion

GC-EAG analyses of volatiles from female D. pyri midges indicated the presence of two compounds eliciting consistence EAG responses from male antennae (Figure 1). The minor component (EAG response with lower intensity) appeared at 18.42 and 19.26 min on polar and non polar columns respectively while the major component (EAG response with higher intensity) appeared at 20.92 and 20.37 min respectively.

0.08 5000 FID EAG standards standards PLM17.dat PLM17.dat 0.07 Retention Time Retention Time

0.06 2500

0.05 * 0.04 * 0 FID - voltsFID - EAG millivolts EAG 0.03

-2500 0.02 4.38 13.17 13.04 14.08

0.01 7.66

-5000 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Minutes

Figure.1. GC-EAG Analysis of volatiles from female D. pyri on polar GC column (upper trace EAG, lower GC; * indicates EAG response)

Although only tiny amounts of the pheromone components were present (approx. 4.3 pg per female), a mass spectrum was obtained on the major component and the MS fragmentation pattern and GC retention indices were compared with those of known compounds. The mass spectrum (Figure 2) showed ions at m/z 43 and m/z 61 which are diagnostic peaks for acetate esters. The fragment of low intensity at m/z 234 is consistent with

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loss of two acetoxy groups from a 17-carbon diacetate with one double bond (molecular weight 354). The rest of the MS fragmentation pattern was difficult to interprete. However, comparison of GC retention times (Table 1) and mass spectra (Figure 2) with those of synthetic standards showed that the data for the naturally-occurring compound were identical with those for (Z)-2,13-diacetoxy-8-heptadecene (Figure 3) prepared during previous work on the pheromone of the closely related apple leaf curling midge, D. mali.

Table 1. Retention time and retention indices (RI) of major pheromone component and synthetic (Z)-2,13-diacetoxy-8-heptadecene on non-polar and polar GC columns in GC-EAG and GC-MS

compound Non-polar GC column Polar GC column RT (min) RI RT (min) RI

GC-EAG Natural compound 20.37 1854 20.92 2087 GC-MS Natural compound 33.86 1854 24.33 2082 GC-MS Synthetic compound 33.88 1857 24.34 2083

Figure 2. Mass spectrum of major pheromone component (upper) and synthetic (Z)-2,13- diacetoxy-8-heptadecene (lower).

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Traps baited with the synthetic (Z)-2,13-diacetoxy-8-heptadecene did not catch males of D. pyri even though these were observed to be present. This may be because only one of the four isomers of (Z)-2,13-diacetoxy-8-heptadecene is attractive and the others inhibit attraction. The synthetic compound also contained 5% of the (E) isomer and this may be inhibitory Furthermore, presence of the minor component in the pheromone blend may be required for attraction.

OAc OAc CH H C 3 3

Figure 3. Structure of (Z)-2, 13-diacetoxy-8-heptadecene

Thus the major component of the female sex pheromone of D. pyri is thought to be (Z)- 2,13-diacetoxy-8-heptadecene which is chemically related to the pheromone of D. mali, (Z)- 13-acetoxy-8-heptadecen-2-one. Further work is required to confirm this structure, to determine which of the four possible isomers is produced by the female midge, and to identify the second “minor” pheromone component. An attractive lure for use in the field will then be developed.

Acknowledgements

We thank UK Horticultural Development Council and the Worshipful Company of Fruiterers for funding this project.

References

Barnes, H.F. 1948: Gall midges of economic Importance. Vol. III Gall midges of Fruit. – Crosby Lockwood, London. Cross, J.V., Hall, D.R. & Shaw, P. 2007: Exploiting the sex pheromone of the apple leaf midge, Dasineura mali, for pest monitoring and control. – IOBC/wprs Bulletin 30(4): 159-167. Cork, A.; Beevor, P.S.; Gough, J.E. & Hall D.R. 1990: Gas chromatography linked to electroantennography: a versatile technique for identifying insect semiochemicals. – In: Chromatography and Isolation of Insect Hormones and Pheromones. A.R. McCaffery & I.D. Wilson (eds.), Plenum Press, London: 271-279.

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 175

The effect of aphid sex pheromone and plant volatiles on the behaviour of Dysaphis plantaginea and its parasitoid Aphidius matricariae

J. Fitzgerald1, C. Jay1, C. James1, L. Wadhams2, S. Dewhirst2, C. Woodcock2, G. Poppy3, A. Stewart-Jones3 1 East Malling Research, East Malling, Kent, ME19 6BJ, UK, [email protected] 2 Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK 3 University of Southampton, Southampton, SO16 7PX, UK

Abstract: The objective of this research was to determine the practicability of exploiting the responses of aphids and their natural enemies to aphid sex pheromone and to plant volatiles in order to enhance biocontrol of the pest. In experiments to determine the composition of the Dysaphis plantaginea Passerini (Homoptera: Aphididae) sex pheromone, coupled GC-EAG analysis of air entrainment samples from D. plantaginea oviparae using male antennae located four physiologically active components. The two major components were shown to be (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone. A third EAG-active component was identified that elicited a strong behavioural response by male D. plantaginea. This demonstrates that the sex pheromone of D. plantaginea comprises at least a three component mixture. Many plants respond to herbivore feeding by modifying their secondary metabolism to produce semiochemicals that act to reduce herbivore colonisation and increase attraction of beneficials, particularly highly mobile parasitoids. In our experiments, production of a number of compounds, from both the primary and secondary host plants, was shown to be increased in response to D. plantaginea infestation. Results from laboratory behavioural studies and from field experiments will be used to outline the effects of several of these compounds on pest and parasitoid behaviour.

Key words: Dysaphis plantaginea, Aphidius matricariae, sex pheromone, plant volatiles

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 177-184

Different host plant odours influence migration behaviour of Cacopsylla melanoneura (Förster), an insect vector of the apple proliferation phytoplasma

Christoph J. Mayer1,2, Jürgen Gross2 1 BBA, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, 69221 Dossenheim, Germany, [email protected] 2 Institute for Phytopathology und Applied Zoology, Justus-Liebig University of Gießen, Gießen, Germany, [email protected]

Abstract: In recent years the hawthorn psyllid Cacopsylla melanoneura (Förster) has been identified as one of the insect vectors of the apple proliferation phytoplasma which causes major damages in apple cultivation. C. melanoneura is univoltine, spending only a short time of its life cycle on apple or hawthorn – during copulation, egg laying and nymphal development. The adults of the new generation migrate soon after emergence to coniferous trees and spend the largest part of their life there. After overwintering, the adults return to apple or hawthorn for reproduction, in early spring. In order to elucidate the migration behaviour and ecology of C. melanoneura, different populations were investigated in field, over a two year period. A detailed description of their life cycle, supplemented by timing and duration of their two migration phases and the involved host plants are presented. Further, hawthorn psyllids of different ages (newly hatched adults and overwintered adults) were investigated in a dynamic Y-olfactometer setup for their preferences towards the odours of different host plants. The behavioural response of the two tested ages of the psyllids corresponds with the two different phases of migratory behaviour in the field. While there was a strong positive response for apple or hawthorn odours in overwintered adults, there was none in freshly emerged adults of the next generation. In contrast, the newly emerged adults showed a strong response for spruce volatiles.

Key words: Cacopsylla melanoneura, psyllids, apple proliferation phytoplasma, insect vector, chemoecology, migration behaviour, life cycle

Introduction

In recent years it was discovered that the univoltine psyllid species Cacopsylla melanoneura (Foerster) and C. picta (Foerster) (Hemiptera: Psyllidae) are the vectors for the apple proliferation phytoplasma, causing major economic yield losses by inducing ‘witches brooms’ and tasteless dwarf fruits (Frisinghelli et al., 2000; Jarausch et al., 2004; Seemüller et al., 2004; Tedeschi et al., 2002; Tedeschi and Alma, 2004). The hawthorn psyllid C. melanoneura spends most of the year on conifers, whereas reproduction and larval development takes place on hawthorn (Crataegus spp. L.) or apple (Malus domestica L.) (Rosaceae) in early spring (Lauterer, 1999; Ossiannilsson, 1992). Thus, during their life time the adults have to switch at least twice between different hosts. Hitherto not much is known about orientation and host finding in psyllids. There have been a few records mentioning attraction of different psyllid species to yellow card traps (Brennan and Weinbaum, 2001; Lapis and Borden, 1995). Furthermore, Soroker et al. (2004) reported an attraction of female pear psyllid C. bidens by volatiles of pear twigs, while Gross and Mekonen (2005) recorded olfactory response in the apple feeding psyllids C. melanoneura and C. picta firstly. In this study we investigated population development of C. melanoneura in two different apple orchards during two consecutive reproductive seasons (2005 and 2006). Additionally,

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population densities were investigated at a hawthorn hedgerow and a lowland stand of spruce. During the migration phases of 2006, a stand of spruce located in a mountainous region was also investigated to determine the time when the last mature adults leave their overwintering hosts for reproduction and when the first young adults of the new generation return. The data of extensive monitoring of C. melanoneura populations allow a detailed description of their life cycle with timing and duration of two migration phases as well as the involved host plants. The roles of chemical cues from different host plants for host finding behaviour of C. melanoneura during their migratory phases were also investigated by Y-tube olfactometer trials.

Material and methods

Population monitoring Monitoring of population dynamics was carried out by beating tray sampling (ten branches with three beats each per sample) at five locations in the vicinity of Heidelberg, Germany: (1) apple orchard without any pest management, located at Neuenheimer Feld (Heidelberg) (NPM orchard); (2) apple orchard with integrated pest management, located at BBA, Dossenheim (IPM orchard); (3) not managed hawthorn hedgerow (Crataegus monogyna), bordering the orchards of the BBA Dossenheim (HH); (4) lowland stand of spruce without management, Neuenheimer Feld (LS); (5) stand of spruce in a mountainous region, located at Weißer Stein (400 m NN) (MS). At all lowland localities (1-4) sampling was done for at least 16 months continuously until September 2006. In 2005 sampling started in March in the apple orchards (1, 2), followed by the hawthorn hedgerow (3, in April) and spruce (4, in June). Additionally in 2006, sampling was done at the mountainous stand of spruce (5) from February to June for monitoring the time when mature psyllids finally leave their overwintering host, and when young adults of the following generation return, respectively. At sites 1-3 and 5 ten samples were taken during each time of sampling while at site 4 three samples were taken. The frequency of sampling was twice a week from March to September 2005, weekly in October and November 2005, as well as from March to September 2006. During winter times (December 2005 to February 2006) sampling was done about every second week. Determination of the collected psyllids was done using the key of (Ossiannilsson, 1992).

Rearing of test insects for biotests Mature adults of C. melanoneura were collected from hawthorn (Crataegus monogyna) by beating tray sampling in early spring and held in a tempered greenhouse chamber under natural light conditions (D:N = 20:15°C) on caged hawthorn except for the psyllids used in experiment 4, which were transferred to apple trees (‘Gala Royal’) 2-4 days before behavioural trials. Mature adult females of C. melanoneura were separated one day before testing and stored in plastic vials at 4°C. The vials were exposed to room temperature 30-120 min prior to start of the behavioural trials. Freshly emerged young adults were obtained from laboratory rearing on caged hawthorn under the same conditions as described for mature psyllids. To ensure a defined age of 0-4 days after hatching for olfactometer trials, all freshly emerged psyllids were collected from the rearing cages every 1-2 day and were kept in separate boxes until testing. Young adult females were separated 30-120 min prior to testing and stored in plastic vials under room temperature until testing. This was done as young adults are much more susceptible to starving and desiccation as mature adults are. Behavioural trials

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Behavioural tests were carried out using a dynamic olfactometer consisting of a Y-shaped glass tube (entrance arm: 12.5 cm, test arms: 21.0 cm, inner diameter: 0.6 cm). All trials were done at room temperature (18-20°C) in a dark environment with one light source (daylight spectrum) above the olfactometer. Charcoal cleaned air was pumped through two glass jars (volume 2 L) containing the volatile sources. In each jar a water filled glass vial was placed. Two different plant twigs (length 10-15 cm) were cut directly before the experiments and placed each in one of the vials. Volatile flow in each arm of the olfactometer was 150 mL/min controlled by a flow meter (Supelco, Bellefonte, PA). Different odours were offered singularly or simultaneously (hawthorn twigs, apple twigs ‘Gala Royal’, spruce twigs, or empty) in the test arms of the olfactometer. The position of the test arms was turned every second trial, and after four trials the glass tube was exchanged, cleaned and heated as described below. For each test ten females were put simultaneously into the entrance arm of the olfactometer and were allowed to walk up and decide for one of the test arms within 15 min. Every individual that had passed a final marking (10.0 cm after the branching) on one of the test arms was counted. Results were analysed statistically by log (x+0.5) transformed dependent t-test (StatSoft, 1999). After each set of trials all parts of the setup behind the charcoal filter were cleaned with ethanol (70 % V/V) and deionised water and after that heated at 110 °C for at least 30 min.

Results and discussion

Population development The monitoring of psyllid populations during two reproductive seasons revealed some details about the population dynamics of C. melanoneura (Fig. 1, 2): In both years the first overwintered adults (“mature adults”) arrived in the orchards and in the hawthorn hedgerow at the end of March. Their abundance over the whole reproductive season was about 10-fold higher on hawthorn than on apple. Additionally, the time span in which mature adults of C. melanoneura were captured on hawthorn was four weeks longer than on apple, lasting until the end of May. This indicates that apple may be a less preferred and even less suitable reproduction host. The former explanation will be discussed below together with the results of the behavioural trials, whereas the latter needs further examination in field and laboratory experiments. The population density in the NPM orchard was higher than in the IPM orchard, in both years. However, it is not clear whether lower density of C. melanoneura in the IPM orchard is on account on pest control applications or whether it is due to different varieties, tree size and other parameter found in the NPM orchard. At all three reproduction sites (NPM, IPM, HH) second instar nymphs were firstly observed in the second week of April, the first adult individuals of the new generation (“young adults”) were observed at the beginning of May (weeks 19 (2005) and 20 (2006)). The young adults completely disappeared from hawthorn and apple after week 25 (end of June) in both years. Based on this observation one could conclude that the developmental time from nymph to adult on apple and hawthorn was equal, indicating that apple may be just a suitable host plant for reproduction like hawthorn. Interestingly, young adults left their two different host plants simultaneously, but their parents were found four weeks longer on hawthorn than on apple. To explain these observations, three different possibilities are feasible: (1) nymphal development on apple is delayed; (2) young adults staid longer on apple than on hawthorn after hatching; (3) mature females on hawthorn stopped laying eggs some weeks before they died. The first mentioned assumption is contradicted by the observation that young adults appear on apple and hawthorn at the same time. Assumption (2) implies a

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higher need of nutrient uptake for the adults on apple, while nymphal development was not delayed. Further experiments are necessary to elucidate this matter.

Figure 1. Population dynamics in 2005 and 2006 of C. melanoneura in two apple orchards (A: NPM, B: IPM). Mature adults arrive from overwintering hosts about week 12 and population reaches its peak 1-2 weeks later. Young adults appeared around week 19- 25. Note different scale of y-axis for the two orchards. Apart from the time period shown, no psyllids were captured on apple.

Figure 2. Population dynamics in 2005 and 2006 of C. melanoneura in a hawthorn hedgerow (A) and on lowland spruce stand (B). Monitoring started in 2005 (week 15 (hawthorn) and 20 (spruce)). A: Population density on hawthorn was higher in 2005. In both years more mature than young adults were found. B: High amounts of mature adults were found in the second half of March just before population was built up on hawthorn. Young adults were found one week after first captures were made on hawthorn.

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In both years at all three sites, the relative numbers of young adults of the following generation were much lower than the relative numbers of the preceding generation in spring. The only exception was observed at the NPM orchard in 2006, where a maximum of 0.5 individuals per sample of mature adults were caught, compared to 2.0 individuals per sample of young adults of the following generation. Oviposition trials with C. picta revealed that a female oviposits more than 200 eggs in a single week (unpublished results). Assuming a similar fecundity for females of C. melanoneura would lead to the expectation of much higher population densities of young adults. But as reported below, the young adults left their reproduction host within a few days after hatching, so that no high densities as expected could be collected at the same time.

Figure 3. Migratory phases and different host plants during the life cycle of C. melanoneura: After development from egg to adult on their reproduction host plants (apple, hawthorn) young adults leave them (migratory phase 1, MP 1) by switching to transitional hosts (lowland conifers), before reaching mountainous conifers as overwintering hosts. After overwintering, returning mature adults (migratory phase 2, MP 2) first go to lowland conifers (transitional host), before finally shifting to the reproductive hosts for oviposition.

Monitoring psyllids on lowland spruces in surroundings of the NPM orchard revealed first appearance of young adults, one week after they were observed on hawthorn and apple. The threefold number of young adults on spruce than on apple was collected in the NPM orchard. The captures of young adults on lowland spruces declined and dropped to zero, during the same time as young adults disappeared from hawthorn and apple. In 2006, sampling of mountainous spruce stands (400 m NN) showed that first young adults of C. melanoneura appeared in week 23, four weeks after the first young adults were observed on their reproduction hosts. In week 24 there was already an average capture of 2.5 individuals per sample in the mountainous spruce stands, while last individuals of young adults were

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found on lowland spruce. Thus, we conclude that young adults move from their reproduction sites (apple, hawthorn) to spruce stands close to them within one week after hatching, then stay some days, before leaving them again, settling on spruce trees at higher locations. After overwintering, the first mating between mature adults of C. melanoneura was observed on mountainous spruce in mid February. Shortly after this observation the first mature adults were caught on lowland spruce (week 8). The captures on lowland spruce had their peak in week 12 with an average of 38.6 C. melanoneura per sample. In week 13 the last mature adults were caught on mountainous spruce and in week 16 the last ones on lowland spruce. They were also simultaneously collected from their reproductive hosts, as described above. This indicates a migration of the mature adults from mountainous spruce (overwintering host) to lowland spruce (transition host) before finally shifting to apple trees or hawthorn bushes (reproduction hosts). Summarizing the results of monitoring of C. melanoneura over a period of two years, we can describe the life cycle of C. melanoneura including their different host plants and migration phases as follows (Fig. 3): After having completed their nymphal development on apple or hawthorn, young adults start their first migration phase (MP1) and leave their reproduction host immediately, switching to surrounding conifers (not only spruce, but as well pine, larch, and Douglas fir (unpublished results)) as transitional host plants. Within four weeks after appearance, the first individuals of this new generation are found on mountainous stands of conifers. These mountainous conifers are used as overwintering hosts. With the arrival on the overwintering host, MP 1 ends. The first incidence of mating, occurring already on the overwintering host, is probably the beginning of the second migration phase (MP 2), before mature psyllids return to the reproduction hosts for further mating and oviposition. During MP 2 the first psyllids are found on transitional host (lowland spruce) in late February, and in the middle of March the first individuals have reached the reproduction hosts. MP 2 leads to the last phase of the psyllids life cycle, whereas the reproductive phase is characterized mainly by mating and oviposition.

Behavioural trials Behavioural responses of the psyllid females were shown for both migratory phases (Table 1). When mature females at the beginning of MP 2 were tested for attraction to host plant odours, they favoured hawthorn odours which were offered simultaneously with either apple odour or an empty control. But they did not distinguish apple odour from an empty control. After having gained experience with apple odours by feeding and egg laying on apple for 2-4 days, apple odour became more attractive than hawthorn odour when offered, simultaneously. Young adult females, 0-2 days after their last moulting, which were supposed to be at the beginning of their MP 1, showed slight preference for volatiles of hawthorn when offered with an empty control, simultaneously. But they did not distinguish between the volatiles of apple and an empty control, or between hawthorn and apple, respectively. When volatiles of their overwintering host (spruce) were tested simultaneously with volatiles of their reproduction hosts (apple or hawthorn), the young females did not distinguish between hawthorn and spruce odours, but significantly preferred spruce to apple odours. These results confirm the findings of Gross and Mekonen (2005) that C. melanoneura is capable to perceive and to orientate towards different host odours. The results of the olfactometer trials fit very well with the findings of the population monitoring, too: Hawthorn is the predominant reproduction host of C. melanoneura. Thus, it is not surprising that during MP 2 mature females, which are in search of a suitable oviposition site, prefer its volatiles to apple volatiles, reflecting the higher attractiveness of hawthorn compared to apple in the field. The field population of mature adults was higher in both years and lasted four weeks longer on hawthorn than on apple. The psyllids used in experiments 3 and 4 were originally collected

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from and kept on hawthorn, but the psyllids in experiment 4 additionally had experienced apple as food source and oviposition site for 2-4 days prior to the trials. In both experiments, the females preferred the volatiles of the last suitable host they experienced: hawthorn in experiment 3 and apple in experiment 4. This shows that C. melanoneura females are able to learn the volatile bouquet of a suitable host.

Table 1: Behavioural response of C. melanoneura females to host plant volatiles in a Y-tube olfactometer. All tested psyllids were collected from (mature adults) or reared on (young adults) hawthorn. The last food source prior to the trials was hawthorn except for the psyllids used in experiment 4, which experienced apple as host for a period of 2-4 days. The mean number +/- SD of psyllids which entered the respective arm of the olfactometer is presented. Plant odours marked grey are significantly more attractive than the alternative offered odour sources. Dependent t- test, n.s.: p > 0.05.

Life Volatile source mean +/- mean +/- stage Exp. # n P < A SD B SD A B 1 apple empty 20 3.80 1.36 3.70 1.72 n.s. 2 hawthorn empty 20 4.70 1.22 2.75 1.12 0.01 3 apple hawthorn 20 3.20 1.44 4.90 1.41 0.01 mature mature females females 4 apple hawthorn 19 3.68 1.38 1.84 1.30 0.001 5 apple empty 17 3.12 1.54 2.88 1.36 n.s. 6 hawthorn empty 20 3.30 1.56 2.50 1.73 0.05 7 apple hawthorn 20 2.30 1.30 1.95 1.47 n.s. young

females females 8 apple spruce 20 1.10 1.07 2.65 2.01 0.01 9 hawthorn spruce 20 2.30 1.17 2.55 1.61 n.s.

The behavioural responses of young females during MP 2, when they left the reproduction host and started to migrate to coniferous overwintering hosts, were the same as for the mature females, if apple or hawthorn volatiles were tested with an empty control, simultaneously . There was no discrimination between apple volatiles and an empty control, but a preference for hawthorn volatiles compared to an empty control. If hawthorn volatiles were tested with apple volatiles simultaneously, the mature females during MP 1 reacted differently than young females during MP 2: they preferred the hawthorn odours, while in contrast the young females did not distinguish between the volatiles of these two plants. Further, these results show that hawthorn, on which nymphal development took place, did not turn repellent for the young females, as it was still preferred to an empty control. But its volatiles lost their attractiveness for young females which did not distinguish between apple and hawthorn volatiles. When volatiles of the overwintering host (spruce, experiments 8 and 9) were tested against volatiles of one of the two reproductive host plants, they were preferred to apple volatiles, but no different response was detected when spruce was offered with hawthorn, simultaneously. The preference for spruce compared to apple volatiles indicates that in field young adults, already 1-2 days after hatching, may orientate towards the volatiles of spruce, if they had developed on apple. As the tests were carried out with very young females (1-2 days old), a possible increase in attraction of spruce odours compared to hawthorn odours has to be tested in future experiments with young females which are some days older. Young psyllids might still be attracted by hawthorn volatiles, if no suitable

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overwintering host is nearby. Finally, these results indicate that MP 1 may be initiated by the loss of attractiveness of their reproduction host combined with an increasing attractiveness of spruce.

Acknowledgements

The authors are grateful to Elke Breitinger, Kai Lukat and Sandra Förmer for their valuable assistance in insect rearing, field sampling and conducting behavioural trials. This study was funded by the German Research Association (Deutsche Forschungsgemeinschaft, GR 2645/1-1). C.J.M. thanks the IOBC fund for a student travel award.

References

Brennan, E.B. & Weinbaum, S.A. 2001: Psyllid responses to colored sticky traps and the colors of juvenile and adult leaves of the heteroblastic host plant Eucalyptus globulus. – Environ. Entomol. 30(2): 365-370. Frisinghelli, C.; Delaiti, L.; Grando, M.S.; Forti, D. & Vindimian, M.E. 2000: Cacopsylla costalis (Flor 1861), as a vector of apple proliferation in Trentino. – J. Phytopath. 148(7- 8): 425-431. Gross, J. & Mekonen, N. 2005: Plant odours influence the host finding behaviour of apple pyllids (Cacopsylla picta; C. melanoneura). – IOBC/wprs Bulletin 28(7): 351-355. Jarausch, B.; Schwind, N.; Jarausch, W. & Krczal, G. 2004: Overwintering adults and spring- time generation of Cacopsylla picta (syn. C. costalis) can transmit apple proliferation phytoplasmas. – Acta Hort. (ISHS) 657: 409-413. Lapis, E.B. & Borden, J.H. 1995: Role of wavelength-specific reflectance intensity in host selection by Heteropsylla cubana Crawford (Homoptera, Psyllidae). – Pan-Pac. Entomol. 71(4): 209-216. Lauterer, P. 1999: Results of the investigations on Hemiptera in Moravia, made by the Moravian museum (Psylloidea 2). – Acta Mus. Morav. 84: 71-151. Ossiannilsson, F. 1992: The Psylloidea (Homoptera) of Fennoscandia and Denmark. – Fauna Entomologica Scandinavica (26). Seemüller, E.; Dickler, E.; Berwarth, C. & Jelkmann, W. 2004: Occurrence of psyllids in apple orchards and transmission of apple proliferation by Cacopsylla picta (syn. C. costalis) in Germany. – Acta Hort. (ISHS) 657: 533-537. Soroker, V.; Talebaev, S.; Harari, A. & Wesley, S. 2004: The role of chemical cues in host and mate location in the pear psylla Cacopsylla bidens (Homoptera: Psyllidae). – J. Insect Behav. 17(5): 613-626. StatSoft (1999). STATISTICA for Windows (Computer programm manual). '99 Edition Tedeschi, R.; Bosco, D. & Alma, A. 2002: Population dynamics of Cacopsylla melanoneura (Homoptera: Psyllidae), a vector of apple proliferation phytoplasma in northwestern Italy. – J. Econ. Entomol. 95(3): 544-551. Tedeschi, R. & Alma, A. 2004: Transmission of apple proliferation phytoplasma by Caco- psylla melanoneura (Homoptera: Psyllidae). – J. Econ. Entomol. 97(1): 8-13.

Insecticide Resistance

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 187-194

Reliability of resistance monitoring on diapausing larvae of codling moth, Cydia pomonella (L.)

M. Reyes1, J. Olivares1, C. Ioriatti2, E. Pasqualini3, P.J. Charmillot4, P. Franck1, B. Sauphanor1 1 PSH - Ecologie de la Production Intégrée, INRA Site Agroparc, 84914 Avignon Cedex, France; [email protected] 2 IASMA Research Center – Plant Protection Department, S. Michele all’Adige, 38010 – Trento. Italy; [email protected] 3 Dipartimento di Scienze e tecnologie Agroambientali (DiSTA), Università di Bologna, Italy; [email protected] 4 Station fédérale de recherches en production végétale de Changins, CH-1260 Nyon, Switzerland; [email protected]

Abstract: Resistance monitoring in Cydia pomonella (L.) (Lepidoptera: Tortricidae) has been mainly performed on non-target instars, and currently topical application on diapausing larvae is a widely used method. However, high differences in resistance ratios according to the developmental stage have already been documented and laboratory results are not ever consistent with observed field situations. Relating the expression of resistance mechanisms with resistance of a given instar to various insecticides could simplify the monitoring procedure. We used topical application on diapausing larvae to evaluate the susceptibility to ten insecticides of four laboratory strains and 47 field populations, on which resistance mechanisms were also analyzed. All populations seemed to be less susceptible than the susceptible laboratory strain for at least one insecticide. The resistance to five of the studied insecticides was significantly correlated with the activity of enzymatic detoxication systems, mainly mixed function oxidases and Glutathion S- transferase, while target site mutations were not related to any reduction of susceptibility. For few populations, assays on first instar larvae and field trials were also performed. Results were not always consistent with those of topical applications. For some compounds, monitoring test using diapausing larvae tends to overestimate the magnitude of resistance. Otherwise, the spectrum of insecticide resistance cannot ever be explained by known resistance mechanisms. Bioassays therefore remain a necessary tool for resistance monitoring and topical application on diapausing larvae allows a rapid and large-scale evaluation. However when a resistance is pointed out by such bioassay, it still has to be confirmed with a more reliable method involving the target instar of the insecticide.

Key words: Cydia pomonella, insecticide resistance, monitoring, bioassay, resistance mechanism

Introduction

Resistance monitoring in Cydia pomonella (L.) has been mostly performed on non-target instars, because they are more convenient for routine detection. Bioassays on adult male moths captured in sexual traps were first developed, using topical application or incorporation of the insecticide in the trap adhesive (Riedl et al., 1985; Varela et al., 1993). Alternative tests involved larvae of different instars collected in infested fruits, then exposed to insecticide residue on treated fruits or artificial diet (Charmillot et al., 2001; Charmillot et al., 2003, Ioriatti et al., 2005). Last, diapausing larvae collected from band traps (Audemard, 1992) were assayed by topical application after a chilling period in controlled conditions (Sauphanor et al., 2000a; Charmillot et al., 2003). However none of these tests are carried out on target

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instars of the pest (i.e. eggs or neonates), and variations in the expression of resistance between developmental stages have often been documented (Bouvier et al., 2002). Moreover, large number of insects is needed for separate assessment of resistance to each insecticide. Therefore, relating the expression of enzyme systems involved in resistance to the susceptibility of a given instar to various insecticides could simplify the monitoring procedure. In the present work, we tried to answer the following questions: • Is there a convergence between the resistance ratios assessed using bioassays on diapausing larvae and neonates? • Is the expression resistance mechanisms related to the magnitude of insecticide resistance? • Are these laboratory evaluations consistent with field observations?

Material and methods

Insects One susceptible (Sv) and three resistant (Rdfb, R∆ and Raz) strains are mass reared on artificial diet at INRA Avignon (France). Sv is maintained without insecticide exposure and Rdfb, R∆ and Raz, are submitted to constant selection pressure, by exposing the larvae to diflubenzuron, deltamethrin and azinphos-methyl, respectively (Sauphanor et al., 2000a, Boivin et al., 2003). Field populations were collected as diapausing larvae from French, Swiss, Italian, Spanish and Armenian orchards, using corrugated cardboard traps in autumn 2004 and 2005 (Audemard 1992). Samples were kept at 2°C and 12:12 light: dark during three months in order to satisfy the chill requirement and extracted from cold storage 24 or 48 (diflubenzuron) hours before insecticide application. For two populations, issuing either from an organic orchard or from a chemically treated orchard, some larvae were reared until adult stage and paired to obtain neonate larvae.

Insecticides Fresh solutions of formulated insecticides were prepared in distilled water for the neonates bioassays, while tests on diapausing larvae were carried out with solutions of technical insecticides in organic solvents. Azinphos-methyl, chlorpyrifos-ethyl, emamectin, spinosad and thiacloprid were evaluated on neonates, while the same compounds plus phosalone, deltamethin, diflubenzuron, tebufenozide, emamectin and fenoxycarb were evaluated on diapausing larvae (Sauphanor et al., 2000a; Pasquier and Charmillot, 2003).

Bioassays Bioassays on both stages were performed using discriminating dose. For diapausing larvae they were previously defined as providing at least 97% mortality in laboratory susceptible strains (Pasquier and Charmillot, 2003). Larvae were topically treated on the middle of the dorsum using a P2 Gilson micropipette, with 1 µl of the diagnostic concentration. The control insects were treated with the same volume of solvent. According to the size sample, 2 to 10 insecticides were assayed on 10 to 60 larvae per sex for each population (6 batches of 10 larvae for laboratory strains). After treatment, batches of 10 larvae were placed in PVC cages provided with corrugated cardboard pieces and transferred under suitable conditions for adult emergency (25°C, 60% RH and 16:8 light: dark). Mortality was recorded in terms of non- emerged adults. Considering the high mortalities induced by the diagnostic dose of emamectin in all field populations and laboratory strains, an additional dose-response relationship was established for this insecticide on the reference laboratory strains (Sauphanor et al., 2000a).

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For neonates the diagnostic concentration was determined from a concentration-mortality curve performed on the susceptible reference strain. Microplates of 96 wells were filled with 150 µl of artificial diet (Stonefly Industries Ltd.), on which was applied 6 µl of insecticide solution. At least 30 newly hatched larvae were assayed in individual wells for each insecticide. Mortality was registered after 4 days. A larva was considered dead when it did not respond to a probe with dissecting forceps. For one population, a field trial was also carried out, to establish the effectiveness of the insecticides evaluated in the laboratory on neonate larvae.

Enzymatic activity The GST, MFO and EST activities were analysed on adults emerged from non-treated or control larvae. Fluorescence and absorbance were measured using a microplate reader (HTS 7000, Perkin Elmer). Enzyme extract. Adult abdomens were individually homogenized on ice in 150 µl of Hepes buffer (50 mM; pH 7,0) and centrifuged at 15,000 g for 15 minutes at 4°C. The supernatant of each sample was used as enzyme source (Reyes et al., 2004). Protein content of each sample was measured according to Bradford (1976), using bovine serum albumin to build the standard curve. Esterases. The total esterase activities were measured using β-naphthyl acetate as substrate (Bouvier et al., 2002). Each well was supplied with 0.1 mM substrate in 50 mM of phosphate buffer (pH 6.5), 0.5 µl of enzyme extract and 89.5 µl of Hepes buffer (50mM, pH 7.0). After 15 min of incubation at 30°C, 20 µl of a staining reagent containing 3 g/l Fast Garnet and 35 g/l sodium dodecyl sulfate were added to the solution and the absorbance measured at 492 nm after 15 min at room temperature. Glutathione-S-transferases. GST activities were determined using monochlorobimane (MCB) as substrate (Nauen and Stumpf, 2002). Each well was supplied with 30 µl of enzyme extract, 168 µl of 100 mM reduced glutathione (GSH) in Hepes buffer (50 mM, pH 7.0) and 2 µl of 30 mM MCB (Reyes et al., 2004). Fluorescence was measured after 20 min of incubation at 22 °C with 380 nm excitation and 450 nm emission filters. Since bimane- glutathione adduct is not commercially available the activity was expressed as fluorescence units par insect (Nauen and Stumpf, 2002). Mixed-function oxidases. The MFO activity was determined using 7-ethoxycoumarin O- deethylation (ECOD) (Ullrich and Weber, 1972) adapted for in vivo analysis in microplate (De Sousa et al, 1995). The abdomens of adults were dissected in NaCl (6‰) and introduced individually into the wells containing 100 µl of phosphate buffer (50 mM, pH 7.2) and ethoxycoumarin (0.4 mM). After 4 hours of incubation at 30°C, the reaction was stopped by adding 100 µl of 0.1 mM glycine buffer (pH 10.4)/ethanol (v/v). The 7-hydroxycoumarin fluorescence was quantified with 380 nm excitation and 450 nm emission filters. Twelve wells receiving glycine buffer previous to incubation were used for controls.

Detection of the kdr and AchE mutations The genetic variability at a fragment of the sodium channel gene and AchE was analysed by PCR-RFLP. The first fragment encompasses the molecular target linked with pyrethroid resistance (L1014F in trans-membrane segment IIS in the amino-acid sequence, Brun-Barale et al., 2005) and the second one, the molecular target linked with organophosphate resistance (F399V in AchE-1, Casanelli et al., 2006). Total DNA was extracted in 200 µl from an adult leg with 10% Chelex 100 (Biorad) solution (Walsh et al., 1991) and 6 µl of proteinase K (10mg/ml). After a four times dilution, this extract was used as DNA template for PCR amplifications (Franck et al., 2006). Five microlitres of the PCR product were subsequently digested with 2 units of Tsp509I (NEB) in 20 µL reaction volume prior to electrophoresis on

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6% polyacrylamide gel. DNA fragments were visualized after silver staining coloration (Creste et al., 2001). After digestion, the kdr and the sensible alleles were respectively identified by DNA fragments at 80 bp and 112 bp (Franck et al., 2006) and 174 bp and 197bp for sensible and muted AchE-1, respectively.

Statistical analysis Mortalities of two stages exposed to the diagnostic concentrations, were corrected for control mortality (Abbott, 1925). Enzymatic activities were subjected to ANOVA and the means compared by protected least significant difference (PLSD) Fisher test. Relationships between insecticide efficacies and resistance mechanisms were investigated through linear regressions.

Results and discussion

Bioassays Results of topical application on diapausing larvae showed a reduced sensitivity to all the tested insecticides, when compared to the laboratory susceptible strain. More than 80% of the analysed populations were classified as resistant for 9 of the tested insecticides. However «resistant populations” exhibited a very large range of mortalities (Table 1).

Table 1. Proportion of resistant population of Cydia pomonella, evaluated using topical application on diapausing larvae.

Diagnostic Percentage of Mortality (%) Number of concentration resistant Resistant field Insecticide populations (ppm) populations Sv pops Azinphos methyl 45 400 84,4 96,9 0 - 85,7 Chlorpyrifos ethyl 30 1200 86,7 96,7 0 - 83,3 Deltamethrin 18 100 100 99,5 0 - 88,9 Diflubenzuron 24 24000 91,7 97,3 0 - 84,2 Emamectin 24 500 16,7 100 83,3 - 92,9 Fenoxycarb 25 1 92,0 97,7 0 - 73,7 Phosalone 15 300 100 98,5 0 - 65,4 Spinosad 23 600 87,0 100 7,1 - 88,9 Tebufenozide 45 300 88,9 97,0 0 - 75,3 Thiacloprid 37 500 92,0 100 0 - 84,6

Bioassays on neonates indicated a reduced efficacy of azinphos-methyl in the chemically treated population, for which more than 70% of larvae survived to the diagnostic concentration. Chlorpyriphos-ethyl and the new insecticides emamectin, spinosad and thiacloprid, were highly effective on both populations, with mortalities similar to those of the sensible strain. The comparison of these two toxicological tests confirmed that resistance might differ according to the developmental stage, and revealed lower mortalities on diapausing larvae (Figure 1). It was not the case for emamectin, presumably due to the high value of the diagnostic concentration. Results are in agreement with Bouvier et al., 2002, who already detected differences in resistance ratios between different larval instars of codling moth.

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100 100

80 80

60 60

40 40

20 20

0 0 Azin Chl-e Ema Spi Thia Azin Chl-e Ema Spi Thia neonates diapausing neonates diapausing

Figure 1. Comparison of toxicological test. Left, population from a chemical treated orchard; right, organic orchard.

Resistance mechanisms Most resistant field populations expressed enhanced MFO and GST activities when compared to the susceptible strain (F = 9.024, p<0.0001, df = 51; F = 10.441, p<0.0001, df = 51), while EST activities values were mostly lower than in S strain (F= 4.836; p<0.0001, df = 51). AchE-1 mutation was present only in the Spanish population, in which no individuals carrying the kdr mutation were detected. In opposition, kdr mutation was found in populations from all studied countries. Some significant correlations were detected when considering the total population sample (Table 2). MFO activity was positively correlated with the resistance to azinphos- methyl, diflubenzuron, spinosad, tebufenozide and thiacloprid, while increased GST activity was related to resistance to azinphos-methyl. For azinphos-methyl, tebufenozide and thiacloprid, we moreover observed a negative correlation with esterase activity.

Table 2. Relationships between enzymatic activity and resistance to 10 insecticides on 4 laboratory reference strains and 47 field populations of Cydia pomonella

GST EST>1 EST<2 MFO Azin-m Chl-e Delta Diflu Ema Feno Phos Spin Tebu Thia GST 1,00 -0,25 -0,11 0,37 0,29 -0,12 0,12 0,08 -0,24 0,24 0,32 0,23 -0,02 0,28 EST> 1,00 -0,47 -0,34 -0,42 -0,18 -0,24 -0,33 -0,19 -0,30 -0,17 -0,17 -0,24 -0,34 EST< 1,00 0,34 0,14 0,21 0,27 0,34 0,42 0,18 0,00 -0,20 0,14 0,17 MFO 1,00 0,63 -0,05 0,26 0,55 0,20 0,21 0,36 0,42 0,21 0,58 fd 50 50 50 50 48 33 21 27 27 28 18 26 48 40 Italics numbers (p< 0.05); underlined numbers (p < 0.01) 1 Frequency of individuals showing an activity higher than that of 90% of Sv individuals 2 Frequency of individuals showing an activity lower than that of 90% of Sv individuals

According to these results, resistance to some compounds could not be explained by the mechanisms that were investigated, suggesting that unknown mechanisms could be involved. The field trial on the chemically treated population showed that it was susceptible to all tested compounds, while the diapausing larvae of this population expressed a significant resistance to four of them (Fig. 2). For this population, the test on neonates provides a better reliability to field situation. Only azinphos-methyl showed a reduced efficacy on neonates

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compared to field experiment, presumably related to the security index of the recommended field dose.

100

0 Azinphos Chlorpyrifos Emamectin Spinosad Thiacloprid

neonates diapausing field

Figure 2. Compared efficacy of 5 insecticides in a field trial, on neonates and diapausing larvae of a single population.

Conclusions These results indicate that some resistances can not be explained by the investigated mechanisms. Bioassays thus remain a necessary tool for resistance monitoring. Results also confirm that topical application on diapausing larvae allows a rapid and large-scale evaluation, but not ever reliable method to monitoring resistance. Finally, laboratory tests on neonates (F1) of field populations may be a useful tool to validate the resistance to some insecticides.

Acknowledgements We would like to thank Jesús Avilla and Dolors Bosch from UDL-IRTA Lerida for providing us the Spanish population, Dominique Beslay, Jean-François Toubon, Julien Delnatte and Denis Pasquier for technical support.

References

Abbott, 1925. A method of computing the effectiveness of an insecticide. – J. Econ. Entomol. 18: 275-277. Audemard, H. 1992. Population dynamics in codling moth. – In: Tortricid pests: Their Biology, Natural Enemies and Control. L.P.S. van der Geest & H.H. Evenhuis (eds.), Elsevier Science Publishers Amsterdam: 329-338. Boivin, T., Bouvier, J.C., Chadœuf, J., Beslay, D. & Sauphanor, B. 2003. Constraints on adaptative mutations in the codling moth Cydia pomonella (L.): measuring fitness trade- offs and natural selection. – Heredity 90: 107-113. Bouvier, J., Boivin, T., Beslay, D. & Sauphanor, B. 2002. Age-dependent response to insecticide and enzymatic variation in susceptible and resistant codling moth larvae. – Arch. Insect Biochem. Physiol. 51: 55-66.

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Bradford, M. 1976. A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding. – Anal. Biochem. 72: 248-254. Brun-Barale, A., Bouvier, JC., Pauron, D., Bergé, JB. & Sauphanor, B. 2005. Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test. – Pest Manag. Sci. 61: 549-554. Cassanelli, S.; Reyes, M.; Rault, M.; Manicardi, G.C. & Sauphanor, B. 2006. Acetylcholin- esterase mutation in an insecticide resistant population of the codling moth Cydia pomonella (L.). – Insect Biochemistry and Molecular Biology 36: 642-653. Charmillot, P.J.; Gourmelon, A., Fabre, A.L. & Pasquier, D. 2001. Ovicidal and larvicidal effectiveness of several insect growth inhibitors and regulators on the codling moth Cydia pomonella L. (Lep., Tortricidae). – J. Appl. Ent. 125: 147-153. Charmillot, P.J., Pasquier, D., Grela, C., Genini, M., Olivier, R., Ioriatti, C. & Butturini, A. 2003. Résistance du carpocapse Cydia pomonella aux insecticides. – Revue Suisse Vitic. Arboric. Hortic. 35(6): 363-368. Creste, S., Tulman Neto, A. & Figueira, A. 2001. Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. – Plant Molecular Biology Reporter 19: 299-306. Franck, P., Reyes, M., Olivares, J. & Sauphanor, B. 2006. Genetic differentiation in the codling moth: comparison between microsatellite and insecticide resistant markers. – Submitted. Ioriatti, C, Charmillot, P.J., Forno, F., Mattedi, L., Pasquier, D. & Rizzi, C. 2005: Control of Codling moth Cydia pomonella L. using insecticides: field efficacy in relation to the susceptibility of the insect. –IOBC/WPRS Bulletin 28 (7): 259-264. Nauen, R. & Stumpf, N. 2002. Fluorometric microplate assay to mesure glutathione-S-transferase activity in insects ad mites using monochlorobimane. – Anal. Biochem. 303: 194- 198. Pasquier, D. & Charmillot, PJ. 2003. Effectiveness of twelve insecticides applied topically to diapausing larvae of the codling moth, Cydia pomonella L. – Pest Manag. Sci. 60: 305- 308. Riedl, H., Seaman, A. & Henrie, F. 1985. Monitoring susceptibility to azinphosmethyl in field populations of the codling moth (Lepidoptera: Tortricidae) with pheromone traps. – J. Econ. Entomol. 78: 692-699. Reveuny, H. & Cohen, E. 2004. Evaluation of mechanisms of azinphos-methyl resistance in the Codling moth Cydia pomonella (L.). – Arch. Insect Biochem. Physiol. 57: 92-100. Reyes, M., Bouvier, J.C.; Boivin, T.; Sauphanor, B. & Fuentes-Contreras, E. 2004. Suscepti- bilidad a insecticidas y actividad enzimática en Cydia pomonella L. (Lepidoptera: Tortricidae) proveniente de tres huertos de manzano de la región del Maule, Chile. – Agricultura Técnica 64(3): 229-237. Sauphanor, B., Bouvier, J. & Brosse, V. 1998. Spectrum of insecticide resistance in Cydia pomonella (Lepidoptera: Tortricidae) in Southeastern France. – J. Econ. Entomol. 91: 1225-1231. Sauphanor, B., Brosse, V., Bouvier, J., Speich, P., Micoud, A. & Martinet, C. 2000a. Monitoring resistance to diflubenzuron and deltamethrin in French codling moth populations (Cydia pomonella). – Pest Management Sci. 56: 74-82. Sauphanor, B., Bouvier, J.C., Beslay, D., Bosch, D. & Avilla, J. 2000b. Mechanisms of azinphos-methyl resistance in a strain of Cydia pomonella from southern Europe. – CR. 21st Int. Cong. Entomol, Iguassu (Brazil), 20-26 Aug. 2000.

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Ullrich, V. & Weber, P. 1972. The O-dealkylation of 7-ethoxycoumarine by liver micro- somes: a direct fluorometric test. – Hope-Serler’s Z. Physiol. Chem. 353: 1171-1177. Varela, L., Welter, S., Jones, V., Brunner, J. & Riedl, A. 1993. Monitoring and characterization of insecticide resistance in codling moth (Lepidoptera: Tortricidae) in four Western States. – J. Econ. Entomol. 86: 1-10.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 195-199

A new bioassay to test insecticide resistance of Cydia pomonella (L.) first instar larvae: results from some field populations of Lleida (Spain)

Dolors Bosch1, Marcela Rodríguez1, Jesús Avilla1,2 1 IRTA.Centre UdL-IRTA, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]; [email protected] 2 Department of Crop and Forest Science, Universitat de Lleida, Rovira Roure 191, 25198 – Lleida, Spain; [email protected]

Abstract: The insecticide resistance of first instar codling moth, Cydia pomonella (L.), larvae from wild female x wild male crosses has been tested using a new test. Neonate larvae were placed on pieces of semisynthetic diet treated topically with the insecticide solution evenly distributed on its surface using a humidified brush. Sixteen newly hatched codling moth larvae were deposited on each piece of diet and individually confined in a gelatine capsule. The mortality was recorded 24 hours and 5 days after the application. The repeatability of the bioassay was good, as the heterogeneity indexes obtained were low. The LC50 of a susceptible population from Lleida (Spain) to the insecticides azinphos-methyl, phosmet and lambda-cyhalothrin were 200 mg/L, 420 mg/L and 0.35 mg/L, respectively. These values were used to assess the resistance of two field populations collected in the apple and pear growing area of Lleida. The populations were collected as larvae in damaged fruits during 2006, in orchards where chemical control had failed. The observed resistance ratios ranged from 1 to more than 38.

Key words: Cydia pomonella, resistance, L1 larvae, azinphos-methyl, phosmet, lambda-cyhalothrin

Introduction

Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a widespread key pest of apple, pear and walnut production. Its control has been based on the use of broad spectrum insecticides, such as organophosphates and pyrethroids. Resistance to different organophosphates and pyrethroids has been recorded in the United States (Welter et al., 1991; Varela et al., 1993) and Europe (Sauphanor and Bouvier, 1995; Charmillot et al., 2001; Charmillot et al., 2005). Many of the studies on C. pomonella resistance have been conducted on adults or on diapausing larvae because they are easy to collect and because it is difficult to obtain high number of individuals from field populations. However, the correlation between the results against those non-target instars and the target one, first instars larvae (L1), is not always clear (see Reyes et al., this volume). Some bioassays for neonate larvae have been developed, either using the Potter Tower (Sauphanor and Bouvier, 1995) or mixing the formulated insecticide with the artificial diet (Reuveny and Cohen, 2004). As the Potter Tower is not always available and when mixing the insecticide in the diet the applied concentration is far from the concentration applied in the field, a new test was developed. The aim of the present work was to test the toxicity of the insecticides azinphos-methyl, phosmet and lambda-cyhalothrin to neonates of susceptible and field codling moth populations using a new and easy bioassay methodology.

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Material and methods

Insects The insects used in the bioassay were a susceptible population (S-Lleida) reared in the Centre UdL-IRTA of Lleida since 1992, when it was collected from an abandoned apple orchard, and 2 field populations (Arcs and Reñé, from the Lleida region), obtained collecting damaged fruits during the 2006 first generation in orchards where chemical control had failed. Wild males and females were crossed to obtain enough progeny to carry out insecticide resistance tests on neonate larvae.

Chemicals Azinphos-methyl (Gusathion, 20% a.i. p/v from Aragonesas Agro, S.A.) was used first to test the methodology because it is widely used in the field. Phosmet (Imidan, 50% a.i. p/p from Comercial Química Massó) and lambda-cyhalothrin (Karate, 10% a.i. p/v from Syngenta) were tested later.

Bioassays One 32 µL drop of a solution of the insecticide in water was deposited using a micropipette on the surface of one 4 x 4 x 1 cm3 piece of the semisynthetic diet used to feed the larvae (Pons, 1992). The product was immediately distributed evenly using a humidified brush, and the diet was allowed to dry for two hours. Sixteen less than 24-h old larvae (L1) codling moth were deposited on each piece of diet and each one was individualized within a gelatine capsule. Each piece of diet with the larvae was placed in a closed plastic box to avoid desiccation, and it was kept in a climatic chamber (22ºC, 16:8 L:D). Mortality was recorded 24 h later, removing the gelatine capsule and observing the diet under a binocular microscope, and 4 - 5 days later, looking for the larvae inside the diet. A larva was considered dead if it did not respond to a gentle touch with a dissecting forceps. Missing larvae were considered as escaped and subtracted from the initial number of treated larvae. Seven to 8 concentrations of each insecticide plus a water control were tested. At least 32 larvae were used for each concentration. The LC50 and LC90 of S-Lleida for the insecticides azinphos-methyl, phosmet and lambda-cyhalothrin were calculated. The response of the field populations (Arcs and Reñé) to the LC50 of the susceptible population was tested treating at least 32 larvae.

Statistical analysis Mortality was corrected using the Abbott’s formula. The POLO-PC program (LeOra Software, 2001-2006) was used for Probit analysis of the susceptible population bioassay. The 2 LC50 and LC90 values, their 95 % confidence limits and the Heterogeneity Factor (χ /df) were estimated. The Resistance Ratio (R.R.) of field populations was computed by comparison of their corrected mortality with the corrected mortality of the susceptible population.

Results and discussion

Only 43 and 70 codling moths adults were obtained from the larvae collected from fruits damaged by the first codling moth generation at Reñé and Arcs, respectively. As the synchronization of the emergence of males and females was not good, only a total of 110 and 554 L1 larvae from Reñé and Arcs, respectively, were treated. The number of eggs per field adult obtained ranged between 3 and 8. It’s easier, although longer in time, to obtain higher progeny using diapausing larvae, due to a better synchronised emergence of adults. Reuveny

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and Cohen (2004) solved the lack of fecundity of codling moth females from field populations keeping the adults in outdoor conditions. The Heterogeneity Factor of the data sets ranged from 0.49 to 1.83 (24-h mortality), and from 0.75 to 1.06 (4 - 5 days mortality) (Table 1). This means that the methodology was repeatable and the results obtained with it, reliable. The mortality of the control 24 hours after the treatment was always 0, and less than 1.8%, 4 or 5 days after it. The LC50 and the LC90 of azinphos-methyl, phosmet and lambda-cyhalothrin of the susceptible population (S-Lleida) 24 h and 4-5 days after the treatment are shown in Table 1. The LC50 values obtained at the two moments of recording the mortality were different for azinphos-methyl and lambda- cyhalothrin, but not for phosmet. The LC90 values were different only for azinphos-methyl. However, the differences were small in all the cases. The Security Index ranged between 0.7 and 1.2 for the organophosphates, while it was higher for the pyrethroid. These values are similar to those obtained by Sauphanor et al. (1998) using benzoylureas against C. pomonella neonate larvae of a susceptible population.

Table 1. Toxicity of azinphos-methyl, phosmet and lambda-cyhalothrin to neonate larvae of a susceptible population of C. pomonella. Topical application of the formulated insecticide on diet. Mortality recorded 24 hour and 4-5 days after the application. n = number of treated larvae. H.F. = Heterogeneity Factor (X2/df). S.I. = Security Index = Recommended rate / LC90.

Control LC50 (mg/L) LC90 (mg/L) Insecticide n mortality (95% confidence (95% confidence H.F. S.I. (%) limits) limits) 24 h after the treatment

Azinphos- 709 0 274.13 680.11 1.36 0.74 methyl (246.03 - 308.98) (562.53 – 879.65)

Lambda- 377 0 0.55 2.06 0.49 4.85 cyhalothrin (0.46 - 0.64) (1.62 – 2.85)

Phosmet 770 0 495.55 996.69 1.83 1.00 (451.64 – 546.81) (857.24 – 1228.62)

4 -5 days after the treatment Azinphos- 709 1.39 202.05 449.27 1.06 1.10 Methyl (180.90 – 223.19) (390.92 – 542.23)

Lambda- 377 1.67 0.35 1.59 0.75 6.70 Cyhalothrin (0.29 – 0.42) (1.23 – 2.29)

Phosmet 770 1.79 421.84 813.78 1.04 1.20 (383.92 – 457.15) (729.95 – 944.27)

Table 2 shows the corrected mortality and the R.R. of the tested insecticides on the two field populations. The Arcs population could be tested only with azinphos-methyl and lambda-cyhalothrin. This population showed a Resistance Ratio below 2.4 for both insecticides, while the Reñé population showed a R.R. of 0.76 for the pyrethroid but near 9 for the azinphos-methyl and higher than 38 for phosmet. This plot has been traditionally

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treated with organophosphates (10 treatments in 2005 and 7 in 2006), what explains the lack of response to this products.

Table 2. Mortality and Resistance Ratio (R.R.) of L1 from two codling moth field populations exposed to the LC50 of the susceptible population (S-Lleida) for azinphos-methyl, phosmet and lambda-cyhalothrin. Topical application of the formulated insecticide on diet. Mortality recorded 4-5 days after application. n = number of treated larvae. R.R.= field population corrected mortality / susceptible laboratory population corrected mortality.

Insecticide Population n Corrected mortality R.R. (%)

S-Lleida 31 26.88 – Azinphos- methyl Reñé 45 3.03 8.87 Arcs 32 11.55 2.33

S-Lleida 63 50.16 – Lambda- cyhalothrin Reñé 32 65.63 0.76 Arcs 32 23.08 2.17

Phosmet S-Lleida 14 38.77 – Reñé 31 0.00 >38

Conclusions

The bioassay used is easy to carry out and gives reliable and repeatable results, and it does not require expensive apparatus to perform the treatment. The way the insecticide contacts the larvae is similar to the way it happens in the field, acting on the target instar. However, it is necessary to standardize a methodology to evaluate the resistance on juvenile instars in order to compare the results between the different groups working on the same subject. The susceptibility of three products has been evaluated only on two populations that were available at that moment, but one of them presented a Resistance Rate greater than 38 for phosmet, and near 9 for azinphos-methyl. These rates were related to a high number of organophosphates treatments carried out in the field. This situation could be not different from the other plots that need to be treated more than 8-10 times against codling moth. Next step will be to find the diagnostic concentration (LC50 or LC90) of the rest of the products that act on juvenile instars and evaluate the levels of resistance presents in the area of Lleida.

References

Charmillot, P.J., Pasquier, D. & Briand, F. 2005: Résistance du carpocapse Cydia pomonella aux insecticides. Tests par application topique sur des larves diapausantes collectées en automne 2003 dans les vergers suïsses. – Revue suisse Vitic. Arboric. Hortic. 37(2): 123- 127. Charmillot, P.J., Pasquier, D., Dessimoz, S., Genini, M. & Olivier, R. 2001: Resistance of the codling moth Cydia pomonella to insecticides. Topical application tests with diapausing larvae collected in autumn 2001. – Revue suisse Vitic. Arboric. Hortic. 34(4): 247-251.

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Pons, S. 1992. Inducció de la diapausa en Cydia pomonella L. (Lepidoptera: Tortricidae) i les seves implicacions en el control integrat a Lleida. – Projecte Final de Carrera. Escola Tècnica Superior d’Enginyeria Agrària de Lleida. Universitat de Lleida. Reuveny, H. & Cohen, E. 2004: Resistance of the codling moth Cydia pomonella (L.) (Lep., Tortricidae) to pesticides in Israel. – JEN 128 (9/10): 645-651. Sauphanor, B. & Bouvier, J.C. 1995: Cross resistance between benzoylureas and bezoyl- hydrazines in the codling moth, Cydia pomonella (L.). – Pestic. Sci. 45: 369-375. Sauphanor, B., Bouvier, J.C. & Brosse, V. 1998: Spectrum of insecticide resistance in Cydia pomonella (Lepidoptera: Tortricidae) in southeastern France. – J. Econ. Entomol. 91(6): 1225-1231. Varela, L.G., Welter, S.C., Jones, V.P., Brunner, J.F. & Riedl, H. 1993: Monitoring and characterization of insecticide resistance in codling moth (Lepidoptera: Tortricidae) in four western states. – J. Econ. Entomol. 86: 1-10. Welter, S.C., Varela, L. & Freeman, R. 1991: Codling moth resistance to azinphos methyl in California. – Resist. Pest Manag. Newslett. 3: 12.

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 201-204

Susceptibility of codling moth, Cydia pomonella (L.) to tebufenozide in Trentino apple growing area

Claudio Ioriatti1, Cristina Tomasi1, Pierre Joseph Charmillot2, Denis Pasquier2, Benoît Sauphanor3, Maritza Reyes3 1 IASMA Research Center - Plant Protection Department, Via E. Mach, 1, 38010 – San Michele all’Adige (TN), Italy, [email protected] 2 Agroscope Changins-Wädenswil ACW, CH-1260 Nyon, Switzerland, [email protected] 3 PSH – Ecologie de la Production Intégrée, INRA Site Agroparc, 84914 Avignon Cedex 9, France, [email protected]

Abstract: The early detection of insecticide-resistant populations is of paramount importance for the correct and effective implementation of integrated resistance management programs. The present study aims to compare three different methods to detect the loss of insecticide susceptibility in field populations of codling moth to tebufenozide: results of the topical applications of a discriminating dose on overwintering larvae are compared with the enzymatic analysis of the adults as well as with the results of the bioassays performed on the neonates larvae. The study was carried out on four populations collected in orchards with different pesticide pressure. The two populations collected in the apple growing areas where the pesticide pressure was higher showed a significant reduction in the mortality when the overwintering larvae were treated topically. The loss of susceptibility was confirmed when the neonate larvae of the same populations were fed with apples treated with a discriminating dose of tebufenozide. According to the results of the enzymatic analyses performed on the emerged adults, the Mixed-Function Oxidase enzymatic system was involved in determining the loss of susceptibility. Approximatively a third of the individuals of these both populations were resistant compared with the susceptible population. No differences were found between the reference strain and the two other populations that were collected in the apple growing areas where the pesticide pressure was lower. The relationship between the results obtained with the time-consuming apple dipping method and with the more simple topical treatments of overwintering larvae validates the second one as standard method for detecting and defining resistance to tebufenozide in codling moth.

Key words: Cydia pomonella, insecticide resistance, detoxifying enzymes, bioassay,

Introduction

Trentino is one of the main Italian apple growing regions. Insecticide resistance to different classes of insecticides has been recently documented in the codling moth collected in the experimental orchard of the IASMA Research Center (Ioriatti et al., 2003; Ioriatti et al., 2005) by using the topical application of the insecticide on overwintering larvae. This bioassay was first defined for Insect Growth Regulators (IGRs) (Sauphanor et al., 1999; 2000) and then developed for a set of insecticides (Charmillot et al., 2003; 2005a; Pasquier and Charmillot, 2003). Topical application of discriminating concentrations of insecticides on the diapausing larvae collected in corrugated band traps in the previous season seems to be a sensitive technique for resistance detection in codling moth populations. The test has already been successfully used for resistance surveys in Switzerland, Italy and France (Charmillot et al., 2003; 2005b; Ioriatti et al., 2000; Sauphanor et al. 2000) and it is recognised as particularly performing in detecting IGR-insecticide resistance.

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Tebufenzoide, a Moulting Accelerating Compound (MAC) widely used in the area, was chosen as reference to evaluate the susceptibility to all the IGRs. Tebufenozide is mainly effective for young larvae, but it is also toxic against eggs when laid on treated leaves and it is able to sterilise adults getting in contact with the residues on the leaves (Pons et al., 1999; Sun and Barrett, 1999; Knight, 2000; Charmillot et al., 2001). Because of its lethal and sublethal effects and its long residual persistence acting on the resting adults, tebufenozide exerts high selection pressure on the codling moth populations. This could accelerate the appearance of resistant strains. The present study aims to compare three different methods to detect the loss of insecticide susceptibility in field populations of codling moth: results of the topical applications of a discriminating dose (DD) on overwintering larvae are compared with the enzymatic analysis of the adults as well as with the results of the bioassays performed on the neonate larvae. The study was carried out on four populations collected in orchards with different pesticide pressure.

Material and methods

Insects Reference strains: Three reference strains were used in this study: the Agroscope ACW Changins strain (RAC_S) for the topical test, the INRA-Avignon strain (INRA_Sv) for the enzymatic analyses, and the Andermatt Biocontrol (Grossdietwil - CH) strain (AB_S) for the dipping test with the neonate larvae. All of them have been kept in mass rearing on artificial diet for several generations and can be considered susceptible to the insecticides. Field population: In the autumn 2003, 2004 and 2005, diapausing larvae were collected in 4 orchards located in different districts of the Trento province. Cardboards containing the overwintering larvae were stored at 6°C until use. The sampled orchards were submitted to a different insecticide pressure. S. Michele and Roncafort orchards are situated in the Adige valley (180 m a.s.l.) and the codling moth control requires 6-7 insecticide applications per year. Revò and Vervò orchards are located in the medium-upper Non valley (altitude 650 and 800 m a.s.l.) where the codling moth control requires 2 and 3 treatments per year respectively.

Tests Topical test with the DD of tebufenozide defined by Charmillot et al. (2005a) were performed on the overwintering larvae of the four field populations. The mortality rate of the different populations was compared to the susceptible one using the chi-square test. Enzymatic analyse: The glutathion-S-transferas (GST), mixed function oxidases (MFO) and esterase (EST) activities were evaluated on emerged adults (Reyes et al., 2004). Biochemical data were subjected to an analysis of variance (ANOVA) and the means compared by protected least significant difference (PLSD) Fisher test. Dipping test: A dose-mortality reference curve was established for tebufenozide (Mimic SC 240 g/l. - Isagro Italia) in the laboratory using the AB_S strain and thinning apples were dipped in 1 litre solution of insecticide at 9 different concentrations (Charmillot et al., 2001). According to the baseline determined with the CIRAD-CA / URBI Montpellier DL50 Version 4.6 programme, a discriminating dose of 1000 ppm (=LC99) was chosen for the detection of resistance in the brood of the field-collected insects. The mortality rate in the field populations was compared with that obtained in the susceptible strain (AB_S) using the chi- square test.

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Results and discussion

Topical test The topical test using a DD of 300 ppm detected a significant (P>0.05) loss in susceptibility to tebufenozide in the populations of S. Michele and Roncafort year 2003 and 2004. The mortality rate ranged from 45.7% to 75.7% in the S. Michele population and from 65.8% to 75% in the Roncafort population respectively in year 2003 and year 2004. The mortality of the two populations did not differ significantly from the reference when an increased DD of 500 ppm was used. All the overwintering larvae collected in the orchards of Revò and Vervò died when treated topically with the lowest DD.

Enzymatic analysis The EST and GST activity ratios between the four field populations and the INRA_Sv strain varied from 0.5 to 1.8 A significant increase of MFO activity was observed only in S. Michele population, resulting in a 3.4-fold ratio when compared to the reference laboratory strain. Considering MFO activity, the frequency of resistant individuals determined according to Reyes et al. (present volume) was significantly higher in both S. Michele and Roncafort populations than in the susceptible. No significant increase in frequency of individuals with enhanced enzymatic activity was found for the Vervò and Revò populations.

Dipping test on neonate larvae The dipping test performed on the neonate larvae showed a highly significant reduction in the susceptibility of the two populations of S. Michele and Roncafort (P=0.001), with a corrected mortality of 74.1 and 71.8 respectively. A less significant reduction (P=0.05) in the mortality was found in the Revò population with a larval mortality of 90%. No statistical difference was found between the mortality of Vervò population and the susceptible strain.

Conclusions

Results of this study showed that early detection of loss in susceptibility of tebufenozide is possible by using the topical bioassay before important control failures occur, in time to implement integrated resistance management strategies. For this reason topical bioassay with overwintering larvae is considered as a useful tool for implementing resistance monitoring programs that are particularly important for the insecticides exerting high selection pressure on codling moth as is the case for tebufenozide. Despite the little value of the resistance ratio, approximately a third of the individuals of the S. Michele and Roncafort populations survives to both bioassays (topical and dipping) and showed a significant increase in the enzymatic activity. These results allow us to conclude that these two populations have developed resistance to tebufenozide. Considering the small number of treatments with tebufenozide that has been done so far, the reduced susceptibility of the two populations is probably due to cross resistance with other insecticides more widely applied in the past. No resistance to tebufenozide was detected in the both Revò and Vervò populations; the two orchards are located at higher altitude than the previous, the life cycle of codling moth being shorter and the pesticide pressure being lower.

References

Charmillot P.J.; Gourmelon A.; Fabre A.L. & Pasquier D. 2001: Ovicidal and larvicidal effectiveness of several insect growth inhibitors and regulators on the codling moth Cydia pomonella L., (Lep. Tortricidae). – J. Appl. Ent. 125: 147-153.

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Charmillot, P.J.; Pasquier, D.; Grela, C.; Genini, M.; Olivier, R.; Ioriatti, C. & Butturini, A. 2003: Résistance du carpocapse Cydia pomonella aux insecticides: Tests par application topique sur des larves diapausantes collectées en automne 2002. – Revue Suisse Vitic. Arboric. Hortic. 35 (6): 363-368. Charmillot, P.J; Pasquier, D. & Briand, F. 2005a: Detection of codling moth Cydia pomonella resistance by topical application of insecticides and validation on a laboratory resistant strain by dipping of apple and incorporating products into artificial diets. – IOBC/WPRS Bulletin 28 (7): 265-269. Charmillot, P.J.; Pasquier, D. & Briand, F. 2005b: Résistance du carpocapse Cydia pomonella aux insecticides: Tests par application topique sur des larves diapausantes collectées en automne 2003 dans les vergers suisses. – Revue Suisse Vitic. Arboric. Hortic. 37 (2): 123-127. De Sousa, G.; Cuany, A.; Brun, A.; Amichot, M.; Rahmani, R. & Berge, J. 1995: A micro- fluorometric method for measuring Ethoxycoumarin-O-Deethylase activity on individual Drosophila melanogaster abdomens: interest for screening resistance in insect populations. – Annal. Biochem. 229: 86-91. Ioriatti, C.; Sauphanor, B.; Cainelli, R.; Rizzi, C. & Tasin, M. 2000: Cydia pomonella (L.): Primo caso di resistenza a diflubenzuron in Trentino. – Atti Giornate Fitopatologiche 1: 319-325. Ioriatti, C.; Boselli, M.; Butturini, A.; Cornale, R. & Vergnani, S. 2003: Integrated resistant management of Codling moth Cydia pomonella L. in Italy – Resistance Pest Manage- ment Newsletter 12 (2): 65-69. Ioriatti, C; Charmillot, P.J.; Forno, F.; Mattedi, L.; Pasquier, D. & Rizzi, C. 2005: Control of Codling moth Cydia pomonella L. using insecticides: field efficacy in relation to the susceptibility of the insect. – IOBC/WPRS Bulletin 28 (7): 259-264. Pasquier, D. & Charmillot, P.J. 2003. Effectiveness of twelve insecticides applied topically to diapausing larvae of the codling moth, Cydia pomonella L. – Pest Manag. Sci. 60: 305- 308. Pons, S.; Riedl, H. & Avilla, J. 1999: Toxicity of the ecdysone agonist tebufenozide to codling moth (Lepidoptera: Tortricidae). – J. Econ. Entomol 92(6): 1344-1351. Reyes, M.; Bouvier, J.C.; Boivin, T.; Sauphanor, B. & Fuentes-Contreras, E. 2004: Suscepti- bilidad a insecticidas y actividad enzimática en Cydia pomonella L. (Lepidoptera: Tortricidae) proveniente de tres huertos de manzano de la región del Maule, Chile. – Agricultura Técnica 64(3): 229-237. Sauphanor, B; Bouvier, J.C. & Brosse, V. 1999: Effect of an ecdysteroid agonist, tebufenozide, on the termination of diapause in susceptible and resistant strains of the codling moth, Cydia pomonella. – Entomol. exp. appl. 90: 157-165. Sauphanor, B.; Brosse, V.; Bouvier, J.C.; Speich, P.; Micoud, A. & Martinet, C. 2000: Monitoring resistance to diflubenzuron and deltamethrin in French codling moth populations (Cydia pomonella). – Pest Manag. Sci. 56: 74-82. Sun, X. & Barrett, B.A. 1999: Fecundity and fertility changes in adults codling moth (Lepidoptera: Tortricidae) exposed to surface treated with tebufenozide and metoxy- fenozide. – J. Econ. Entomol. 92: 1039-1044.

Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 205

Evolution of codling moth, Cydia pomonella (L.), resistance in Swiss orchards, tested by topical application of insecticides

P.J. Charmillot, F. Briand, C. Salamin, D. Pasquier Agroscope Changins-Wädenswil ACW. CP 1012. 1260 Nyon. Switzerland. [email protected]

Abstract: Since 1996, resistant strains of codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae) have appeared in insecticides treated orchards in Switzerland. From then, observations of more or less resistant populations have been recorded, on an increasing surface. Since the first resistant moth appears in an orchard, further treatments with insecticides (mainly IGRs) keep on killing sensitive moths when the population of resistant codling moth increases in time, sometimes reaching high levels of insensitivity to classical treatments. A good pest control management combining mating disruption and CpGv (granulosis virus) can efficiently reduce population size within a few years. Resistance levels of C. pomonella diapausing larvae have been evaluated for several years using topical application of different insecticides. Larvae collected in autumn in sensitive and resistant populations from several orchards are treated with one µl drop of insecticide at a discriminating dose, and laid on the back of the larvae. Larvae are then allowed to hide in corrugated cardboards and reared into climatic chambers until adult emergence. Survival rate is calculated compared to control. This method allowed a survey of orchards populations over several years, often from quite resistant stages until after some years of mating disruption. At that time, population sizes often reach extremely low levels, making difficult the collecting of diapausing larve for tests. However, topical application tests made possible the observation of changes in sensitivity since the release of insecticide selection pressure on populations. Indeed, obvious recuperation was visible within a 5-years period of mating disruption technique.

Key words: Codling moth, Cydia pomonella, insecticide, resistance, topical application

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 207-213

Resistance to insecticides and baseline sensitivity in codling moth populations from Pennsylvania, USA orchards

Greg Krawczyk, Larry A. Hull Pennsylvania State University, Department of Entomology, Fruit Research and Extension Center, Biglerville, PA 17307, USA, [email protected]

Abstract: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), after years of relatively low importance recently become one of the most significant internally feeding apple pests in the mid- Atlantic region of United States. Codling moth male moths collected from commercial and abandoned fruit orchards in Pennsylvania, USA were tested for their sensitivity levels to azinphos-methyl and methomyl using adult topical bioassays. Larval sensitivity to older and newly introduced insecticides (i.e., acetamiprid, novaluron and rynaxypyr) was also assessed using diet surface topical bioassays. Adult codling moth populations expressed differences up to 13 fold in their sensitivity to azinphos- methyl and up to 5 fold towards methomyl. During codling moth larval bioassays we observed differences in larval sensitivity to various compounds as well as differences between compounds based on the timing of mortality readings.

Key words: codling moth, insecticide resistance, baseline insecticide sensitivity.

Introduction

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is one of the most important internally feeding apple pests in the mid-Atlantic region of United States. In a research paper published in 1934 Hodgkiss et al. stated “the distribution of codling moth in Pennsylvania is statewide” and “the topography of Pennsylvania has a modifying effect on the nature of the infestations which renders the insect more virulent in the south-central counties”. During observations conducted between 1925 and 1933, authors documented the differences in the number of generations in the different parts of the state and concluded that codling moth in orchards located in south-central Pennsylvania have two complete and a partial third generation (Hodgkiss et al., 1934). After years of relatively low or almost non- existent fruit infestations from this pest, the importance of codling moth started to gradually increase in recent years. During the period of 1998 to 2005 the codling moth was responsible for 760 rejections of fruit loads delivered to Pennsylvania fruit processors. The presence of one live larva during the sampling process for a truckload of fruit (e.g., 3000-20000 kg) is a cause for rejection of the entire fruit load. From 1998-2000, the number of fruit loads rejected due to the presence of codling moth was not higher than 26 per year (2000 season), but from 2001-2005 the numbers of fruit loads rejected increased from 62, 121, 118, 187 to 207, respectively. While the number of codling moth rejected fruit loads is still lower than the number of fruit loads rejected for the presence of the Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) (i.e., 1991 rejected fruit loads during 1998-2005), it is important to note the ascending trend related to the occurrence of codling moth (Krawczyk, 2006). Numerous factors such as lack of proper pest(s) monitoring, problems with correct timings or delivery (i.e., coverage) of insecticides, and insecticide resistance phenomena have

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probably contributed to this rapid development of codling moth related problems in some Pennsylvania orchards. For almost 40 years, the organo-phosphate (OP) insecticides have been the cornerstone of apple and peach insect management programs. They have provided excellent control of most direct insect pests and, despite their relatively broad-spectrum activity, are of relatively low toxicity to many important natural enemies, particularly mite predators (Hull et al., 1997). Despite this long-term reliance on OPs, there have been relatively few instances of pests developing pesticide resistance. Notable exceptions include the tufted apple bud moth Platynota idaeusalis (Walker) (Knight et al., 1990), obliquebanded leafroller Choristoneura rosaceana (Harris) (Lawson et al., 1997), and more recently, the oriental fruit moth (Usmani and Shearer, 2001). Reduced susceptibility of codling moth male moths to azinphos-methyl, phosmet, and methomyl has been also documented in Pennsylvania apple and peach orchards (Krawczyk and Hull, 2004). Various instances of resistance to organo-phosphate insecticides in codling moth populations were documented previously in California (Varela et al., 1993, Dunley and Welter, 2000), Washington (Knight et al., 1994) and North Carolina (Bush et al., 1993). In Europe Sauphanor et al. (1998) observed resistance to insect growth regulator insecticides in codling moth populations and reported possible cross-resistance among insect growth regulators, organo-phosphate and pyrethroid insecticides. In recent years there have been a number of new, “reduced-risk” or “OP replacement” insecticidal products that have become available to the tree fruit industry that have shown promise as alternatives to OPs, carbamates, and pyrethroid insecticides. The four newer classes of insecticides include insect growth regulators (IGR’s), naturalytes, neonicotinoids and oxadiazines. Two IGR’s, methoxyfenozide and novaluron, have shown excellent activity against leafrollers and codling moth in orchard trials (Hull and Krawczyk, 1999; Hull personal information). The naturalyte insecticide, spinosad, provided excellent control of leafrollers and leafminers. The neonicotinoids or chloronicotinyls (imidacloprid, thiamethoxam) are primarily active against leafhoppers, aphids and leafminers. However, other compounds from this group appear to have a broader range of activity, with potential efficacy against some lepidopteran pests (acetamiprid, thiacloprid or clothianidin) (Hull personal information). In this report we discuss the sensitivity of naturally occurring codling moth adult populations to azinphos-methyl and methomyl, two commonly used broad-spectrum insecticides in Pennsylvania. We also report the larval baseline sensitivity of various laboratory reared codling moth populations to some newly registered and unregistered compounds.

Material and methods

Codling moth adult bioassays During the 2001-2005 growing seasons multiple populations of adult codling mothwere collected from commercial and abandoned apple orchards and bioassayed for their sensitivity to frequently used broad-spectrum insecticides: azinphos-methyl and methomyl. Bioassay procedures followed those described by Shearer and Riedl (1994). Scenturion delta traps (Suttera, OR) baited with regular codling pheromone (1 mg) were deployed in commercial orchards and checked on a daily basis. Collected adult moths, after remaining for less than 24 hours in a trap and attached ventrally to the adhesive surface were topically treated on their dorsum with 1.0 µl of solution of insecticide dissolved in acetone or acetone alone (control). The insecticides were applied using a repeating microsyringe dispenser (Hamilton, Reno, NV). At least five different concentrations of each insecticide were used for testing adult

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moth response during each bioassay. Treated moths were left on the sticky floor at the temperature of 22°C. The mortality readings were conducted at 48 hours. Moths that did not respond to a gentle touch by a camel brush were considered dead. Mortality data was subjected to probit analysis (POLO PLUS) (LeOra Software, 2003). The slopes of the probit regression, and LC50 and LC90 values were estimated for each insecticide.

Larval baseline bioassays For the larval bioassays, neonate larvae originating from laboratory established colonies were used. The larval bioassays were performed using a lima bean diet as a food source (Shorey and Hale, 1965). Formulated insecticides were diluted into six to eight concentrations, and 0.5 ml of insecticide solution was applied to the surface of lima bean diet in a 28 ml cup. Five neonates were transferred to the treated diet, and larval mortality was assessed at 1, 4, 5 and/or 7 days for each tested insecticide. Mortality data was subjected to probit analysis (POLO). The slopes of the probit regression, and LC50 and LC90 values were estimated for each insecticide.

Results and discussion

Various numbers of codling moth populations collected from commercial and abandoned Pennsylvania apple orchards were tested during each year of the project. The sensitivities of the tested populations to azinphos-methyl and methomyl insecticides are presented in Table 1 and Table 2.

Table 1. Codling moth male adult sensitivity data to azinphos-methyl using adult topical insecticide bioassays. Codling moth populations were collected from various commercial and abandoned apple orchards in Pennsylvania, US.

LC50 (ppm) LC90 (ppm) Year Population* N slope (FL at 0.95) (FL at 0.95) L 144 1.3 ± 0.4 16.8 157.8 (5.1 – 28.9) (81.3 – 955.1) 2001 H 397 3.5 ± 0.5 215.7 503.8 (147.7-277.7) (381.8 –842.8) L 108 2.6 ± 0.7 16.9 53.0 (7.7 – 25.0) (36.1 -112.3) 2002 H 207 3.2 ± 0.5 108.5 275.9 (84.3-135.0) (210.3 -431.2) L 131 2.2 ±0.5 32.4 127.4 (15.9 – 49.1) (81.1 – 320.2) 2003 H 156 2.9 ± 0.5 37.6 101.9 (27.7 – 49.6) (73.2 – 179.3) L 83 2.1 ± 0.6 16.5 66.4 (7.3 – 26.1) (40.0 – 233.5) 2005 H 137 1.1 ± 0.3 225.2 2980.2 (65.7 – 567.2) (995-96621) * L - denotes the codling moth population with the lowest LC50 value during a given season; H – denotes the population with the highest LC50 value.

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Table 2. Codling moth male adult sensitivity data for methomyl using adult topical insecticide bioassays. Codling moth populations were collected from various commercial and abandoned apple orchards in Pennsylvania, US.

LC50 (ppm) LC90 (ppm) Year Population* n slope (FL at 0.95) (FL at 0.95) L 122 2.1 ± 0.7 17.5 71.7 (5.5 – 28.9) (41.1 – 338.1) 2001 H 152 2.4 ± 0.6 96.1 324.9 (63.8 – 130.7) (216.1 –811.6) L 112 3.7 ± 0.9 21.2 46.9 (12.7 – 27.8) (35.7 – 80.8) 2003 H 179 2.2 ± 0.4 85.1 333.1 (55.8 – 115.1) (233.4 – 612.5) L 165 1.8 ± 0.4 20.9 108.5 (11.9 – 30.2) (69.6 – 250.4) 2005 H 121 2.2 ± 0.6 72.1 274.9 (32.5 – 111.3) (165.6 – 1174) * L - denotes the codling moth population with the lowest LC50 value during a given season; H – denotes the population with the highest LC50 value.

For the azinphos-methyl bioassays, the biggest difference at the LC50 level between the most and the least sensitive populations was observed during the 2001 (12.8 fold) and 2005 seasons (13.6 fold) (Table 1). The differences in methomyl sensitivities were smaller and varied from 3.4 to 5.5 fold during the 2005 and 2001 seasons, respectively (Table 2). During similar studies conducted in the Western US, Varela et al. (1993) observed differences in the field efficacy of insecticides when the differences between populations at the LC50 level were no greater than 2-3 fold. Our adult bioassay results support the hypothesis about the presence of various levels of azinphos-methyl, and to lesser extent methomyl resistance in Pennsylvania codling moth populations. At the same time it is worthy to note, that many Pennsylvania growers still use low rates of azinphos-methyl (average of less than 0.84 kg AI per hectare) and it is possible that in many orchards a higher rate of this compound would still be effective for controlling codling moth populations. In most cases, the field rate of the applied insecticide is higher than the rate needed to achieve acceptable pest control, but other elements of the application process such as water volume, equipment, or weather also can affect an insecticide’s efficacy. In general, if a lack of insecticide efficacy is observed and all the other factors impacting the application are the same, it is a good idea to stop using the compound (or group of compounds with the same mode of action) and replace it with a compound with a different mode of action. The codling moth baseline larval sensitivity data to various new and older insecticides are presented in Table 3. During our observations, each tested compound exhibited greater toxicity (lower LC50 and LC90 values) with increasing time after the initial assessment (one day) (Table 3). The differences between these two readings (either 1 and 4 or 4 and 7 days) were very visible for slower working compounds such as IGRs (novaluron and methoxyfenozide) or spinosad. During the 1 day reading (4 day for novaluron) the estimated LC90 values for methoxyfenozide, novaluron and spinosad were higher than the recommended field rates: for methoxyfenozide 308 ppm (284 g AI//946 liters of water/ hectare), for novaluron 160 ppm (147 g AI/946 liters of water/ hectare) and for spinosad 135

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ppm (124 g AI/946 liters of water/hectare). Although it is very difficult to directly relate the information collected using laboratory larval bioassays to the field performance of insecticides, the comparison with recommended field rates should provide an index of relative expected field efficacy. When the larvae were allowed to feed on methoxyfenozide, novaluron or spinosad treated diet for a longer period, the LC50 and LC90 values dropped to much lower values, well below recommended field rates. However, it is important to note that in an orchard situation, by four days codling moth larvae are already inside the fruit and are no longer exposed to the insecticides.

Table 3. Baseline sensitivity data of neonate codling moth larvae to various insecticides assessed using a diet surface topical bioassay. Mortality readings were conducted at 1, 4, 5 and/or 7 days after the larval placement on the treated diet. Bioassays were conducted using various codling moth populations reared at least for 1 generation in the laboratory on thinned fruit.

Mortality LC50 (ppm) LC90 (ppm) Compound at day: N Slope (FL at 0.95) (FL at 0.95) acetamiprid 1 299 1.5 ± 0.3 1.1 7.6 (0.5 - .6) (5.3 – 13.9) 7 264 1.9 ± 0.4 1.0 4.7 (0.4 – 1.6) (3.3 – 8.7) azinphos-methyl 1 400 2.4 ± 0.3 15.4 52.4 (11.9 – 19.1) (39.9 – 78.4) 4 300 3.2 ± 0.5 4.9 12.4 (3.9 – 6.0) (9.9 – 17.7) fenpropathrin 1 300 1.2 ± 0.3 0.1 1.08 (0.01 – 0.23) (0.68 – 2.02) methoxyfenozide 1 450 0.8 ± 0.2 132.9 4690 (61.6 – 652.8) (861 – 213408) 4 450 1.9 ± 0.2 4.2 19.7 (3.2 – 5.2) (14.6 – 29.7) novaluron 4 700 1.5 ± 0.4 353.0 2553.0 (234.0 – 836.0) (1000 – 30852) 7 700 0.7 ± 0.1 0.6 49.6 (0.2 – 1.2) (21.5 – 168.2) rynaxypyr 1 400 1.6 ± 0.2 0.8 4.9 (0.5 – 1.0) (3.7 – 7.7) 5 250 3.1 ± 0.6 0.4 1.1 (0.3 – 0.5) (0.9 – 1.5) spinosad 1 240 1.1 ± 0.4 9.9 132.8 (4.0 – 546.2) (20.0 – 1521655) 4 240 2.6 ± 0.4 1.0 3.1 (0.7 – 1.3) (2.2 – 5.6)

For azinphos-methyl, despite the 3 fold (at the LC50 level) differences observed during 1 and 4 day readings, the toxic effect of this compound on codling moth neonate larvae was very rapid. The recommended field rate of azinphos-methyl of 923 ppm (850 g of AI/946

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liters of water/hectare) is much higher than the estimated LC90 values. No differences were observed at 1 and 4 day LC50 values for acetamiprid suggesting rapid kill for this compound. The differences in the LC50 and LC90 levels between 1 day and later mortality readings are very important from a practical perspective. Under normal orchard conditions the newly hatched codling moth larva will feed on the surface of fruit for less than 24 hours before entering the fruit. To prevent a larva from causing fruit injury the insecticide has to kill the larva before it enters the fruit. Even partial feeding expressed as “stings” can decrease the value of fruit. Our bioassays suggest that only azinphos-methyl and acetamiprid were able to rapidly kill the larvae after 1 day of feeding. Methoxyfenozide, novaluron and spinosad were very active against neonate codling moth at extended mortality readings but their LC50 and LC90 values at the 1 day reading (or 4 day for novaluron) were much higher than at the 4 (or 7 for novaluron) day assessment. Also, for the recently discovered but un-registered compound, rynaxypyr the 1 day LC50 and LC90 values were higher than LC values at 5 days. Information generated during these studies on current levels of resistance to organophosphate and carbamate insecticides throughout Pennsylvania may help avoid or delay the development of further resistance by allowing pest managers the opportunity to fashion more-assiduously-tailored spray programs as well as to test anti-resistance strategies.

Acknowledgements

The authors want to thank the State Horticultural Association of Pennsylvania and the Pennsylvania Apple Marketing Board for their financial support during multiple years of this project. We also want to thank various Adams County fruit growers who allowed us to use their orchards for collection of codling moth adults for the resistance and baseline sensitivities study. The credits are also extended to the whole group of our summer assistants without whose efforts this study could not have been conducted. Special thank to Teresa Krawczyk for her involvement in rearing and conducting laboratory bioassays on codling moth.

References

Bush, M.R.; Abdel-Aal, Y.A.L. & Rock, G.C. 1993: Parathion resistance and esterase activity in codling moth(Lepidoptera: Tortricidae) from North Carolina. – J. Econ. Entomol. 86: 660-666. Dunley, J.E. & Welter, S.C. 2000: Correlated insecticide cross-resistance in Azinphosmethyl resistant codling moth (Lepidoptera: Tortricide). – J. Econ. Entomol. 93: 955-962, Hodgkiss, H.E. & Haley, D.E. 1934: Codling moth in Pennsylvania. – J. Econ. Entomol. 27:232-239. Hull, L.A.; McPheron, B.A. & Lake, A.M. 1997: Insecticide resistance management and integrated mite management in orchards: can they coexist? – Pesticide Science 51: 359- 366. Hull, L.A. Krawczyk, G. 1999: Concentrate airblast insect evaluation, 1998. – Arthropod Management Tests 24: 19-23. Knight, A.L.; Brunner, J.F. & Alston, D. 1994: Survey of azinphosmethyl resistance in codling moth (Lepidoptera: Tortricidae) in Washington and Utah. – J. Econ. Entomol. 87: 285-292. Knight, A.L.; Hull, L.; Rajotte, E.; Hogmire, H.; Horton, D.; Polk, D.; Walgenbach, J.; Weires, R. & Whalon, J. 1990: Monitoring azinphos-methyl resistance in adult male Platynota idaeusalis (Lepidoptera: Tortricidae) in apple from Georgia to New York. – J. Econ. Entomol. 83: 329-334.

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Krawczyk, G. 2006: Monitoring Insecticide Resistance in Field Populations of Oriental Fruit Moth and Codling Moth Collected from Pennsylvania Fruit Orchards. – Pennsylvania Fruit News 86(2): 31-35. Krawczyk, G. & Hull; L.A. 2004: Utilization of various technologies for understanding, monitoring and controlling codling moth in PA apple orchards. – Pennsylvania Fruit News 84: 21-39. Lawson, D.S.; Reissig, W.H. & Smith, C.M. 1997: Response of larval and adult obliquebanded leafroller (Lepidoptera: Tortricidae) to selected insecticides. – J. Econ. Entomol. 90: 1450-1457. Shorey, H.H. & Hale, R.L. 1965: Mass rearing of the larvae of some noctuid species on a simple artificial medium. – J. Econ. Entomol. 58: 52-54. Souphanor, B.; Brosse, V.; Monier, C & Bouvier, J.C. 1998: Differential ovicidal and larvicidal resistance to benzoylureas in the codling moth, Cydia pomonella. – Entomol. Exp. Appl. 88: 247-253. Usmani, K.A. & Shearer, P.W. 2001: Susceptibility of male Oriental fruit moth (Lepidoptera: Tortricidae) populations from New Jersey apple orchards to azinphos-methyl. – J. Econ. Entomol. 94(1): 233-239. Varela, L.G.; Welter, S.C.; Jones, V.P.; Brunner, J.F. & Riedl, H. 1993: Monitoring and characterization of insecticide resistance in codling moth (Lepidoptera: Tortricidae) in four western states. – J. Econ. Entomol. 86: 1-10.

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 pp. 215-219

Baseline toxicity of new insecticides for Grapholita molesta management

Peter W. Shearer1, James F. Walgenbach2, Greg Krawczyk3 1 Department of Entomology, Rutgers University, Rutgers Agricultural Research & Extension Center, 121 Northville Road, Bridgeton, New Jersey 08302-5919, USA, [email protected] 2 Department of Entomology, North Carolina State University, Mountain Horticultural Crops Research & Extension Center, Fetcher, North Carolina, 28732-9244, USA, [email protected] 3 Pennsylvania State University, Department of Entomology, Fruit Research and Extension Center, 290 University Drive, Biglerville, PA 17307. [email protected]

Abstract: In the mid-late 1990’s, the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), became a serious pest of apples grown in the eastern U.S.A. Several factors were associated with these outbreaks, but insecticide resistance was the major reason for G. molesta control failures that resulted in infested apples at harvest. Resistance monitoring programs revealed that G. molesta populations had developed resistance to organophosphorous (OP), carbamate, and pyrethroid insecticides. In anticipation of new insecticides being used against this pest, laboratory bioassays were conducted to generate baseline susceptibility data for future resistance management efforts. Using a surface-treated lima bean diet assay, we determined base-line concentration-response parameters for acetamiprid, azinphosmethyl, esfenvalerate, indoxacarb, methozyfenozide, novaluron, pyriproxyfen, spinosad, and thiacloprid for G. molesta neonates.

Key words: insecticide resistance, resistance monitoring, apple, bioassay

Introduction

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is the most important pest of peaches grown in the northeastern USA and eastern Canada, but has not generally been considered a significant pest of apple (Pree, 1985). However, during the mid 1990’s, apple growers in New Jersey, USA, experienced severe crop losses because of high numbers of G. molesta infested fruit. By the late 1990’s through 2002, apple growers from numerous eastern USA states, including those in the mid-Atlantic region plus North Carolina and Michigan, reported that G. molesta were infesting apple fruit resulting in load rejections at processor facilities and causing large economic losses (Shearer et al., 2003). There are numerous reasons for these outbreaks, including growing apples in proximity of peaches, a preferred host, because G. molesta can disperse from peaches to apples during the growing season (Chapman and Lienk, 1971). Once populations are established in apple blocks, they can continue to develop and cause problems (Rothschild and Vickers, 1991). Peaches and apples are often planted in close proximity in New Jersey (Usmani and Shearer, 2001) and Pennsylvania (Krawczyk, personal observation), but peaches are generally not planted in the apple regions of North Carolina (Walgenbach, personal observation). Additionally, poor spray coverage and application timing, low insecticide rates, and growers switching to selective insecticides for leafroller pests (Hull et al., 2003) have also contributed

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to G. molesta problems in the eastern USA. However, studies have revealed that G. molesta populations in problem apple orchards had developed resistance to commonly used organophosphate insecticides (Shearer et al., 2001; Usmani and Shearer, 20001) in addition to several pyrethroids and carbamates (Krawczyk, 2006). Fruit growers in the USA are currently transitioning to new tactics to manage G. molesta in eastern USA apple orchards. The single most important reason for these changes to apple IPM programs is a result of implementation of the Food Quality Protection Act (FQPA) of 1996. This law specifically requires the U.S. Environmental Protection Agency (EPA) to develop pesticide tolerances that place greater emphasis on the safety of infants and children, and that also address safety concerns related to farm workers and environment. Essentially, organophosphate insecticides are being regulated such that their uses are more restrictive or forbidden. Increased instances of insecticide-resistant pest populations is also contributing to changes in apple pest management programs. In addition to using mating disruption (Kovanci et al., 2005), growers are incorporating newly registered insecticides (Hull et al., 2002). Several of these new insecticides have unique modes of action and do not have a history of use against G. molesta. This provides researchers and extension personnel with the opportunity to generate base-line sensitivity data and monitoring protocols for these new products that allows for the development of resistance management programs and subsequent evaluations of their success (Brent, 1986). The purpose of this paper is to report on our initial results from laboratory assays designed to generate base-line sensitivity data for several new insecticides against G. molesta. We anticipate using these results in future resistance-monitoring programs.

Material and methods

Insect rearing The G. molesta colonies used for testing were established from field populations collected from minimally sprayed orchards in each state. Larva were collected from infested fruit and shoots then reared on either green thinning apples using a method similar to the one described by Pree (1985) or a lima bean-based diet (Shorey and Hale, 1965). Larvae were allowed to develop in environmental chambers (25 0C, 70% RH, and a 16:8h L:D photoperiod). Adults were kept in cages in the laboratory under ambient conditions where they oviposited on wax- paper sheets placed in their holding containers. Wax paper sheets with G. molesta eggs were either placed back on the various food substrates for colony maintenance or cut into 3 cm squares, placed in covered Petri dishes inside environmental (same conditions as above) to allow for neonate emergence for larval assays.

Insecticides tested All insecticides tested were commercially available formulations of the following insecticides: acetamiprid (Assail® 70WP), azinphosmethyl (Guthion® 50WSB), esfenvalerate (Asana® XL), indoxacarb (Avaunt® 30DG), methozyfenozide (Intrepid® 2F), novaluron (Rimon® 0.83EC), pyriproxyfen (Esteem® 35WP), spinosad (Spintor® 2SC), and thiacloprid (Calypso® 4F). Each product was mixed with water to make serial dilutions for testing.

Surface-treated diet assays Clear plastic cups (30 ml) were filled 1/3 from the bottom with freshly made hot lima bean diet and allowed to cool and set. The surface of the diet cups was then treated with 0.5 ml of formulated insecticide dissolved in water. Control cups were treated with water only. The treated cups were allowed to dry for about 2-3 hrs. Five neonate G. molesta were placed on

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the surface of the diet in each cup using a fine sable/synthetic brush. The treated cups were covered with sized paper insert lids. The treated cups with G. molesta larvae were placed in environmental chambers set at conditions specified above. The larvae were evaluated at 1, 4, and 7 days, or evaluated after successful development to the adult stage. On days 1 and 4, the surface of the diet, sides of the cup and lid were checked for dead larvae. On day 7, the diet was examined for presence of live larvae or left to evaluate successful completion to the adult stage.

Statistical analyses All concentration-response parameters were calculated using POLO Plus (LeOra Software 2003). Mortality data that was less than 5% or greater than 95% were excluded from the analyses.

Results and discussion

Extending post-treatment mortality assessment from 1, to 4, to 7d lowered the LC values considerably (Table 1). The LC50 values decreased about 21- and 6-fold when the evaluation time increased from 1 to 4, and 4 to 7d, respectively. Lengthening the evaluation period allowed more time for mortality to occur in addition to allowing more toxicant to be ingested, thus lowering the LC values. On a practical note, delaying the evaluation time until a week after treatment allowed surviving larvae additional time to grow, making them easier to find in the diet.

Table 1. Effect of post-treatment evaluation time on concentration-response line parameters for G. molesta neonate survival on novaluron-treated diet.

Post-treatment N slope LC50 LC90 assessment (ppm) (ppm) 1 day 450 0.6 51.0 5182.0 4 day 350 0.3 2.4 569.0 7 day 300 0.8 0.4 13.0

The concentration-response parameters for G. molesta neonates tested against various insecticides revealed that several non-organophosphate insecticides were more toxic than azinphosmethyl on a dose-relationship basis when evaluated seven days after treatment (Table 2). This relationship holds true for field rates, because these products are applied at rates that are considerably lower than the typical application rates for azinphosmethyl.

Table 2. Concentration-response line parameters for G. molesta neonates for various insecticides using the surface-treated diet assay, 7-day post-treatment evaluation.

Material n slope LC50 (ppm) LC90 (ppm) Thiacloprid 235 1.8 0.5 2.5 Indoxacarb 599 1.6 0.2 1.0 Spinosad 300 1.7 0.5 2.6 Azinphosmethyl 605 2.8 4.2 12.0 Esfenvalerate 451 2.4 0.3 0.8

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When surface-treated diet assays were evaluated based on survival to adult stage, concentration-response parameters also showed that some of the newer insecticides were more toxic at lower doses than the standard azinphosmethyl comparison (Table 3). In this instance, only pyriproxyfen and acetamiprid had LC values higher than azinphosmethyl.

Table 3. Concentration-response line parameters for G. molesta neonates for various insecticides using the surface-treated diet assay, survival to adult stage.

1 Material slope LC50 (ppm) LC90 (ppm) Novaluron 2.6 0.4 0.9 Methoxyfenozide 3.9 0.4 1.2 Pyriproxyfen 2.0 13.8 36.3 Spinosad 3.3 1.0 2.6 Acetamiprid 3.2 4.2 11.1 Azinphosmethyl 6.0 2.7 7.2 1 n = 720 neonates tested per material

As growers switch to new products because of governmental regulatory decisions, loss of efficacy of standard products due to insecticide resistance, or economic factors, it is useful to provide growers with recommendations on how to manage their pests so they remain susceptible to various treatment options as along as possible. Insecticide resistance is essentially susceptibility management and it is imperative to manage product use to protect these insecticides from improper use and subsequent loss of efficacy in the field. One way to monitor changes in pest population susceptibility is to generate base-line susceptibility data for insecticides. Ideally, bioassay methodology is relatively easy, provides rapid results, and is standardized across wide areas. The information from these efforts can be used for future evaluations of resistance management programs by tracking changes in population response to these products. If the assay is sensitive enough and populations are monitored at a frequency that allows for early detections of declining susceptibility, then growers can be advised to change their pest management program. These assays can also be used to help explain whether field-failures are a result of insecticide resistance development or other factors.

Acknowledgements

The authors would like to thank Ann Rucker (Rutgers University) and Steve Schoof (North Carolina State University) for their technical assistance and the following companies for providing support for this work: Bayer Crop Sciences, Cerexagri, Inc., Chemtura Corp., Dow AgroSciences LLC, E.I. du Pont de Nemours and Co., Makhteshim Agan of North America, and Valent Agricultural Products. This is New Jersey Agricultural Experiment Station Publication No. E-08-08184-03-06 and was supported, in part, by Hatch Act funds.

References

Brent, K.J. 1986: Detection and monitoring of resistant forms: An overview. – In: Pesticide Resistance: Strategies and Tactics for Management. National Academy of Sciences (eds.), National Academy Press, Washington, DC: 298-312.

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Chapman, P.J., & Lienk, S.E. 1971: Terraced fauna of apple in New York (Lepidoptera: Tortricidae) including an account of apples occurrence in the State, especially on a naturalized plant. – New York State Ag. Expt. Sta. Special Publication: 122 pp. Hull, L.A.; Myers, C.; Ellis, N. & Krawczyk, G. 2003: Management of the internal lepidopteran complex in Pennsylvania. – Intl. Dwarf Fruit Tree Assoc. Compact Fruit Tree. 36: 21-25. Hull, L.A.; Krawczyk, G.; Ellis, N. & Myers, C. 2002: Management tactics for the oriental Fruit moth (Grapholita molesta) in Pennsylvania apple orchards. – Pennsylvania Fruit News 82(2): 29-35. Kovanci, O.B.; Schal, C.; Walgenbach, J.F.; & Kennedy, G. 2005: Comparison of mating disruption with pesticides for management of oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) in North Carolina apple orchards. – J. Econ. Entomol. 98: 1248-1258. Krawczyk, G. 2006: Monitoring insecticide resistance in field populations of Oriental fruit moth and Codling moth collected from Pennsylvania fruit orchards. – Pennsylvania Fruit News 86(2): 31-35. Pree, D.J. 1985: Grapholita molesta. – In: Handbook of Insect Rearing. Vol. 2. Singh, P. & Moore, R.F. (eds.), Elsevier, Amsterdam: 307-311. Rothschild, G.H.L. & Vickers, R.A. 1991: Biology, ecology and control of the Oriental fruit moth. – In: Tortricid Pests: Their Biology, Natural Enemies and Control, Vol. 5. Van der Geest, L.P.S. & Evenhuis, H.H. (eds.), Elsevier, Amsterdam: 389-412. Shearer, P.W.; Usmani, K.A.; Krawczyk, G.; Hull, L.; Gut, L.; Reissig, H. & Agnello, A. 2001: Toxicological response of male Oriental fruit moth collected from Eastern apple orchards to Azinphosmethyl. – Proceedings of the 75th Ann. Western Orchard Pest and Disease Mgt. Conf. Portland, OR. January 10-12, 2001: 117-118. Shearer, P.W.; Atanassov, A.; & Krawczyk, G. 2003: The invasion of internal fruit feeders: Efforts by Rutgers and Penn State Universities to face the challenge. – Intl. Dwarf Fruit Tree Assoc. Compact Fruit Tree. 36: 28-29. Shorey, H.H. & Hale, R.L. 1965: Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. – J. Econ. Entomol. 58: 522-524. Usmani, K.A. & Shearer, P.W. 2001: Topical pheromone trap assays for monitoring susceptibility of male Oriental fruit moth (Lepidoptera: Tortricidae) populations to azinphos- methyl in New Jersey. – J. Econ. Entomol. 94: 233-239.

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Pome Fruit Arthropods IOBC/wprs Bulletin Vol. 30 (4) 2007 p. 221

Evaluation of pear psylla, Cacopsylla pyri (L.), susceptibility to cypermethrin in pear orchards of Lleida, Spain

Xavier Miarnau1,2, Miquel Artigues2, María José Sarasúa1,2 1Universitat de Lleida, Department of Crop and Forest Science, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]; [email protected] 2IRTA.Centre UdL-IRTA, Rovira Roure 191, 25198 – Lleida, Spain, [email protected]

Abstract: Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in the fruit growing area of Lleida (NE Spain). Chemical control is the most common method used against pear psylla, but the number of insecticides registered to control it has been reduced in the last years. The high selection pressure with these few insecticides applied repeatedly over the whole area can result in the appearance of resistance, as it has happened in France. With the aim of monitoring changes in the susceptibility of C. pyri to cypermethrin, we used topical application bioassays with adults. The bioassays were carried out for four years (2003-2004-2005-2006). We collected populations from different areas of Lleida, where heavy use of insecticides is the common practice and we monitored the variation in time of the susceptibility within the season. At the end of the evaluation period we tried to detect the resistance mechanisms of the different populations with the application of synergists. We obtained the baseline data, LC50, LC90 of all the populations. No evidence of a high level of resistance has been found. However when we applied the recommended field concentration, mortality never reached 100 %, and after the application of the insecticide with synergists there was an increase of mortality in all the populations. Our results show that all the populations of our area have some level of resistance or at least a low level of susceptibility and that the susceptibility to cypermethrin was even lower in the summer adults

Key words: Cacopsylla pyri, Pear psylla, cypermethrin, resistance, bioassays, synergists

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