The Effects of Spinosad on Beneficial Insects and Mites Used in Integrated Pest Manage- Ment Systems in Greenhouses Miles, M

IOBC / WPRS

Working Group „Pesticides and Beneficial Organisms“

OILB / SROP

Groupe de Travail „Pesticides et Organismes Utiles“

Proceedings of the meeting

Dębe, Poland

27th – 30th September 2005

editors: Heidrun Vogt & Kevin Brown

IOBC wprs Bulletin Bulletin OILB srop Vol. 29 (10) 2006

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

The IOBC/WPRS Bulletin is published by the International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palearctic Regional Section (IOBC/WPRS)

Le Bulletin OILB/SROP est publié par l‘Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles, section Regionale Ouest Paléarctique (OILB/SROP)

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. Phili ppe C. Nicot INRA – Unité de Pathologie Végétale Domaine St Maurice - B.P. 94 F-84143 Monfavet Cedex France

ISBN 92-9067-193-7 http://www.iobc-wprs.org

Preface

This Bulletin contains the contributions presented at the meeting of the IOBC WG „Pesticides and Beneficial Organisms“ held in Dębe near Warsaw, Poland, from 27th to 30th September 2005, in the Training Centre of the Ministry of Environmental Protection. The meeting was attended by 55 participants from 14 countries. 22 presentations were given, covering diverse aspects of side-effects of pesticides on beneficial organisms: questions about IPM (integrated pest management), how to improve it, problems occuring due to changes in the pesticide choice, effects of selected pesticides on beneficials (parasitoids and predators), methodical aspects, comparison of sensitivity of several ladybird species to pesticides etc.. The Meeting included a technical visit to a private apple grower, the Research Institute of Vegetable Crops and the Research Institute of Pomology and Floriculture in Skiernewice. At the end of the meeting a discussion took place to summarize the workshop and to define further main objectives of the WG as well as possible topics for collaborations. These mainly were: – interpretation and importance of side-effect results in the field; – sublethal effects and their importance on population level of the beneficials; – effects of pesticides with special modes of actions (e.g. IGRs); systemic pesticides may need adoption of methods and prolongation of the observation period, more data about their persistence needed; – effects of pesticides in protected crops; – extrapolation of data between species; – more guidance in field methods needed, especially sampling techniques. The meeting in Dębe was a very succesful one and demonstrated once again that the WG offers an excellent forum for bringing together experts working in the field of “side-effects” of pesticides and for elaborating the basic information necessary for IPM and the preservation of beneficial organisms. The meeting was characterized by lively discussions and intense information exchange among the participants. Many thanks go to the local organizers and their team for the excellent choice of the meeting place, their great hospitality and the perfect organization of the excursion with its technical and cultural aspects.

Heidrun Vogt (Convenor)

Dossenheim, 28 July 2006 ii

iii

List of Participants

Barbara BAIER, Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Ecotoxicology and Ecochemistry in Plant Protection, Königin-Luise Str.19, D-14195 Berlin, GERMANY. E-mail: [email protected] Bozena BARIC, Faculty of Agriculture, Svetošimunska 25, Zagreb 10 000, CROATIA. E-mail: [email protected] Anja BARTELS, AGES, Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1226Vienna, AUSTRIA. E-mail: [email protected] Markus BARTH, BioChem agrar, Kupferstr. 4, D-04827 Gerichshain, GERMANY. E-mail: [email protected] Kevin BROWN, Ecotox Limited, P.O. Box 1, Tavistock PL19 0YU, UK. E-mail: [email protected] Nicola DAVIES, CEMAS, Glendale Park, Fernbank Road, North Ascot, Berkshire, SL5 8JB, North Ascot, UK. E-mail: [email protected] Zbigniew DĄBROWSKI, Dept. of Applied Entomology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warszawa, POLAND. E-mail: [email protected] Cora DRIJVER, Plant Protection Service, Geertjesweg 15, P.O. Box 9102, Wageningen 6700 HC, THE NETHERLANDS. E-mail: [email protected] Gillian FERGUSON, Ontario Ministry of Agriculture, Food & Rural Affairs, 2585 County Rd. 20, Harrow N0P2G0, CANADA. E-mail: [email protected] Marion GAGNIARRE, Phyteurop, Courcellor 2, 35,rue d’Alsace, 92300 PewalbisPerret 92531, France. E-mail: [email protected] Darek GAJEK, Research Institute of Pomology and Floriculture, Pomologiczna str. 18. 96- 100 Skierniewice, POLAND. E-mail: [email protected] Ine GEUIJEN, NOTOX B.V., P.O. Box 3476, ‘s-Hertogenbosch 5203 DL, THE NETHERLANDS. E-mail: [email protected] Bruno GOBIN, PCF - Research Centre for fruit growing, De Brede Akker 13, Sint Truiden 3800, BELGIUM. E-mail: [email protected] iv

Julia GÓRECKA, Katedra Entomologii Stosowanej, WOiAK, SGGW, Nowoursynowska 159, Warszawa 02-776, POLAND. E-mail: [email protected] Bilgin GÜVEN, Bornova Zirai Mücadele Araştırma Enstitüsü, Gençlik sokak No: 6, Izmir 35040 TURKEY. E-mail: [email protected] Nigel HALSALL, Insect Investigations Ltd., (I2L), Capital Business Park, Wentloog, Cardiff, CF3 2PX, UK. E-mail: [email protected] Rufus ISAACS, Michigan State University, CIPS 202, East Lansing, MI 48824, USA. E-mail: [email protected] Jean - Pierre JANSEN, Department of Biological Control and Plant Genetic Ressources - Agricultural Reasearch Centre, Gembloux, 2 Chemin de Liroux, Gembloux 5030, BELGIUM. E-mail: [email protected] Iris KÖNINGS, GEP Trial Manager, Le Haut Serre, F-26570 Montbrun-les-Bains FRANCE. E-mail: [email protected] Danuta KROPCZYŃSKA – LINKIEWICZ, Dept. of Applied Entomology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warszawa, POLAND. E-mail: [email protected] Hristina KUTINKOVA, Fruit Growing Institute, Plovdiv – 4004, kv.\"Ostromila \"12, BULGARIA. E-mail: [email protected] Barbara ŁABANOWSKA, Research Institute of Pomology and Floriculture, Pomologiczna str. 18, 96-100 Skierniewice, POLAND. E-mail: [email protected] Thomas MACDONALD, MGS Horticultural, 50 Hazelton Street, Leamington N8H3W1, CANADA. E-mail: [email protected] Alicja MACIESIAK, Research Institute of Pomology and Floriculture, Pomologiczna str. 18. 96-100 Skierniewice, POLAND Pilar MEDINA, E.T.S.I. Agrónomos. Universidad Politécnica de Madrid, Ciudad Universitaria s/n, Madrid 28040, SPAIN. E-mail: [email protected] Benoit MÉGEVAND, IMPACTEST, Avenida Almirante Reis, 204, 7º Dto, Lisboa 1000- 056, PORTUGAL. E-mail: [email protected] Mark MILES, Dow AgroSciences, 3 Milton Park, Abingdon OX14 4RN, UK. E-mail: [email protected] Ed MOERMAN, Koppert Biological Systems, PO Box 155, Berkel en Rodenrijs 2650 AD, THE NETHERLANDS. E-mail: [email protected] v

Monika MOLL, IBACON GmbH, Arheilger Weg 17, 64380 Roßdorf, GERMANY. E-mail: [email protected] Edmund NIEMCZYK, Research Institute of Pomology and Floriculture, Pomologiczna str. 18, 96-100 Skierniewice, POLAND. E-mail: [email protected] Karin NIENSTEDT, Springborn Smithers Laboratories (Europe) AG, Seestrasse 21, 9326 Horn, SWITZERLAND. E-mail: [email protected] Remigiusz W. OLSZAK, Research Institute of Pomology and Floriculture, Pomologiczna str. 18, 96-100 Skierniewice, POLAND. E-mail: [email protected] Juliette PIJNAKKER, PPO Praktijkonderzoek Plant & Omgeving, Postbus 8, Naaldwijk 2671 KT, THE NETHERLANDS. E-mail: [email protected] Zofia PŁUCIENNIK, Research Institute of Pomology and Floriculture, Pomologiczna str. 18. 96-100 Skierniewice, POLAND. E-mail: [email protected] Delphine POULLOT-JUAN, ENIGMA. Hameau de Saint Véran, Beaumes de Venise 84190, FRANCE. E-mail: [email protected] Stefan PRUSZYŃSKI, Institute of Plant Protection, Miczurina str. 20, Poznań 60-318, POLAND. E-mail: [email protected] Ellen RICHTER, Institute for Plant Protection in Horticulture, Federal Biological Research Centre, Messeweg 11/12, 30104Braunschweig, GERMANY. E-mail: [email protected] Uta RÖHLIG, BioChem agrar, Kupferstr. 4, 04827Gerichshain, GERMANY. E-mail: [email protected] Susanne SCHMITZ, German Federal Environmental Agency, Wörlitzer Platz 1, 06844 Dessau, GERMANY. E-mail: [email protected] Hans Jürgen SCHNORBACH, BayerCropScience AG, Agronomic Development, Geb 6100, Alfred Nobel Str. 50, 40789Monheim, GERMANY. E-mail: [email protected] Leen SCHOEN, Sica CENTREX, Ancien chemin de Pia, Torreilles 66440, FRANCE. E-mail: [email protected] Małgorzata SEKRECKA, Research Institute of Pomology and Floriculture, Pomologiczna str. 18, 96-100 Skierniewice, POLAND. E-mail: [email protected] Amanda SHARPLES, Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK. E-mail: [email protected] vi

Peter SMYTHEMAN, Biological Crop Protection Ltd, Occupation Road, Ashford, TN25 5EN, UK. E-mail: [email protected] Marc VAN DE VEIRE, Faculty of Bio-engineering Sciences, University of Ghent, Coupure Links 653, Ghent 9000, BELGIUM. E-mail: [email protected] Russel VAUGHAN, Mambo-Tox Ltd., 2 Venture Road, Chilworth Science Park, Chilworth, Southampton SO16 7NP, UK. E-mail: [email protected] Stephen VINALL, Mambo-Tox Ltd., 2 Venture Road, Chilworth Science Park, Chilworth, Southampton SO16 7NP, UK. E-mail: [email protected] Elisa VIÑUELA, Polytechnic University of Madrid, Protección de Cultivos, E.T.S.I. Agrónomos, Ciudad Universitaria S/n, Madrid 28040, SPAIN. E-mail: [email protected] Heidrun VOGT, BBA, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str.101, 69221 Dossenheim, GERMANY. E-mail: [email protected] Anna WALTERSDORFER, BayerCropScience AG, Industriepark Höchst, H872, 65926 Frankfurt am Main, GERMANY. E-mail: [email protected] Wojciech WARABIEDA, Research Institute of Pomology and Floriculture, Pomologiczna str. 18. 96-100 Skierniewice, POLAND. E-mail: [email protected] vii

Contents

Preface...... i

List of participants...... iii

Protection of beneficial organisms in research and practice in Poland Pruszyński, S...... 1

Does implementation of a reduced-risk blueberry insect control program enhance biological control? Isaacs, R., Mason, K.S., Brewer, M., Noma, T. & O'Neal, M...... 7

Harmful and beneficial entomofauna in apple orchards grown under different management systems Andreev, R., Olszak, R. & Kutinkova, H...... 13

Building a selectivity list of plant protection products on beneficial arthropods in open field: a clear example with potato crop Hautier, L., Jansen, J.-P., Mabon, N. & Schiffers, B...... 21

Influence of plant protection measures on the carabid fauna of sugar beet and potato fields in Poland Nijak, K. & Pruszyński, S...... 33

How much precision does a regulatory field study need? Brown, K. & Miles, M...... 43

The effects of Spinosad on beneficial insects and mites used in integrated pest management systems in greenhouses Miles, M...... 53

A method to prove long term effects of neonicotinoids on whitefly parasitoids Richter, E...... 61

Mancozeb: A profile of effects on beneficial and non-target arthropods Miles, M...... 67

Side effects of insecticides used in cotton and vineyard areas of Aegean Region of Turkey on the green lacewing, Chrysoperla carnea (Steph.) (Neuroptera: Chrysopidae) under semi field conditions Güven, B. & Göven, M.A...... 81 viii

Effects of botanical insecticides on two natural enemies of importance in Spain: Chrysoperla carnea (Stephens) and Psyttalia concolor (Szépligeti) Medina, P., Budia, F., González, M., Rodríguez, B., Díaz, A., Huerta, A., Zapata, N. & Viñuela, E...... 85

Comparative sensitivity of four ladybird species to five pesticides Jansen, J.-P. & Hautier, H...... 95

Natural enemies of plum brown scale Parthenolecanium corni Bouché (Homoptera: Coccidae) in plum orchards in the region of Plovdiv Arnaoudov, V., Olszak, R. & Kutinkova, H...... 105

ABSTRACTS

Consideration of side effects on beneficial organisms during product development in the agrochemicals industry Schnorbach, H.J...... 113

Effects of Imidacloprid on Poecilus cupreus larvae depending on the mode of application Baier, B., Norr, C., Schenke, D. & Scharnhorst, T...... 114

Side effects of pesticides on Aphelinus mali and other antagonists of the woolly apple aphid Vogt, H. & Ternes, P...... 115

Effects of toxins in transgenic crops on natural enemies Dąbrowski, Z.T. & Górecka, J...... 116

Influence of the treated media on the residual toxicity of several insecticides to Chrysoperla carnea and Chrysoperla externa in laboratory Contreras, G., Medina, P. & Viñuela, E...... 117

Side effects of various pesticides on Feltiella acarisuga Altena, K. & Moerman, E...... 118

Side effects of various pesticides on Amblyseius swirskii Vanaclocha Arocas, P., Hoogerbrugge, H. & Moerman, E...... 118

Side-effects of IGR on development of an aphid-parasitoid, Aphidius colemani (Hymenoptera: Braconidae) Viereck Juan, D. & Ferré, J. B...... 119

The loss of earwig populations in Belgian orchards: testing side-effects on orchard management Gobin, B...... 120

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 1-5

Protection of beneficial organisms in research and practice in Poland

Stefan Pruszyński The Institute of Plant Protection, Miczurina 20, 60-318 Poznań, Poland, e-mail: [email protected]

Abstract: Polish research units belonging to Ministry of Agriculture, higher education and Polish Academy of Science developed in the second half of nineteenth century research programmes concerning the occurrence and biology of important beneficial organisms, selectivity of plant protection products to beneficial organisms, effects of chemical treatments on various components of agrocenosis, protection of beneficial organisms during chemical treatments and elaboration of integrated protection of agriculture and horticulture crops. The results of this work led to the implementation of integrated production of several horticultural crops, biological and integrated protection of glasshouse crops and recommendations for integrated protection of some agricultural crops.

Key words: Poland, beneficial organisms, research programmes, practical application of biological methods.

Introduction

Research and other activities regarding plant protection in Poland have a long and rich tradition. However after the Second World War in the early 1950’s a mass introduction of Colorado potato beetle (Leptinotarsa decemlineata Say) into Polish territory became an important issue concerning environmental and human safety (Pruszyński and Węgorek 1991). At this time in Poland the area of land under potato cultivation was 2.3 million ha and the occurrence of this pest posed a serious economic threat. Mass application of DDT to control the Colorado potato beetle demonstrated many negative aspects, such as resistance of the pest and the presence of DDT in fatty tissue of the Polish population following unreasonable use of chemical plant protection products. Due to worldwide criticism and the results of investigations the Polish government established the Act of the Council of the Prime Minister No 64/70 of 18th May 1970 regarding organising research on toxicology and safe application of pesticides and control of their residues in food and the human environment. This resulted in the withdrawal of DDT, mercury dressing and other hazardous substances from the market. Summarising that time it should be underlined that, unlike in other communist countries, collectivisation never took place in Poland and because of this it was possible to safeguard beautiful agricultural landscape, rich in forests, lakes, streams and natural habitats important for the development of beneficial organisms.

Some facts from the history

In parallel with optimising chemical crop protection measures in Poland there has been continuing research into biological methods of pest control. In 1951 the Polish translation of I. Rubcow’s book “Biological method in pest control” was published (Rubcow 1951). In 1964 the Conference “Present state of research on

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beneficial organisms from the point of view of the necessity of plant protection in Poland” organised by the Working Group of Biological Method in Plant Protection called by the Committee of Plant Protection Polish Academy of Science took place. During that Conference 27 lectures were given in which the state of research of particular beneficial organisms and their meaning for plant protection were evaluated. Lecturers were mainly scientists from institutes of the Polish Academy of Science and Agricultural Universities, but than in next years the fast development of scientific groups working on biological control in the Institute of Plant Protection in Poznań and the Institute of Pomology (nowadays Institute of Pomology and Floriculture) in Skierniewice took place. In those Institutes belonging to the Ministry of Agriculture and responsible for recommendation for the protection of agricultural and horticultural crops from the beginning in research programmes the principle was taken to join together pure and applied research. From the University of Agriculture biological control research was most strongly developed in the University of Agriculture (SGGW) in Warsaw. Additionally, other research programmes were also developed in research units in the country. The research programme at Winna Góra concerned the influence of intensive plant protection on the different elements of agriculture environment (Węgorek 1993, Węgorek at al 1990). In 1963, during the Scientific Session of Institute of Plant Protection, Lipa presented a lecture “Chemical or biological control in plant protection” (Lipa 1963). At the same time, in the Pomology Institute in Skierniewice, experiments on the influence of chemical plant protection application on the beneficial organisms were started (Niemczyk & Wiąckowski 1965). In 1967-1978 several valuable coursebooks were written by Polish authors (Sandner 1972, Lipa 1967, Koehler 1968, Boczek & Lipa 1978). The results of the research programmes on biological control in Poland and its implementation into practice was summarised by Lipa & Pruszyński (1985) A conference entitled “Parasitic and predatory insects and their use in biological method of plant protection” was organised in 1971 at Jabłonna near Warsaw. In Skierniewice two scientific conferences organised by the Biological Method Section of the Plant Protection Committee took place: in 1993 “Actual and potential use of biological pest control on plants” (Proceedings 1995) and in 1997 “Effectiveness and practical application of biological control in plant protection” (Proceedings 1997). The conferences were organised by Professor Dr. E. Niemczyk.

International cooperation

Polish scientists from the beginning actively participated in the work of the international organisation for biological control. In the 1970’s they attended in the meetings of the IOBC/WRPS Working Group of Biological and Integrated Control in Glasshouses. In 1991 Poland was the organiser of the International Conference “Plant Protection in Glasshouses” (Poznań – Naramowice). When the East Paleartic Regional Section of IOBC was established in Moscow in 1975, Poland became a member and her scientists held positions of office in the section. Several meetings of the Commission and Working Groups of this section were organised in Poland, but conferences were also organised in Poland at which members of East and West Sections participated. The good examples could be two Conferences: Microbial Pest Control (1995 Poznań) and Pest and Weeds Control in Sustainable Fruits Production (1995 Skierniewice).

The situation of Poland in the centre of Europe and present political situation gives a good possibility for treating Poland like a bridge between West, Central and East Europe and as a good place for scientific meetings for the members of both sections. In this place it should be pointed out that in the 1990’s under the Soviet Union biological methods were widely applied throughout Poland on a large scale (Pruszyński 1997).

Present state

The research concerning beneficial insects and their protection are currently carried out in several research units in Poland, the most important being; Research Institute of Pomology and Floriculture, Skierniewice – research concerning occurrence of beneficial organisms in orchards and other horticultural crops, introduction of predators and parasites, microbiological control, selectivity of pesticides, elaboration of integrated production programmes and integrated production. Institute of Plant Protection, Poznań – influence of chemical treatment on beneficial insects in agricultural crops, occurrence of beneficial species, introduction of predators and parasites, elaboration of integrated protection programmes and integrated production on agriculture crops, biological control in glasshouses, microbiological control. University of Agriculture (SGGW), Warszawa – research on predatory Phytoseiidae, selectivity of plant protection products to predatory mites, biological control in glasshouses, integrated protection and production. Academy of Agriculture, Wrocław – biological control of winter rape pests, biological control of aphids. Academy of Agriculture, Kraków – biological and nonchemical control of pests in horticulture crops especially in small gardens. Academy of Agriculture, Poznań – research on natural enemy of aphids and some orchard pests. Research Institute of Forestry, Warszawa – biological control of forest pests. Some research programmes concerning biological control are also carried out in Institute of Vegetable Crops (Skierniewice), University of Warmia and Mazury (Olsztyn), Academy of Agriculture in Lublin and Szczecin and some others. To give a full picture collection and systematic activity on different family of beneficial insects should be mentioned. Looking from the practical point of view integrated apple production was introduced to commercial production more than 15 years ago (Niemczyk 2002). Developed in the Research Institute of Pomology and Floriculture the control programme included introduction of Typhlodromus pyri and application of selective plant protection products. The situation changed in 2004 when the new Plant Protection Act came to the power in Poland. According to the Art. 5 of that Act the National Plant Protection Service is authorised for certification on integrated production. Presently the methods for integrated production on more than 20 horticultural crops (including early potatoes) are described and that system of production technology is more and more common among producers in Poland. Biological control of glasshouse pests is also widespread.

Conclusions

Looking into the future several positive circumstances can influence the further development on the research programmes concerning influence of chemical control on beneficial insects in Poland. To those belong: − very rich agricultural landscape; − very low usage of chemical plant protection products; − well-prepared and experienced group of researches and teachers; − very good results from previous experiments; − obligatory training for chemical plant protection product users and technical examinations of sprayers; − implementation of integrated production technology based on the Plant Protection Act. The negative factors should be also be mentioned, including: − very low subsidy for science; − very low subsidy for advisory service and a shortage of well-prepared advisors in the subject of biological methods and integrated programmes; − very difficult economic situation of Polish agriculture and horticulture and frequent changes in the production technology between farmers. There are much more positive circumstances but still the negative ones will play the significant role in the further development of the research and implementation of biological control method in Poland. Being an optimist I would like to finish with the words of Professor Dr Joop van Lenteren, the president of IOBC: “Biological control is most successful, most cost effective, safest pest control method; we – biocontrol workers – are strongly needed”.

References

Boczek, J. & Lipa, J.J. 1978: Biologiczne metody walki ze szkodnikami. – PWN Warszawa: 593 pp. Koehler, W. 1968: Biologiczne Metody Ochrony Lasu. – PWRiL: 199 pp. Lipa, J.J. 1963: Chemical or biological methods of plant protection. (in polish, english summary). – Biuletyn Instytutu Ochrony Roślin Nr 24: 213-227. Lipa, J.J. 1967: Zarys patologii owadów. – PWRiL: 342 pp. Lipa, J.J. & Pruszyński, S. 1985: Biological plant protection in Poland during last 25 years and its developing tendencies for the future years. (in polish, english summary). – Materiały XXV Sesji Naukowej Instytutu Ochrony Roślin, Poznań: 41-62. Niemczyk, E. 2002: Eleven years of integrated fruit production in Poland. (in polish, english summary). – Progress in Plant Protection/Postępy w Ochronie Roślin 42(1): 33-38. Niemczyk, B. & Więckowski, S.K. 1965: An attempt of evaluation of the population density of Panonychus ulmi Koch and predatory mites (Acarina Phytoseiidae) in the prune orchards sprayed and unsprayed with winter sprays. (in polish, english summary). – Prace Instytutu Sadownictwa 9: 263-280. Proceedings of the Conference: “Actual and potential use of biological pest control on plants”. Skierniewice, Nov. 22-23, 1993. Research Institute of Pomology and Floriculture. Skierniewice 1995: 172 pp.

Proceedings of the Symposium: “Effectiveness and practical application of biological control in plant protection”. Skierniewice, March 18-19, 1997. Research Institute of Pomology and Floriculture. Skierniewice 1997: 161 pp. Pruszyński, S. 1997: Practical application of biological method in plant protection in several countries of Central-Eastern Europe. – In: Proceedings of the Symposium: “Effectiveness and practical application of biological control in plant protection”. Skierniewice, March 18-19,1997. Research Institute of Pomology and Floriculture. Skierniewice 1997: 9-16. Pruszyński, S. & Węgorek, W. 1991: Control of Colorado beetle (Leptinotarsa decemlineata) in Poland. – Bulletin OEPP/EPPO Bulletin 21: 11-16. Rubcow, I. 1951: Biologiczna metoda walki ze szkodliwymi owadami. – PWRiL. Warszawa: 346 pp. Sandner, H. 1972: Biologiczne metody ochrony roślin. – PWRiL: 202 pp. Węgorek, W. 1993: Influence of pesticides on agroecology. – Roczniki Nauk Rolniczych S.E. 23(1/2): 117-123. Węgorek, W., Trojanowski, H., Dąbrowski, J. & Rudny, R. 1990: Effect of intensive pesticide application on the yield and on some components of agricultural environment. Part II. Researches on side effects on pesticides protected cultures and on some components of agricultural environment. (in polish, english summary). – Prace Naukowe IOR Poznań XXXII (1/2): 117-128.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 7-11

Does implementation of a reduced-risk blueberry insect control program enhance biological control?

Rufus Isaacs, Keith S. Mason, Michael Brewer, Takuji Noma, and Matthew O'Neal* Department of Entomology, Michigan State University, East Lansing, MI 48824, USA. *Present address: Department of Entomology, Iowa State University, Ames, IA 50011, USA.

Abstract: A reduced-risk insect control program was implemented at commercial blueberry farms in Michigan, USA and the side effects on two important groups of natural enemies was monitored. The aphid parasitoid community responded after two years of implementation, with parasitism rates approx. 30% higher in the fields receiving reduced-risk insecticides. Total ground beetle captures were not statistically different between programs, but two dominant species, Harpalus erraticus and Amara aenea, increased in abundance in the reduced-risk program fields. Results are discussed in terms of the benefits growers may expect from adopting insect control programs based on new insecticides.

Key words: Reduced-risk, IPM, carabid, aphid.

Introduction

Changes to pesticide registration requirements in response to recent legislation have restricted availability of broad-spectrum insecticides for many United States food crops, and stimulated registration of insecticides designated as reduced-risk by the US-Environmental Protection Agency. For minor acreage crops, such as highbush blueberry (Vaccinium corymbosum L.), these changes provide the first opportunity for growers to implement an insect IPM program founded on insecticides expected to have lower impact on natural enemies than conventional chemistries. Insecticides recently registered for use in blueberry in the US include imidacloprid, spinosad, and tebufenozide. Although not always benign to natural enemies (Williams et al., 2003), these insecticides have lower human toxicity and potential for environmental contamination. Growers will expect enhanced natural enemy activity if using products with lower toxicity than conventional pesticides, but this data must be gathered from field conditions before making recommendations regarding improved biological control. Our Blueberry RAMP Project is testing the hypothesis that natural enemy populations will increase when growers adopt reduced-risk insecticides in blueberry. Here, we report the response of ground- dwelling carabid beetles and aphid parasitoids. Increasing carabid activity/density has been shown to increase prey removal in blueberry (O’Neal et al., 2005a), and many blueberry insect pests spend a significant portion of their life cycle on or in the ground. Parasitism of blueberry aphids can reach over 60%, and high aphid populations are associated with use of broad-spectrum insecticides (Whalon & Elsner, 1982).

Material and methods

Study sites This project was conducted at six blueberry farms in west Michigan. In spring of 2003, two 2- 4 Ha fields of V. corymbosum, cv. Bluecrop or Jersey, with similar insect pest pressure were

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selected at each farm. Both fields at each farm were scouted weekly during 2003 and 2004 for insect pests, and natural enemies were sampled as described below. One of the fields was managed with the grower’s conventional insecticide program based on broad spectrum insecticides, while the other field was managed in response to the weekly scouting results and was treated with reduced-risk insecticides (Table 1). The same fungicides and herbicides were applied to each field at each farm, and all applications were made by the growers using standard application technology.

Table 1. Conventional and reduced-risk insecticides registered for use against key insect pests in Michigan blueberry fields through the growing season.

Month Crop stage Target Conventional Reduced-risk Pest* April Pre bloom Leafrollers Methomyl, esfenvalerate Tebufenozide May Bloom CBFW B.t. Tebufenozide CFW B.t. Tebufenozide June-July Post bloom CBFW Azinphosmethyl, esfenvalerate Tebufenozide OBLR Phosmet, methomyl Tebufenozide BBA Methomyl Imidacloprid (foliar) White grubs Imidacloprid (soil) July-August Mid-season JB Phosmet, carbaryl, esfenvalerate Imidacloprid BBM Malathion, phosmet Spinosad, imidacloprid BB aphid Methomyl, imidacloprid Imidacloprid July-Sept. Pre-harvest JB Phosmet, carbaryl Spinosad BBM Phosmet, malathion Spinosad, imidacloprid *CBFW = cranberry fruitworm, Acrobasis vaccinii; CFW = cherry fruitworm, Grapholitha packardii; OBLR = obliquebanded leafroller, Choristoneura rosaceana; BBA = blueberry aphid, Illinoia pepperi; JB = Japanese beetle, Popillia japonica; BBM = blueberry maggot, Rhagoletis mendax.

Aphid and parasitoid sampling Weekly scouting was used to determine when the percentage of bushes with aphids present reached ~20%. Thereafter, aphid densities were intensively sampled approximately every two weeks (5 June to 7 August 2003, and 9 July to 12 August 2004). Within each of four sub- sections of the fields, we located 5 bushes infested with aphids. On each bush, the number of aphids and mummies on each branch of first-year growth was recorded. All leaves with mummies were collected and held individually in 2 oz plastic cups until a parasitoid wasp emerged. Parasitoids were identified to genus or in the case of some hyperparasitoids, to family. Mean percent aphid parasitism was calculated for each field on each sampling date and the mean number of aphids per branch and percent parasitism were compared between programs using ANOVA (Statview v 4.57, Abacus Concepts, Berkeley, CA.). Carabid sampling Adult ground beetles were monitored using the methods of O’Neal et al. (2005b). Briefly, pitfall traps (13.5 cm height by 11 cm diameter plastic cups) were placed in the soil between bushes with the rim 1 cm below the soil surface, and covered by a rain guard supported by nails. Approx. 200 ml of ethylene glycol was placed in each trap and refilled as needed. Six traps were deployed in each field; three along a wooded field edge and three 50 m within the interior, and traps were evenly spaced 6-8 rows apart across the plot. Traps were emptied once 9

a week from 8 May – 12 Oct. 2003 and 20 April – 13 Oct. 2004. All ground beetles were identified to morpho-species and voucher specimens were collected and identified to species. To describe ground beetle activity throughout the season, we combined beetle catches by month and report mean captures per field per month. To determine the effects of insecticide program on carabid beetle activity/density, a mixed-model repeated measures ANOVA was used. This analysis was performed on the entire carabid community and on the five most abundant species.

Results and discussion

Aphid parasitoids In the first year of transition to a reduced-risk insecticide program, aphid abundance was not significantly different between programs at any sampling date (P>0.05 for all dates), and the rate of aphid parasitism was also not significantly different between programs for any date (Fig. 1). Responses to the changing spray program were apparent in Year 2; aphid abundance was significantly lower in fields treated with the reduced-risk insecticide program, and aphid parasitism was significantly greater in the same fields by the Aug 9 sample (Fig. 1). Aphid parasitism was by the Praon spp. or Aphidius spp., and there was also some hyperparasitism by one Asaphes spp., one Alloxysta spp., and two Pteromalidae species. There was no consistent response between years to the management program.

Figure 1. Parasitism of blueberry aphid, Illinoia pepperi in blueberry fields managed using two insecticide programs. The asterisk denotes a sample in which parasitism was significantly different between the two programs (P<0.05).

Carabids During Year 1, 32 species of ground beetles were identified with Harpalus pensylvanicus, Harpalus erraticus, Amara aenea, Pterostichus mutus, and Patrobis longicornis being the five most common. Overall community abundance was similar in the first year, and only H. erraticus responded to the difference in insecticide programs, with greater abundance in the reduced-risk than the conventional fields (O’Neal et al., 2005b). A similar ground beetle community was observed in Year 2, that did not vary in overall species richness or abundance 10

between the two insecticide programs. In Year 2, both H. erraticus and A. aenea were more abundant in the reduced-risk than in the conventional program fields. As a species that emerges as an adult late in the season (Fig. 2), it is likely that larval H. erraticus mortality is greater in fields receiving conventional rather than reduced-risk insecticides. However, it is not clear why other fall emerging species, such as H. pensylvanicus, did not respond to the difference in the insecticide program. Differences in carabid biology and behavior are likely to determine the degree of sensitivity under field conditions.

Figure 2. Captures of two carabid species in fields managed with reduced-risk or conventional insecticides. Dates with asterisks were significantly different between the two programs (P<0.05).

Taken together, the carabid and aphid parasitoid results indicate that some components of the natural enemy community in blueberry will respond positively to adoption of reduced-risk insecticide programs. Depending on the natural enemy in question, these changes may take more than one year to become apparent, and they may not be sufficient to provide a high degree of pest control. Availability of reduced-risk insecticides that provide pest control with low impact on natural enemies is expected to enhance the degree of pest suppression provided by natural enemies. Based on our current data, however, the increase in biological control will be small and may take multiple growing seasons to develop. The Blueberry RAMP project will continue to monitor the response of pests and natural enemies to the changing insecticide program for a further two years. In addition to this, measuring the degree of pest control achieved by increasing populations of natural enemies should be a priority, to provide growers with confidence that the additional cost of reduced-risk insecticides provides additional benefits above direct pest control.

Acknowledgements

Our thanks to the many undergraduate students who assisted with this project, particularly Erica Zontek and Joe Asteriou of Kalamazoo College. We acknowledge the assistance of the six blueberry growers who collaborated on this project. We also thank Dave Trinka of MBG Marketing for technical assistance. This was funded in part by USDA RAMP program (grant 2001-51100-11514), Michigan Ag. Experiment Station, and MSU Extension.

References

O'Neal, M.E., Zontek, E.L., Szendrei, Z., Landis, D.A. & Isaacs, R. (2005a): Ground predator abundance affects prey removal in highbush blueberry (Vaccinium corymbosum) fields and can be altered by aisle ground covers. – Biocontrol 50: 205-222. O'Neal, M.E., Mason, K.S., & Isaacs, R. (2005b): Seasonal abundance of ground beetles in highbush blueberry (Vaccinium corymbosum) fields and response to a reduced-risk insecticide program. – Environmental Entomology 34: 378-384. Whalon, M.E. & Elsner, E.A. (1982): Impact of insecticides on Illinoia pepperi and its predators. – Journal of Economic Entomology 75: 356-358. Williams, T., Valle, J. & Viñuela, E. (2003): Is the naturally derived insecticide spinosad compatible with insect natural enemies? – Biocontrol Science and Technology 13: 459- 475.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 13-19

Harmful and beneficial entomofauna in apple orchards grown under different management systems

Radoslav Andreev1, Remigiusz Olszak2, Hristina Kutinkova3 1 Agricultural University, 12 D. Mendeleev str., 4000 Plovdiv, Bulgaria 2 Research Institute of Pomology and Floriculture, 18 Pomologiczna str., 96-100 Skierniewice, Poland 3 Fruit-Growing Institute, 12 Ostromila str., 4004 Plovdiv, Bulgaria

Abstract During the period 1996-2004, the harmful and beneficial insects were observed in apple orchards of the Agricultural University – Plovdiv, Bulgaria, grown under different management systems: biological, integrated and conventional (chemical). A total of 43 pests, belonging to 27 families and 5 orders were recorded in the orchard under biological pest management (BPM). In the orchards under IPM and chemical pest management (CPM) 35 and 26 species were found, respectively. The codling moth, Cydia pomonella, is the main pest of all apple orchards in Bulgaria. Other pests with a high population density in the BPM-orchard were the apple sawfly Hoplocampa testudinea, the pear lace bug Stephanitis pyri, tortricid-moths, the apple clearwing Synanthedon myopaeformis, the leopard moth Zeuzera pyrina and the weevils: Phyllobius oblongus, Rhynchites bacchus and R. aequatus. The populations of aphids, leafminers, Epicometis hirta and leaf-eating caterpillars increased occasionally. The populations of harmful insects in the IPM-orchard (aphids, leafminers, leopard moth and apple clearwing) increased occasionally. A high population density of harmful insects in the CPM-orchard (leafminers, aphids, Epicometis hirta, leopard moth and apple clearwing) was periodically observed. Beneficial insects were very abundant in the BPM-orchard. A total of 30 predators were found, belonging to 4 orders and 7 families. The ladybirds presented the highest population density and were significant as natural regulators of the small pests. Parasitoids from 7 families of Hymenoptera were important natural regulators of aphids, scale insects, leafminers, and tortricids. The population density of beneficial insects was lower in the IPM-orchard, but their importance as natural regulators of pests was still significant. In the CPM-orchard they were found occasionally.

Key words: apple, pest insects, beneficial insects, predators, parasitoids, organic farming, IPM

Introduction

The first environment-friendly pest management systems – integrated and biological – were elaborated in Bulgaria in the years 1967-1980. The basis for organic farming was, however, introduced into practice as late as in 1987 when the Agro-Ecological Centre at the Agricultural University in Plovdiv was created (Karov & Andreev, 2000). Many observations were carried out there, but they were fragmentary and related only to a specific group of insects. (Angelova et al 1995; Babrikova et al 1995; Lecheva et al 1995). Other pest management systems – integrated and conventional (chemical) were used at the same time in the neighbouring orchards. In this paper, the results of nine-year observations on composition of species as well as on population density of pest and beneficial insects in apple orchards grown under biological, integrated and chemical management systems are presented.

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Material and methods

The observations were carried out in three 0.5-ha apple orchards, created in 1988 within the Experimental Field of the Agricultural University – Plovdiv (Central South Bulgaria), in the years 1996-2004. The orchards include 7-9 cultivars but only Primrouge, Cooper Sel.4 and Granny Smith were observed. Biological pest management (BPM) was used in the first orchard according to the rules of organic farming (Karov & Andreev, 2000). Integrated pest management (IPM), according to the rules of integrated apple production (Pelov et al, 1996), was applied in the second orchard. Selective insecticides were applied when the pest population density exceeded the economic injury levels (Zaharieva et al, 1997). A conventional (chemical) pest management (CPM) was employed in the third plot. Aggressive insecticides organophosphates, pyrethroids and carbamates were used there, up to 14 treatments per season.

Table 1. Insecticides used in the apple orchards of the Agricultural University, Plovdiv, grown under different Pest Management (PM) systems in the period 1996 – 2004.

Biological PM Azadirachtin, pyrethrin, B. thuringiensis, granulosis virus, sex pheromones Integrated PM Pyrethrin, B. thuringiensis, granulosis virus, propargit, diflubenzuron, triflumuron, flufenoxuron, hexaflumuron, endosulfan, fosalone, chlorpyrifos-methyl, bensultap Chemical PM Chlorpyrifos-methyl, chlorpyrifos-ethyl, fenitrotion, fosalone, bensultap, tiodicarb, endosulfan, fipronil, deltamethrin, cypermethrin, zeta- cypermethrin, bifenthrin, propargit

The population density of the harmful and beneficial insects was assessed using standard entomological methods: shaking down of 100 branches in every orchard into an entomological net for beetles, bugs, sawflies and caterpillars, pheromone traps for moths and visual observations of branches, leaves and fruits on 10 representative trees for small insect as aphids, psyllids, leaf miners and scale insects (Mikhailova et al, 1982). The observations were done mainly during the growing season and only for the scale insects – in the dormant period. Samples of pest stages were isolated in the laboratory, in order to detect to obtain parasitoids. Three levels for evaluation of density of insects were applied – high, moderate and low. Population density of the pests was compared with the Economic Injury Levels (EIL): high – above EIL; moderate – about or slightly below EIL; low – not bringing any economic losses. The density of parasitoids was evaluated according to the following scale: high – parasitoid affected above 10% of the host’s population; moderate – 2-10% of the hosts affected; low – less than 2% of the hosts affected. Population density of the predators was compared with the overall number of the coccinellids found in BPM-orchard: high – the number of individuals (for a particular species) making above 10% of the coccinellids’ population; moderate – the species constituting 2-10% of the coccinellids’ population; low – the species constituting less than 2% of the coccinellids’ population.

Results and discussion

A total of 43 pests, from 27 families and 5 orders were recorded in the BPM-orchard (Table 2). The species of the orders Lepidoptera (17) and Hemiptera (16) were the most abundant there. 35 species were found in the IPM-orchard and 26 species – in the CPM-orchard. The codling moth, Cydia pomonella, is the main pest of apple in Bulgaria. This species has two generations per year and causes damage to fruits from May till harvest time. The population density of codling moth was permanently extremely high in all orchards and it was necessary to control it using insecticides and other plant protection tactics as well. Other pests with a high population density in the BPM-orchard were the apple sawfly Hoplocampa testudinea, tortricid-moths, the weevils Phyllobius oblongus, Rhynchites bacchus and R. aequatus, the pear lace bug Stephanitis pyri, the apple clearwing Synanthedon myopaeformis and the leopard moth Zeuzera pyrina. The populations of the chafer Epicometis hirta and leaf-eating caterpillars increased occasionally. Usually one or another species of leaf-eating caterpillars prevailed for a couple of years. In the years 1996-1998 it was gypsy moth Lymantria dispar, in the years 1998-2000 brown-tail moth Euproctis chrysorrhoea, in the years 2001-2003 the lackey moth Malacosoma neustria, and in the years 2003-2004 small ermine moth Yponomeuta padellus. A tendency to a slow increase of the population of Orthosia sp. was observed in the last years. In the BPM-orchard a natural regulation of apple blossom weevil Anthonomus pomorum, scale insects, aphids and leafminers was satisfactory, although sometimes their densities increased. No significant economic losses from other insect pests were noted. In the IPM-orchard the populations of apple sawfly, aphids, leafminers, leopard moth and apple clearwing increased occasionally. A high population density of leafminers was observed in CPM-orchard, in addition to codling moth. An increase in the population densities of aphids, Epicometis hirta, as well as of leopard moth or apple clearwing was periodically observed. The highest population density and biodiversity of beneficial insects was noted in the BPM-orchard (Table 3). A total of 30 species of predators were found, belonging to 4 orders and 7 families. The ladybirds presented the highest population density and played a significant role in natural regulation of aphids, scale insects and spider mites. Thirteen species of them were identified. The most common was the 7-spot ladybird, Coccinella septempunctata. Other species with high density were the 14-spot ladybird Propylea quattuordecimpunctata, 2-spot ladybird Adalia bipunctata and Stethorus punctillum. The green lacewings (order Neuroptera) were other significant predators. The species Chrysopa carnea was at high density every year. In the BPM-orchard seven species of predatory bugs were recorded, but at a low density. Larvae of predatory flies (order Diptera) – gall midge Aphidoletes aphidimyza and some hover-flies (family Syrphidae) were found at high density in colonies of aphids every year. Important natural regulators were also parasitoids from the order Hymenoptera (Table 4). They referred to 7 families and controlled aphids, scale insects, leafminers, tortricids and apple blossom weevil Anthonomus pomorum. The exact number of these species is still unknown. The beneficial insects in the BPM-orchard were not able to control efficiently either codling moth, leopard moth, pear lace bug or the weevils R. bacchus and R. aequatus. The leaf-eating caterpillars and Epicometis hirta appeared out of control in some years too.

Table 2. Pest insects in apple orchards in Plovdiv region, grown under different management systems, occurring in the years 1996-2004.

Order / Pest management Family Species Suborder biological integrated chemical Hemiptera / Aphididae Aphis pomi De Geer *(*) (*) (*) Homoptera Dysaphis mali Ferr. **(*) *(*) *(*) Dysaphis devecta Walk. (*) (*) *(*) Pemphigidae Eriosoma lanigerum Hausm. * o o Diaspididae Diaspidiotus perniciosus Comst. ** * * Parlatoria oleae Colv. * * o Lepidosaphes ulmi L. * o o Coccidae Eulecanium mali Schr. * * o Psyllidae Psylla costalis Flor. ** * * Cicadellidae Cicadella viridis L. * * o Thyphlocyba rosae L. *(*) (*) (*) Empoasca sp. *(*) (*) (*) Membracidae Ceresa bubalus F. * o o Hemiptera / Tingidae Stephanitis pyri F. *** * o Heteroptera Miridae Lygus sp. * * o Pentatomidae Carpocoris sp. * * o Diptera Itonididae Dasyneura mali Kieff. **(*) ** * Coleoptera Curculionidae Anthonomus pomorum L. ** * * Phyllobius oblongus L. *** ** * Phyllobius argentatus L. * o o Attelabidae Rhynchites aequatus L. *** ** * Rhynchites bacchus L. *** * * Scarabaeidae Epicometis hirta Poda. **(*) ** ** Cerambycidae Tetrops preusta L. ** * * Lepidoptera Tortricidae Cydia pomonella L. *** *(**) *(**) Hedia nubiferana Haw. ** * * Pandemis sp. * * o Archips sp. * o o Lyonetiidae Leucoptera scitella Zell. * *(*) *(*) Lyonetia clerkella L. * * * Nepticulidae Stigmella malella Stt. **(*) *(*) *(*) Lithocolletidae Phyllonoricter corylifoliella Hb. **(*) *(*) *(*) Phyllonoricter blancardella F. **(*) *(*) *(*) Gelechiidae Recurvaria nanella Hb. ** * o Yponomeutidae Yponomeuta padellus L. * o o Lasiocampidae Malacosoma neustria L. (**) o o Lymantriidae Euproctis chrysorrhoea L. (***) (**) (*) Lymantria dispar L. (**) (*) o Noctuidae Orthosia sp. *(*) * o Cossidae Zeuzera pyrina L. *** **(*) *(*) Cossus cossus L. * * * Sesiidae Synanthedon myopaeformis Bork. *** ** ** Hymenoptera Tenthredinidae Hoplocampa testudinea Klug *** *(*) *(*) Legend to population density symbols: *** – high; ** – moderate; * – low; o - not present; (*) – species with fluctuating population density.

Table 3. Predatory insects in apple orchards in Plovdiv region, grown under different management systems, occurring in the years 1996-2004.

Order / Family Species Pest management Suborder Biological Integrated Chemical Hemiptera / Nabidae Nabis ferus L. ** * o Heteroptera Nabis feroides Rem. * o o Anthocoridae Anthocoris nemorum L. ** * * Anthocoris nemoralis F. * o o Orius niger Wolff. * * o Deraeocoris ruber L. * * o Atractotomus mali Fieb. * * * Diptera Syrphidae Scaeva sp. ** ** * Syrphus sp. ** ** o Episyrphus sp. *** ** * Cecidomyiidae Aphidoletes aphidimyza Rond. *** *** * Coleoptera Coccinellidae Coccinella septempunctata L. *** * o Stethorus punctillum Ws. *** *** * Propylaea quadrodecimpunctata L. *** ** * Adalia bipunctata L. *** ** * Adonia variegata Gz ** ** o Syncharmonia conglobata L. ** * * Calvia quadrodecimgutatta L. ** ** * Calvia decimgutata L. * * o Scymnus frontalis F. * ** * Scymnus subvilosus Gz. * * * Thea vigintiduopunctata L. * * * Hipodamia tredecimpunctata L. * o o Chilocorus bipustulatus L. * * o Neuroptera Chrysopidae Chrysopa carnea Stef. *** *** ** Chrysopa perla L. ** * o Chrysopa septempunctata Wesm. * o o Chrysopa formosa Br. * * o Chrysopa prasina Burm. * * o Hemerobiidae Hemerobius humulinus L. * * o Legend to population density symbols: *** - high; ** - moderate; * - low; o - not present.

The population density of beneficial insects was lower in the IPM-orchard, but their importance as natural regulators of aphids and scale insects was still significant. The ladybirds were the most common again, but the prevalent species was Stethorus punctillum. Other predatory species with higher population density were gall midge, hover-flies, green lacewing Chrysopa carnea and parasitoids. In the CPM-orchard beneficial insects were found occasionally. Adults of Chrysopa carnea and ladybirds were the most common. Occurrence of some parasitoids and parasitised aphids or scale insects was noted.

Conclusions

More than 40 pest insects and a great number of their parasitoids and predators occur in apple orchards grown under biological pest management in Bulgaria. The organic farming causes excellent premises for protection of biodiversity in the orchards and for realising the process of natural regulation of the pests. The beneficial insects in a BPM-orchard are not able to 18

control efficiently the codling moth, the leopard moth, the pear lace bug or the weevils R. bacchus and R. aequatus. The populations of aphids, leafminers, chafer E. hirta and leaf- eating caterpillars may increase unexpectedly.

Table 4. Insect parasitoids from the order Hymenoptera in apple orchards in Plovdiv region, grown under different management systems, occurring in the years 1996-2004.

Family Species Hosts Pest management biological integrated chemical Ichneumonidae Scambus pomorum Ratz. A. pomorum *** ** * Pimpla sp. Tortricidae *** * o Braconidae Ascogaster quadridentatus Wesm. Tortricidae ** * o Microdus sp. Tortricidae ** * o leafminers; Apanteles sp. leaf-eating ** * * caterpillars Pteromalidae Dibrachis sp. Tortricidae ** ** * Eulophidae Chrysocharis sp. leafminers ** * * Pediobius sp. leafminers *** * * Sympiesis sp. leafminers ** ** * Achrysocharella sp. leafminers * * o Aphelinidae Aphelinus mali Hald. E. lanigerum ** * o Aphytis sp. Diaspididae *** ** * Prospaltella sp. Diaspididae *** ** * Encyrtidae Blastotrix confusa Erd. Coccidae *** * o Aphidiidae Aphidius sp. Aphididae ** ** * Lysiphlebus fabarum Marsh. Aphididae ** ** * Praon sp. Aphididae *** *** * Legend to population density symbols: *** – high; ** – moderate; * – low; o – not present.

Numerous species of pests and beneficial insects may be present in IPM-orchards. The natural regulators may support the insecticide treatments for control of harmful insects as aphids, leafminers, apple sawfly and other pests. The high number of insecticide treatments in the CPM-orchards reduces the number of pest and beneficial insects. The absence of natural regulators, may result in an increase of the population density of leafminers, aphids and other pests.

References

Angelova, R., Babrikova, T., Lecheva, I., Andreev, R. & Dimitrov, Y. 1995. Influence of different plant protection systems on entomo- and acarofauna in apple orchards. – Scientific Works of Higher Institute of Agriculture, Plovdiv, Bulgaria 40 (3): 213-217. Babrikova, T., Angelova, R., Lecheva, I., Andreev, R. & Dimitrov, Y. 1995. Study on population dynamics of predatory species in apple orchard. – Scientific Works of Higher Institute of Agriculture, Plovdiv, Bulgaria 40 (3): 231-234. Karov, S. & Andreev, R. 2000. Plant protection in organic and integrated gardening. – Agro- Ecological Center, Higher Institute of Agriculture, Plovdiv, Bulgaria: 151 pp. Lecheva, I., Babrikova, T., Dimitrov, Y. & Andreev, R. 1995. Predatory ladybirds (Coccinell- idae, Coleoptera) inhabit apple and pear agrocenoses. – Scientific Works of Higher Institute of Agriculture, Plovdiv, Bulgaria 40 (3): 235-239. 19

Mikhailova, P., Straka, F. & Apostolov, I. 1982. Plant protective prognosis and signalization. – Sofia, Zemizdat.: 342 pp. Pelov, V., Angelova, R., Karov, S., Nikolova, G., Borovinova, M., Balinova, A., Mavrodiev, S., Velcheva, N., Makariev, Z., Dzhuvinov, V., Doichev, K., Stoilov, G., Stamatov, I., Zelev, I., Velkov, L., Ivanov, S., Simova, S., Radeva, K. & Strelkova, D. 1996. General principles, guidelines and standards for integrated production of apple and pear. – National Service of Plant Protection, Quarantine and Agrochemistry. Sofia, Bulgaria: 78 pp. Zaharieva, T., Krasteva, H., Grigorov, P., Nikolov, N., Atanasov, N. & Valkov, G., 1997. Economic injury levels for the main pests on agricultural crops. – National Service of Plant Protection, Quarantine and Agrochemistry. Sofia, Bulgari: 23 pp.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 21-32

Building a selectivity list of plant protection products on beneficial arthropods in open field: a clear example with potato crop

Hautier, L.1, Jansen, J-P.1, Mabon, N.2, Schiffers, B.2

1 Department of Biological Control and Plant Genetic Resources, Walloon Centre of Agricultural Research, Chemin de Liroux, 2, 5030 Gembloux, Belgium 2 Analytical Chemistry and Phytopharmacy Unit, Agricultural Faculty of Gembloux, Passage des Déportés, 2, 5030 Gembloux, Belgium

Abstract: In order to promote IPM and the use of selective pesticides in open fields, a program was initiated to provide a selectivity list to pesticide users. The first approach was with potato crop, because of intensive use of pesticides and interest of IPM in this crop in Belgium. For this, the following beneficial arthropods species were selected: Aphidius rhopalosiphi (De Stefani-Perez) (Hym.; Aphidiidae), representative of parasitic Hymenoptera, Adalia bipunctata L. (Col.; Coccinellidae) and Episyrphus balteatus (De Geer) (Dipt.; Syrphidae), both representative of leaf dwelling predators. These are all aphid specific enemies, the main pest problem in potato in Belgium. The toxicity of 20 fungicides and 12 insecticides used in potato during the period of potential exposure of these beneficials were assessed on these species according to methods previously developed. The tests included a glass plate test on inert surface according to IOBC standard and an extended-lab test on natural substrate (barley seedlings for A. rhopalosiphi and French bean seedlings for E. balteatus and A. bipunctata). The spray apparatus was calibrated to deliver a pesticide residue deposit similar to a field application. A chemical dosage of residue was realized at each test on natural substrate to validate the application and follow pesticide degradation during exposure. According to results of both tests, products were rated as “Green” (harmless), “Yellow” (slightly harmful), “Orange” (moderately harmful) and “Red” (harmful). List were build-up according to toxicity results of the products and split in 4 periods of use depending on, aphid natural enemies presence and their importance in the field: period one (until 10 June) and four (after 31 July), no or limited, period 2 (10-30 June) exposure of aphid parasites and period 3 (July), exposure of leaf dwelling predators. These periods were based on field observations of aphids and natural enemies carried out since 1994 in the context of potato pest advisory systems. A first list was compiled and distributed to farmers in 2004 and updated in 2005 with new compounds. The results show that it is currently possible to combine throughout the growing season an effective plant protection program with pesticides that are selective to main aphid natural enemies.

Keywords: Selectivity list, Aphidiidae, ladybird, hoverfly, Aphidius rhopalosiphi, Adalia bipunctata, Episyrphus balteatus, potato, insecticide, fungicide, potato aphids, plant protection products.

Introduction

The use of non-selective pesticides towards beneficial arthropods can have serious consequences on the efficiency of biological pest control. Parasites and predators suppression can lead to pest outbreak and an increase of the insecticides treatments (Ripper, 1956; Pimentel, 1961; Besemer, 1964; Vickerman & Sunderland, 1977; Shires, 1985; Borgemeister & Poehling, 1989; Croft & Slone, 1998).

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Resurgence of secondary pests can also be the consequence of the suppression of beneficial arthropods by non-selective insecticides, herbicides or fungicides (Adams & Drew, 1965; Nanne & Radcliffe, 1971; Brown, 1978; Sotherton et al., 1987; Sotherton & Moreby, 1988; Lagnaoui & Radcliffe, 1998). As a result, non-selective pesticides can lead to a multiplication of pesticides treatments, an increase of production cost and finally a negative impact on health and the environment. In the context of sustainable agriculture and implementation of integrated production systems, the use of selective pesticides towards pests natural enemies becomes necessary. Moreover, it’s required for agricultural specifications and certification standards such as EUREPGAP, PERFECT and GIQF. Aphids are the main insect pest problems encountered in ware potatoes. However, they are most of the time regulated by natural enemies, such as parasitic Hymenoptera, mainly Aphidiidae and aphidophagous predators, such as hoverflies, ladybirds and to a lesser extent lacewings (Jansen, 2000; Jansen & Warnier, 2004). On basis of 1994-2005 observations on more than 200 commercial potato fields, the economic threshold value for aphids has only be reached in only more or less 1 field out of 8 (Jansen, 2005a). On the other fields, aphid populations were naturally controlled by beneficial arthropods. In this context, the use of selective pesticides during aphid natural enemy activity period is of particular importance because the disruption of aphid natural control lead to severe aphid outbreaks and increase of insecticide use. The aim of this research was to assess toxicity of pesticides currently used in potato towards natural enemies of aphids and to provide information to the farmers with help of selectivity lists. These lists can easily be integrated to IPM and potato inputs reduction programs. They can bring an additional support to aphids and potato blight advisory systems and allow a potato qualitative integrated production.

Material and methods

Selectivity lists were derived from a pesticides acute toxicity towards selected natural enemies and coincidence between beneficial arthropods activity and pesticides application periods, according to normal agricultural practices and phenology observed in Belgium.

Beneficials tested and phenology According to the beneficial fauna encountered in potato and the main key pest, aphids, three aphid natural enemies were selected as representative species: - Aphidius rhopalosiphi De Stefani-Perez (Hym.; Aphidiidae) - Adalia bipunctata (L.) (Col.; Coccinellidae) - Episyrphus balteatus (De Geer.) (Dipt ; Syrphidae) A. rhopalosiphi is not encountered in potato but is the representative species for Aphidiidae and parasitic Hymenoptera in the context of product registration at European level. This species is more sensitive to pesticides than Aphidius ervi and Aphidius picipes (Maise et al., 1997) that are the main species in potato (Jansen, 2005b). A. bipunctata is one of the four ladybird species found in potato and probably the most sensitive one (Jansen, in press). E. balteatus is the commonest hoverfly species in potato with up to 80% of the syrphid populations (Jansen & Warnier, 2004). Phenology of beneficials met in potato fields was based on 162 field observations carried out between 1994 and 2002 in the context of aphid advisory system (Jansen, 2002).

Tested Products 20 fungicides, including products with 2 active ingredients and 12 insecticides were tested in their commercial forms. These products correspond to all registered products used in June and

July, when aphid natural enemies are active in the field, according to Belgian conditions. Insecticides were tested at the maximum recommended field rate on basis of a single application and fungicides were tested at 1.5 x recommended field rate for one application to take into account possible multiple applications of the products at short intervals. Tested products and doses are listed in table 1.

Application of products The toxicity of each product towards beneficial arthropods was assessed according to SETAC guidelines (Barrett et al., 1994) and an original methodology developed by Copin et al. (2001). This original method is based on comparison of pesticide residue deposits on plants treated in the field with spray ramps (Azo 110) and laboratory aerograph Caussin sprayer. The laboratory sprayer was calibrated to have a more or less similar pesticide residue repartition as in the field, with part of the plants, as underside of leaves and basis of the plants receiving less pesticides than upper side of leaves and top of the plant. Spray volume was 200 l/ha ± 10%. All applications of plants were validated by pesticide residue dosage for the repartition and gravimetry.

Testing scheme The acute toxicity was assessed according to a sequential testing scheme, as developed by the IOBC working group “Pesticides and Beneficial Organisms”. The pesticide toxicity was evaluated first on an inert substrate (glass plates, A test). If the product induced a mortality higher than 30%, toxicity assessment was continued on a natural substrate (French bean for Syrphidae and Coccinellidae, barley for Aphidiidae, B test). According to the beneficial arthropods corrected mortality (Mc) observed after 48 h (glass tests and parasitoid tests on plants) or after 72 h (predators on plants), agrochemicals were classified in 4 categories: 1 - harmless product : Mc ≤ 30 % on glass or on plant 2 - slightly harmful product : 30% < Mc ≤ 60% on plant 3 - moderately harmful product : 60 % < Mc ≤ 80% on plant 4 - harmful product : Mc > 80% on plant The limits of each category were selected to get a good discrimination of products in the final lists and to label red products that were known before to make problems in the field (pest resurgence, secondary pest outbreaks, ....).

Selectivity list building The selectivity list will be build by a combination of toxicity test results and classification and insect phenology. Potato vegetation periods will be defined according to phenology of the beneficials and product classification will be done by period, according to final toxicity test results for the main beneficial encountered at this period. For periods when two species are of equal importance (by example ladybirds and hoverflies in July), the geometric mean of both corrected mortality test will be retained for product classification.

Toxicity tests A. rhopalosiphi was exposed to fresh pesticide residues applied to glass plates and to barley seedlings in a similar way to that described by Mead-Briggs (Mead-Briggs & Longley, 1997; Mead-Briggs et al., 2000) except minor changes. For glass plates, there were 5 units of 10 wasps per product and for control instead of 4 x10. On plants, these were 10 x10 wasps per product instead of 5x5 females. Sugar solution was substituted by aphids with more than 100 aphids added to the plant 24h before spraying. Mortalities were assessed after 48h for both tests. A. bipunctata exposure units were made of a circular glass plate (∅ 5cm) treated with pesticide and covered with a plastic ring coated with Fluon GP1 to prevent ladybird escape.

There were a total of 30 larvae/product and 30 larvae for control. For E. balteatus, two treated glass plates were used to form the ceiling and the floor of exposure units, with the treated faces turned inside the units. A plastic ring was inserted between the two glass plates and the units were connected to a pump to renew the air. There were 20 larvae per product and 20 larvae for control at each test. 2-3 day old hoverfly and ladybird larvae were used for the test. They were fed ad lib with pea aphids (Acyrtosiphon pisum). Mortalities were recorded after 48h of exposure.

Table 1: List of tested products. Commercial name, formulation type, active(s) ingredient(s) and tested dose.

active ingredient tested rate Commercial name Formulation (g a.s./ha)

GALBEN M WP benalaxyl + mancozeb 300 + 2438 CLORTOSIP SC chlorothalonil 2250 TATTOO C SC chlorothalonil + propamocarb 1519 + 1519 RANMAN SC cyazofamid 120 CURZATE M WP cymoxanil + mancozeb 135 + 1950 AVISO WG cymoxanil + metiram 216 + 2880 TANOS WG cymoxanil + famoxadone 225 + 225 ACROBAT EXTRA WG dimetomorph + mancozeb 225 + 2513 SERENO WG fenamidone + mancozeb 225 + 1125 SHIRLAN SC fluazinam 300 KOCIDE WG copper hydroxid 2400

Fungicide DEQUIMAN MZ WG mancozeb 4800 UNIKAT PRO WG mancozeb + zoxamide 1801 + 224 TRICARBAMIX EXTRA WG maneb 4800 EPOK 600 EC metalaxyl-m + fluazinam 150 + 300 RIDOMIL GOLD SPECIAL 68 WP metalaxyl-m + mancozeb 150 + 2400 CUPRAVIT FORTE WP copper oxychlorid 3750 ANTRACOL WP propineb 3150 CUPRO-ANTRACOL WP propineb + copper oxychlorid 2775 + 1313 MACC80 Bo. BORDELAISE WP copper sulfate 3750 FASTAC EC alpha-cypermethrin 12.5 CARBISAN WP carbaryl 768 CYMTOP 100 EC cypermethrin 25 DECIS 2,5 EC deltamethrin 7.5 HERMOOTROX EC dimethoate 200 SUMI ALPHA EC esfenvalerate 7.5 KARATE ZEON SC lambda-cyhalothrin 7.5 lambda-cyhalothrin + Insecticide OKAPI EC pirimicarb 7.5 + 150 ZOLONE FLO SC phosalone 750 PIRIMOR WG pirimicarb 200 PLENUM WG pymetrozin 150 FURY 10 EW zeta-cypermethrin 10

Exposure of ladybird and hoverfly larvae to pesticides on plants was made with the same exposure units. Approximately after one week of emergence, young French bean plants were pinched out in order to keep approximately the two first leaves and 7-10 cm of stem, to produce a kind of “standardised” plant. The plants were thus treated and two larvae and aphids were added to the plants when the pesticide residue had dried. The plants were grown on ∅ 9 cm seedlings pots, the substrate was covered with sand and a plastic device with the inner walls coated with fluon was inserted around the French bean. With this device, if the larvae fell from the plant, they were unable to escape and had to stay on the sand or to climb on the plants where the aphids were. For ladybird and hoverfly, there were 2 larvae /plant and 15 plants treated and 15 for control by product. Mortality was recorded after 72h of exposure. All test organisms used for the test were produced by the mass rearing of the laboratory. The three rearings were initiated with organisms collected at field margins and hedges, in an agricultural landscape. Aphidius rearing started in 1994, Adalia in 1995 and Episyrphus in 1996. New organisms were collected yearly to replenish the rearing. Observed mortalities were corrected with control mortality according to Abbott (Abbott, 1925). The validity criteria for acceptance of the results was control mortality below 10% on glass plates and 13% on plants.

Results and discussion

Beneficial phenology and pesticides periods application In potato, aphid parasitoids and predators (ladybirds and syrphids) are the main beneficial insects controlling aphid populations (Jansen, 2000 ; Jansen, 2002). The phenology of these beneficial arthropods, based on field observations between 1994 and 2002, is illustrated in Figure 1. Hymenoptera Aphidiidae were the first active aphids enemies arriving in the field at the same time as the first aphids. The presence of winged parasitised aphids at the beginning of the season is very common, indicating that they arrived in the crop in a same time as aphids. The action of parasitic wasps is clearly very important at the beginning of the aphid infestation period and parasitic wasps are the key beneficial for aphid control in June. Ladybirds and hoverflies generally arrive later. The first eggs were detected around the end of June when aphid populations were sufficient to allow the offspring development These aphidophagous insects remain in the field until aphid populations decline, from July 15 till the end of the month. Their action is curative and a rapid decline of aphid populations is generally observed at the middle of June, when aphid specific predators populations are at their maximum. On the basis of beneficial insects phenology and the periods of application of plant protection products, the farming year was split in four periods, each corresponding to a particular situation: – Period I (... – 10 June): Before aphid and the first beneficial arrival into the field, no restrictions concerning use of products towards their toxicity, except for products which would have a long duration of activity and interfered with other periods. Fungicides and herbicides are used during this period. – Period II (10 June – 30 June): Main activity period of parasitic Hymenoptera. Selectivity list based on Aphidius test results. This period is particularly concerned with fungicides, that are applied weekly during this period. Insecticides can sometimes be used, but only for particular situations. – Period III (1 July – 30 July): Peak aphid populations and the main activity period of aphid specific predators. Selectivity lists are based on A. bipunctata and E. balteatus toxicity results (geometric mean of both tests). Fungicides are routinely applied during this period

and, if needed, insecticides to control aphids. No Colorado beetle treatments are needed according to Belgian conditions. – Period IV (1 August – ...): No aphids and beneficials in the field, there are no restrictions concerning use of products towards their toxicity. Normally only fungicides are used during this period and herbicides at the end of the growing season for potato foliage desiccation before harvesting.

ul g une J y y y y l l u l l

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J J J J 03 J 01 5 A

8 J 9 J 0 - 15 22 J 0 2 e - y 10 17 24 - - - - - a y un

4 - 1 - 8 - 02 23 ul 09 16 M 0 1 1

5 J

0 J 28 2

Aphids

Parasites

Syrphids

Coccinellids

: 1 / 9 years : 2 / 9 years : 3 / 9 years m ore than 4 / 9 years Figure 1. Frequency of aphids and aphid natural enemies arrival in potato in Belgian conditions (1994 to 2002).

Acute toxicity assessment Results of glass plates tests (A test), extended lab-test (B test) and pesticide classification are listed in Table 2 (Aphidius test) and 3 (Adalia and Episyrphus tests). Results with A. rhopalosiphi showed that all fungicides registered in June were harmless on plants, even if several were toxic on glass plates. With insecticides, results were contrasted but finally, several insecticides were rated as harmless: cypermethrin, esfenvalerate, lambda- cyhalothrin, pirimicarb, pymetrozin, zeta-cypermethrin and mixture lambda-cyhalothrin + pirimicarb. Pymetrozin was harmless even on glass plates. The others were still either slightly harmful (alpha-cypermethrin, carbaryl and phosalone), moderately harmful (deltamethrin) or harmful (dimethoate). There were sometimes great differences between pyrethroids, that are unexplained. The formulation type (EW against EC) and the recommended field dose (25g a.i./ha for cypermethrin but only 10 or 12.5 g a.i./ha for zeta and alpha-cypermethrin) could probably have had an influence on toxicity. All fungicides were harmless for A. bipunctata except the mixture metalaxyl-M + fluazinam which was slightly harmful. For insecticides, only pirimicarb and pymetrozin were selective towards A. bipunctata; all the others were very toxic with mortality on plant higher than 80 % and often equal to 100%. E. balteatus was not affected by fungicides used in potato. With insecticides, three products (alpha-cypermethrin, lambda-cyhalothrin and pymetrozin) were harmless on glass plates and two other ones, carbaryl and esfenvalerate, were harmless on plants. The others were slightly harmful as phosalone, moderately harmful (cypermethrin and deltamethrin), or

harmful as dimethoate, pirimicarb and lambda-cyhalothrin + pirimicarb. If results obtained on A. bipunctata and E. balteatus are combined, pymetrozin was the only harmless product for both aphid predators. The other products were slighty harmful or totally harmful for at least one of the two species. Dimethoate, cypermethrin, deltamethrin and the mixture lambda- cyhalothrin + pirimicarb, with one compound highly toxic for ladybird and the other one toxic for hoverfly, were the most toxic insecticides.

Table 2: Result of toxicity test with A. rhopalosiphi, corrected mortality (A=glass plate, B=extended lab) and selectivity class: 1–harmless, 2-slightly harmful, 3-moderately harmful and 4-harmful.

Aphidius test

A B Final Benalaxyl + Mancozeb 0% – 1 Chlorothalonil 10% – 1 Chlorothalonil + Propamocarb 68% 1% 1 Copper hydroxide 48% 23% 1 Copper oxychlorid 20% – 1 Copper sulfat 0% – 1 Cyazofamide 32% 0% 1 Cymoxanil + famoxadone 0% – 1 Cymoxanil + Mancozeb 4% – 1 Cymoxanil + Metiram 23% – 1 Dimetomorph + Mancozeb 2% – 1 Fungicide Fluazinam 8% – 1 Mancozeb 6% – 1 Mancozeb + Zoxamide 0% – 1 Maneb 0% – 1 Metalaxyl-M + Fluazinam 6% – 1 Metalaxyl-M + Mancozeb 45% 4% 1 Propineb 0% – 1 Propineb + Copper oxychlorid 0% – 1 Alpha-cypermethrin 100% 38% 2 Carbaryl 100% 51% 2 Cypermethrin 100% 22% 1 Deltamethrin 100% 80% 3 Dimethoate 100% 100% 4 Esfenvalerate 91% 6% 1 Lambdacyhalothrin 100% 1% 1

Insecticide Lambdacyhalothrin + Pirimicarb 100% 3% 1 Phosalone 68% 50% 2 Pirimicarb 100% 12% 1 Pymetrozin 4% – 1 Zeta-cypermethrin 100% 3% 1

Table 3: Result of toxicity test with A. bipunctata and E. balteatus, corrected mortality (A=glass plate, B=extended lab) and selectivity class: 1–harmless, 2-slightly harmful, 3-moderately harmful and 4-harmful.

Adalia test Episyrphus test Final

A B A B class Benalaxyl + Mancozeb 0% – 0% – 1 Chlorothalonil 0% – 21% – 1 Chlorothalonil + Propamocarb 0% – 0% – 1 Copper hydroxide 3% – 0% – 1 Copper oxychlorid 0% – 0% – 1 Copper sulphate 10% – 0% – 1 Cyazofamide 7% – 0% – 1 Cymoxanil + Famoxadone 0% – 0% – 1 Cymoxanil + Mancozeb 0% – 0% – 1 Cymoxanil + Metiram 10% – 5% – 1 Dimetomorph + Mancozeb 0% – 17% – 1

Fungicide Fenamidone + mancozeb 40% 0% 26% – 1 Fluazinam 100% 20% 0% – 1 Mancozeb 3% – 0% – 1 Mancozeb + Zoxamide 14% – 11% – 1 Maneb 0% – 16% – 1 Metalaxyl-M + Fluazinam 86% 47% 0% – 1 Metalaxyl-M + Mancozeb 33% 0% 45% – 1 Propineb 10% 0% 0% 15% 1 Propineb + Copper oxychlorid 20% – 0% – 1 Alpha-cypermethrin 100% 100% 16% – 2 Carbaryl 100% 100% 100% 0% 2 Cypermethrin 100% 100% 85% 80% 4 Deltamethrin 100% 100% 75% 63% 4 Dimethoate 100% 100% 100% 100% 4 Esfenvalerate 100% 100% 47% 13% 2 Lambdacyhalothrin 100% 100% 0% – 2

Insecticide Lambdacyhalothrin + Pirimicarb 100% 100% 100% 100% 4 Phosalone 96% 100% 63% 56% 3 Pirimicarb 21% – 80% 94% 2 Pymetrozin 0% – 0% – 1 Zeta-cypermethrin 100% 100% 65% 0% 2

Selectivity lists According to the toxicity test results, beneficial arthropods phenology and normal use of the products, 4 selectivity lists, each corresponding to a different period, were built-up (Table 4). These lists were also printed and distributed to farmers and potato industry, with a colour key for toxicity, from green (harmless) to red (harmful) with yellow and orange category. Examination of the lists indicates that fungicides actually used in Belgium are not a problem for aphid natural enemies and do not interfere with aphid natural control. However, as fungicides are widely used throughout the growing season, a great attention must be given to this aspect in the future for new compounds. With insecticides, the situation is not so easy, but several products can be listed as harmless or slightly harmful, at least during some

periods. Pymetrozin has a very good selectivity for both parasitic wasp and aphid specific predators. This new compound is very interesting in the context of IPM, compared to other products. Dimethoate was particularly toxic to beneficials and its use must be avoided at any time. The other compounds had variable effects on the different tested species. Some of them

Table 4: Selectivity lists of products used in potato according to their toxicity towards main aphid natural enemies. 1 – harmless, 2 – slightly harmful, 3 – moderately harmful, 4 – harmful, X – not registered at this period.

Periods I ( -10/06) II (10-30/06) III (1-31/07) IV (1/08-..) No exposure Aphidius tests Episyrphus + No exposure Adalia tests Benalaxyl + Mancozeb 1 1 1 1 Chlorothalonil 1 1 1 1 Chlorothalonil + Propamocarb 1 1 1 1 Copper hydroxide 1 1 1 1 Copper oxychlorid 1 1 1 1 Copper sulfate 1 1 1 1 Cyazofamide 1 1 1 1 Cymoxanil + Famoxadone 1 1 1 1 Cymoxanil + Mancozeb 1 1 1 1 Cymoxanil + Metiram 1 1 1 1 Dimetomorph + Mancozeb 1 1 1 1

Fungicides Fenamidone+ Mancozeb 1 X 1 1 Fluazinam 1 1 1 1 Mancozeb 1 1 1 1 Mancozeb + Zoxamide 1 1 1 1 Maneb 1 1 1 1 Metalaxyl-M + Fluazinam X 1 1 X Metalaxyl-M + Mancozeb X 1 1 X Propineb 1 1 1 1 Propineb + Copper oxychlorid 1 1 1 1 Alpha-cypermethrin – 2 2 – Carbaryl – 2 2 – Cypermethrin – 1 4 – Deltamethrin – 3 4 – Dimethoate – 4 4 – Esfenvalerate – 1 2 – Lambda-Cyhalothrin – 1 2 –

Insecticide Lambda-cyhalothrin + Pirimicarb – 1 4 – Phosalone – 2 3 – Pirimicarb – 1 2 – Pymetrozin – 1 1 – Zeta-cypermethrin – 1 2 –

were selective to parasitic wasps and can be used in June without restriction. In July, because of toxicity on ladybirds and/or hoverflies, use of several products, as cypermethrin, deltamethrin and the mixture lambda-cyhalothrin + pirimicarb, is not recommended. They were great differences in term of toxicity between cypermethrin and the two isomers alpha and zeta-cypermethrin. These differences are probably coming from a combination of tested dose, higher for cypermethrin than for isomers, formulation type and active ingredient toxicity itself.

Conclusions

The results obtained in this study show that it is possible to have a good pest and disease control with products that are selective towards main aphid natural enemies, during all the periods where these beneficial insects are active in the field. Fungicide applications for late blight control are not a problem for selectivity and it is possible, by avoiding the use of several insecticides at particular periods, to maintain aphid natural enemy activity. These selectivity lists can help the farmers to choose the product to spray; and they can also complete the information given by potato advisory systems for aphids control.

Acknowledgements

This study was funded by the Belgian ministry “SPF SANTE PUBLIQUE, Sécurité de la Chaîne alimentaire et Environnement, Direction générale Animaux, Végétaux et Alimentation, Division Matières premières et protection des végétaux”. We thank A.M. Warnier, S. Mahiat, C. Torrekens and J. Vase for their contribution to this study.

References

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Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 33-41

Influence of plant protection measures on the carabid fauna of sugar beet and potato fields in Poland

Katarzyna Nijak & Stefan Pruszyński Department of Ecology and Agricultural Enviromental Protection,Institute of Plant Protecion, Miczurina 20, 60-318 Poznań, Poland. E-mail: [email protected]

Abstract: Beetles belonging to the family Carabidae are surface-active arthropods found in both natural and anthropogenic modified environments. This group of insects is very often considered as bioindicator of changes in the environment. The aim of these experiments was to determine changes arising among fauna of useful arthropods in agrocenosis due to agrochemicals recommended in accordance with good agricultural practice. Field experiments were conducted on commercial fields of sugar beet and potato which were divided in two parts: one intensively protected and one an untreated control part. Barber traps were used for catching fauna during the growing season. The most numerous group of arthropods were beetles, mainly predators belonging to the family Carabidae. Seasonal changes in the quantity of insects showed similar trends on both treated and control fields. Comparison of carabid abundance was carried out with respect to weather conditions and plant protection product treatments. Results showed that chemical treatment did not always influence carabid abundance.

Key words: Carabidae, epigeal arthropods, plant protection, Barber traps

Introduction Chemical plant protection can influence the agricultural environment. Plant protection product development, in terms of selectivity, degradation and application technique can influence the impact on the non-target predatory fauna (Häni et al. 1998). In the 1970’s Professor W. Węgorek started, in the Institute of Plant Protection Field Experimental Station at Winna Góra, investigations concerning the influence of chemical plant protection on the yield of agriculture crops and different elements of the environment (Węgorek et al. 1982, Węgorek et al 1990). The results in this paper are a continuation of these studies at the Plant Protection Institute. Beetles from the family Carabidae are ground-active arthropods inhabiting both natural and anthropogenic modified environments. That group of insects is often called bioindicators of changes in environment because analysis of their occurrence and the species structure of carabid populations can be used as an indicator of environmental degradation (Kabacik 1962, Puszkar 1980, Trojanowski 1983). Presented results are from an investigation concerning the side-effects of chemical plant protection products applied in sugar beet and potato production. The aim of the experiments was to determine changes arising among the fauna of useful arthropods in case of using agrochemicals recommended for the crop protection. Presented results are a part of large four year long rotation experiment.

Material and methods

The investigations were carried out in Winna Góra, 60 km of south east of Poznań, in Poland during 2000 and 2004, on blocks of untreated and treated fields. One combination used 0.45 ha field area. Fields were in an experimental design as shown in Table 1. On treated fields a

33 34

plant protection program was applied according to the Plant Protection Institute recommendation (Zalecenia IOR 2003). Between treated and untreated blocks was an insulation area 21 m wide.

Table 1. Experimental design

Combination Potato Sugar beet 2000 2004 2000 2004 Treated 2 herbicides 4 herbicides 4 herbicides 3 herbicides 3 insecticides 2 insecticides 2 insecticides 4 insecticides 4 fungicides 3 fungicides 1 fungicides Untreated ------

The investigations of predatory ground beetles and other arthropods were performed using Barber’s pitfall traps filled with a solution of ethylene glycol. On each field 10 traps were placed in two rows of five. The distance between adjacent traps was 15 m. (Fig.1). Traps were checked and re-set every week. Collected arthropods were then identified in laboratory according to Reitter (1908) and Hurka (1996). The same cultivation was performed on both treated and untreated fields according to good agricultural practice. The plant protection program applied on treated fields is presented in Tables 2-5.

potato sugar beet potato sugar beet

100m

45m 21m 45m 21m 45m 45m

Treated fields block Untreated fields block

Fig. 1. Layout of Barber’s pitfall traps in experimental fields.

On untreated (control) fields no agrochemicals were used. In 2000, 780 samples were collected from Barber traps – 400 from sugar beet fields and 380 from potato fields. In 2004 the number of samples collected was 600, of which 360 were from sugar beet and 240 from potato fields. The investigations were carried out from May to September in each of the two years. In Winna Góra the soil is a sandy loam, pH 5.6, with humus 1.4%. On experimental area 4 years rotation was used according to scheme in Table 6. 35

Table 2. Plant protection products applied in sugar beet protection in 2000.

Treatment Active ingredients in % Rate kg Name of product date or g/L (L)/ha fenmedipham 60 desmedipham 60 24.04. Betanal Quattro 380 SE (H) 1.50 L ethofumesate 60 metamitron 200 fenmedipham 60 desmedipham 60 Betanal Quattro 380 SE (H) 1.50 L ethofumesate 60 29.04. metamitron 200 fenmedipham 60 desmedipham 60 09.05. Betanal Quattro 380 SE (H) 1.50 L ethofumesate 60 metamitron 200 11.05. Fusilade Super 125 EC (H) fluazifop-P-butyl 125 2.75 L

15.05. Bi 58 Nowy (I) dimethoate 400 0.8 L

14.07. Bi 58 Nowy (I) dimethoate 400 0.8 L

Table 2-54: F – fungicides I – insecticides H – herbicide

Table 3. Plant protection products applied in potato protection in 2000.

Treatment Active ingredients in % or Rate kg Name of product date g/L (L)/ha 17.05. Titus 25 WG (H) rimsulfuron 25 45 g

17.05. Sencor 70 WG (H) metribuzin 70 0.3 kg alpha- 30.05. Alfazot 050 EC (I) 50 0.25 L cypermethrin chloropyrifos 500 16.06. Nurelle D 550 EC (I) 0.50 L cypermethrin 50 14.07. Fury 100 EC (I) zeta-cypermethrin 100 0.1 L

30.05. Bravo 500 S.C. (F) chlorothalonil 500 2.0 L oxadixyl 8 16.06. Sandofan Manco 64 WP (F) 2.0 kg mancozeb 56 14.07. Clortosip 500 SC (F) chlorothalonil 500 2.0 L cymoxanil 4.5 01.08. Curzate M 75,2 WP (F) 2.4 kg mancozeb 68 36

Table 4. Plant protection products applied in sugar beet protection in 2004.

Treatment Active ingredients in % or Rate kg Name of product date g/L (L)/ha fenmedipham 60 desmedipham 60 27.04. Betanal Quattro 380 SE (H) 1.50 L ethofumesate 60 metamitron 200 fenmedipham 91 06.05. Betanal Progress 274 OF (H) desmedipham 71 1.0 L ethofumesate 112 fenmedipham 91 18.05. Betanal Elite 247 EC (H) desmedipham 71 1.0 L ethofumesate 112 03.06. BI 58 Nowy (I) dimethoate 400 0.8 L

14.06. Fury 100 EC (I) zeta-cypermethrin 100 0.1L chloropyrifos 500 15.06. Nurelle D 550 EC (I) 0.6 L cypermethrin 50 alpha- 24.06. Fastac 10 EC (I) 10 0.15 L cypermethrin flusilazole 125 27.08. Alert 375 SC (F) 1.0 L carbendazim 250

Table 5. Plant protection products applied in potato protection in 2004.

Treatment Name of product Active ingredients in % or g/L Rate kg (L)/ha date 27.05. Titus 25 WG (H) rimsulfuron 25 30 g 27.05. Sencor 70 WG (H) metribuzin 70 0.25 kg 31.05. Titus 25 WG (H) rimsulfuron 25 20 g 31.05. Senkor 70 WG (H) metribuzin 70 0.25 kg 01.07. Regent 200 SC (I) fipronil 200 100 ml cymoxanil 25 01.07. Tanos 50 WG (F) 0.5 kg famoxadone 25 cymoxanil 4.5 20.07. Curzate M. 72,5 WP (F) 2.0 kg mancozeb 68 21.07. Mospilan 20 SP (I) acetamiprid 20 0.15 kg cymoxanil 25 02.08. Tanos 50 WG (F) 0.75 kg famoxadone 25

Experimental fields were surrounded by many places where carabids can survive and which could provide refugia for recolonisation, such as woodland (800 m from field), bushes and rows with wild grasses and weeds. Obtained results were subjected to analysis of variance.

Results

In 2000, in untreated sugar beet fields, the total quantity of Carabidae was lower (2956 individuals) than in treated field (3272 individuals)(Table 9). Fewer Staphylinidae (14.57%) and Opiliones (7.8%) were recorded from treated fields. Beetles from other families and the representatives of other insects orders were collected in relatively low numbers and their abundance was not analyzed. Beetle catches from treated and untreated fields showed similar seasonal changes with the highest numbers being recorded on 25th May, 13th June and 3rd July (Figure 2). According to the observations made oin potato fields in 2000, the total number of Carabidae oin the treated field was significantly lower (1376) compared to untreated (1687) field (Fig. 3, Table 7 and 9).

Table 6. Scheme of four year rotation.

Rotation for potato Rotation four sugar beet Winter weat Winter rape Blue lupin Fodder pea Spring barley Corn

Table 7. The incidence of Carabidae in 2000 and statistical analysis.

Crops Mean per plot T student test LSD Potato treated field 137.2 A 0.026937 Potato untreated field 168.1 B Sugar beet treated field 347.5 A 0.447733 Sugar beet untreated field 328.7 A

Table 8. The incidence of Carabidae in 2004 and statistical analysis.

Crops Mean per plot T student test LSD Potato treated field 62.0 A 0.000552 Potato untreated field 37.2 B Sugar beet treated field 126.0 A 0.007582 Sugar beet untreated field 78.1 B

180

160

140

120

100

N. of individuals 60

5 .05 0 .06 .06 07 .08 08 09 3. 4. 4. 11 18. 25.05 01 08.06 15.06 21 29.06 06.07 1 20.07 27.07 03 10.08 17.08 2 31.08 07.09 1 21.09 Date

untraeted field treated field

Fig. 2. Seasonal changes in the number of Carabidae in sugar beet fields in 2000.

350

300

250

200

150 No. of individuals 100

5 .05 0 .06 .06 07 .08 08 09 3. 4. 4. 11 18. 25.05 01 08.06 15.06 21 29.06 06.07 1 20.07 27.07 03 10.08 17.08 2 31.08 07.09 1 21.09 Date

untreated field treated field

Fig. 3. Seasonal changes in the number of Carabidae in potato fields in 2000. 39

180

160

140

120

100

No. of individuals 60

. 5. 6. 8. 9. 0 06 0 .07. 0 .08. 0 .09. 7. 8. 6. 24.05. 31. 0 14.06. 21.06. 2 05.07. 12.07. 19.07. 26 02.08. 09.08. 1 23 30.08. 06. 13 20.09. Date

untreated field treated field

Fig. 4. Seasonal changes in the number of Carabidae in sugar beet fields in 2004.

120

100

No.of individuals 40

. . 5. 6. 8. 0 0 06 .06. 0 08 4. 9. 24.05. 31. 07. 1 21 28.06. 05.07. 12.07. 19.07. 26.07. 02. 0 Date

untreated field treated field

Fig. 5. Seasonal changes in the number of Carabidae in potato fields in 2004. 40

The number of Carabidae on treated and untreated sugar beet field was 50% more numerous than on potato fields in the comparable observation period in 2000 (Table 9). The fluctuation of beetles numbers during the season was comparable on both fields. The highest number of beetles was observed from 25th May to 8th June and from 8th July to 27th July (Fig. 2-3). After insecticide treatment with Alfazot 050 EC (30th May) and Nurelle D550EC (16th ) a decrease of beetle abundance was not noted. After Fury treatment (14th July) a decrease in beetles catch size was noted. However, a greater decrease occurred over the same period on the untreated control field. The occurrence of Carabidae in 2004 was lower then in 2000, with twice as many individuals on beet fields than on potato fields. Only in 2000 on the potato field, the number of caught Carabidae was higher on the untreated field. On remaining experimental fields (sugar beet 2000 treated, 2004 potato and sugar beet treated) Carabidae were more numerous in the treated than in the untreated plots. In our previous experiments the best measurement for the difference between beetles catches in the crops was the mean number of Carabidae caught per trap and checking date. Because of this we also discussed the occurrence of beetles by means of that measurement (Table 9). In 2000 on treated sugar beet field were 16.38 individuals per trap and checking date, and on untreated field 14.73, in 2004 were 6.7 and 4.8 individuals per trap and checking date, respectively. The notable increase of population number was observed in May and between July and August. The lowest occurrence of beetles was observed in June. This situation could be explained by the reproduction characteristic. The emergence (occurrence) of spring and autumn generation caused population fluctuation. But as considered the number of beetles in catch the weather conditions could influence that situation. The main reason for population decrease was probably cold nights in May 2004 and heavy rainfalls in July. On potato field in 2004 seed-potatoes were sparcely placed and additionally late germination decreased the amount of ground cover. It is probable that this led to reduced beetle number (Table 9).

Table 9. The occurrence of Carabidae in 2000 and 2004.

Number of individuals Per trap and checking date Crops Untreated field Treated field Untreated field Treated field 2000 2004 2000 2004 2000 2004 2000 2004 Potato 1687 392 1376 662 8.43 3.3 6.88 5.5

Sugar beet 2956 864 3272 1200 14.76 4.8 16.38 6.7

The numerous occurrences of insects on treated fields may have been due to immigration and movement between plots. Good plant health conditions may have caused better living conditions and breeding habitat for insects. Smaller weed incidence may also have caused better breeding conditions for carabid colonization. It is also possible that surrounding habitats were favorable for carabid colonization. The dominant species of Carabidae on investigated fields were Agonum assimile Payk., A. dorsale (=Platynus dorsalis)(Pont.), Bembidion properans Steph., Calathus ambiguus Payk., Carabus cancellatus Ill., C. granulatus (L.), C. hortensis (L.), Pterostichus caerulescens (L.), Poecilus cupreus (L.), P. vulgaris (L.). Most of these observed species are regarded indicators of agricultural intensity (Pałosz 1996). 41

Conclusions

The problem of side effects of crop protection treatments in agro-ecosystems is very important ( Jaworska, 1996 and 1997; Pałosz, 1995 and 1996). Our results suggest that modern chemical treatments had no significant influence on populations of Carabidae in potato and sugar beet fields. The agrochemicals use in potatoes and beet did not appear to lead long term reduction in numbers of carabid beetles. The situation could be influenced by the field architecture in Poland compared to other European countries, with narrow fields promoting rapid recovery from treatment due to immigration. It is very important that good agriculture practice and Plant Protection could positively influence not only on crop yield but also decreased risk for the agroecosystem environment.

References

Häni, F., Popow, G., Reinhard, H., Schwarz, A., Tanner, K. & Vorlet, M. 1998: Ochrona roślin w uprawie integrowanej. – PWRiL, Warszawa. Hurka, K. 1996. Carabidae of the Czech and Slovak Republics. – Kabourek, Zlin. Jaworska, T. 1996: Zgrupowanie biegaczowatych (Carabidae, Coleoptera) pszenicy ozimej i jarej odchwaszczanej herbicydami. – Progress in Plant Protection / Postępy w Ochronie Roślin 36 (2): 76-78. Jaworska, T. 1997: Wpływ odchwaszczania na dynamikę populacji biegaczowatych (Carab- idae, Coleoptera). – Progress in Plant Protection / Postępy w Ochronie Roślin 37 (2): 235-237. Kabacik, D. 1962: Beobachtungen über die Quantitatsveränderungen der Laufkäfer (Carab- idae) auf verschiedenen Feldkulturen. – Ekol. Pol. (A) 10 (12): 307-323. Pałosz, T. 1995: Skład gatunkowy biegaczowatych (Col., Carabidae) na plantacjach rzepaku ozimego o różnej technologii i intensywności uprawy. – Mat. 35 Sesji Nauk. Inst. Ochr. Rośl. Cz. I: 108-115. Pałosz, T. 1996: Skład gatunkowy biegaczowatych ( Col. Carabidae) na plantacji rzepaku ozimego w sezonie 1994/1995. – Progress in Plant Protection / Postępy w Ochronie Roślin 36 (2): 79-81. Puszkar, T. 1980: Zmiany wybranych elementów zoocenoz w agroekosystemach poddawanych silnej presji emisji przemysłowych. – IUNG Puławy R. 157. Reitter, E. 1908: Familie Carabidae. – Fauna Germanica. Die Käfer des Deutschen Reiches. T. 1: 71-201. Trojanowski, H. 1983: Zmiany środowiska naturalnego lasu „Ruda” pod wpływem emisji Zakładów Azotowych ze szczególnym uwzględnieniem biegaczowatych (Carabidae). – Prace Nauk. Inst. Ochr. Rośl. 25(2): 5-93. Węgorek, W., Dąbrowski, J., Trojanowski, H. & Rudny, R. 1982: Ekonomiczne i środowiskowe skutki intensywnego stosowania chemicznej ochrony roślin. – Materiały 22. i 23. Sesji Inst. Ochr. Roślin:11-40. Węgorek, W., Trojanowski, H., Dąbrowski, J. & Rudny, R. 1990: Wpływ intensywnego stosowania pestycydów na plony i wybrane elementy środowiska rolniczego. – Prace Nauk. Inst. Ochr. Roślin 32 (1/2): 117-128. Zalecenia Ochrony Roślin na lata 2004/05: Rośliny Rolnicze IOR (2).

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 43-52

How much precision does a regulatory field study need?

Kevin Brown1 and Mark Miles2 1 Ecotox Limited, Tavistock, Devon, PL19 0YU, UK 2 Dow AgroSciences, European Development Centre, 3 Milton Park, Abingdon, Oxon, OX14 4RN, U.K.

Abstract: Regulatory field studies with Plant Protection Products are designed to determine which species are affected by simulated commercial use and whether their numbers recover within the season. Such studies involve large scale applications of products to replicated experimental plots with a water treated control and a positive reference treatment included in the experimental design. Taxonomy is a time-consuming and expensive component of these studies and not all arthropod groups can be identified with equal ease. With many thousands of specimens it is not usually possible or sensible to identify all specimens to species level. This paper looks at analysis of the results from a large-scale field study conducted in winter wheat in Devon, UK, using univariate techniques at family level and at species level for carabid beetles, staphylinid beetles, linyphiid spiders and Collembola. The conclusions of this analysis are compared with those made using Principal Response Curves (PRC). When data is summarised at the family level genuine effects on arthropod species could be overlooked. PRC analysis indicates that some non-target arthropods may be more important indicators of treatment effects than others.

Key words: field studies, cereal ecosystem, Principal Response Curves, Carabidae, Staphylinidae, Linyphiidae.

Introduction

Field studies are usually the final stage in the evaluation of a plant protection product, which has been found to be harmful to non-target arthropods in laboratory or in semi-field studies. Whilst large-scale fieldwork can be expensive it is often the identification of the specimens and the recording of the data that is the most time consuming. In a large-scale study in a cereal ecosystem there may be almost half a million specimens collected. Because of the origins of regulatory testing in beneficial arthropod evaluation there is a greater recognition of those species which are predatory or parasitic. In cereal studies carabid beetles and linyphiid spiders have received a great deal of attention, largely because there are clear and well illustrated taxonomic keys, whereas staphylinid beetles have been relatively under represented, possibly because their identification is more difficult and their identification keys are not complete. The purpose of this paper is not to report the results of a single study but rather to look at the relative importance of the information gained by species level taxonomy. Given the large amount of time taken is it worth identifying specimens to species level?

Material and methods

A large-scale field study was conducted in winter barley fields in South West England in 2002 to evaluate the effects of two insecticides on non-target arthropods. The study was of randomised block design with four treatments and four replicates. Treatments consisted of the

43 44

test item at its commercial application rate, a reduced test item rate (2% of field rate) calculated so as to simulate drift at a distance of 1 m, a water treated control and a second insecticide of known effects applied at its field rate as a positive reference. All treatments were applied three times during the summer of 2002 (3, 15 and 31 May) to represent worst realistic use of the product. Plot size ranged from 1.0 to 1.8 ha. Invertebrates were sampled weekly using pitfall traps established in the central 50 m x 50 m area of each plot. Six traps were established in each plot and when in use they contained 50% ethylene glycol as a preservative. Specimens from each plot were identified in the laboratory to a pre-determined level of taxonomic precision. Carabid beetles and the more abundant staphylinid beetles were identified to species level. Linyphiid spiders were sexed and the males of most species recorded to species level whereas the females were recorded to genus. Certain more easily recognised Collembola were identified to species level, whilst others were recorded to family or even super-family level. There were 34 carabid species, 35 staphylinid species and 20 spider species identified. A total of 146 taxonomic units were recognised. Data analysis Results for each taxon were analysed from each sampling date by univariate techniques (Analysis of variance followed by Tukey Test, Zar, 1999). Principle Response Curve (PRC) method described by Van den Brink et al. (1996) and Van den Brink and Ter Braak (1999) was used to interrogate the same dataset using all of the species data collected and presenting the output in a visual form. PRC analysis is based on partial Redundancy Analysis (RDA). This revised analysis included data on all 146 taxonomic units representing a diverse arthropod community. The advantage of multivariate methods over univariate methods is that they use and summarize all information on the investigated populations simultaneously, and in doing so they evaluate the effects of pesticide treatments at the community level. This ordination technique assumes a linear relation between species catch sizes and environmental variables (treatments). Therefore, before analysis, the data on the number of individuals of a species were log transformed so that if n individuals were caught in pitfall traps from a particular field plot the analysis was performed on log (n+1). The added advantage of this transformation is that it reduces the influence of a few species of large catch size on the analysis. For the analysis the software package Canoco for Windows 4.5 was used.

Results and discussion

Results from such studies are normally analysed using univariate techniques and considered for each taxon individually. However, there is no accepted level of precision and it is not uncommon for regulatory studies to report results at the family level only. Table 1 shows the magnitude of the initial impact of each treatment (Abbott, 1925) and the length of time before recovery had occurred when the data for Carabidae, Staphylinidae and Linyphiidae are summarised at the family level. Linyphiid spiders were the most affected with a 90% initial impact for both insecticides and no recovery until the following year. Carabid beetles were relatively robust with impacts of 20% and 33% for insecticides 1 and 2 respectively but both showed recovery after 8 weeks. When the results are presented for several species of carabid, staphylinid and linyphiid (Table 2) the nature of the effects appear rather different. Within the Carabidae, Asaphidion curtum was apparently unaffected by insecticide 1 at either field or drift rate whereas insecticide 2 resulted in an 89.5% effect with no recovery occurring. Bembidion lampros experienced a 96% and a 99% reduction in catch size relative to control samples in each of the two insecticides, with recovery occurring after six weeks.

Table 1: Percentage reduction in catch size due to treatment and time to recovery when analysed at the family level.

Insecticide 1 Insecticide 1 Insecticide 2 Family Drift rate Field rate Field rate

Carabidae Reduction 3% Reduction 20% Reduction 33% Coleoptera Recovery 1 week Recovery 8 weeks Recovery 8 weeks

Staphylinidae Reduction 1% Reduction 77% Reduction 82% Coleoptera Recovery 1 week Recovery 8 weeks Recovery 8 weeks

Linyphiidae Reduction 18% Reduction 90% Reduction 90% Araneae Recovery 6 weeks Recovery 1 year Recovery 1 year

Table 2: Percentage reduction in catch size due to treatment and time to recovery of example species when analysed at the species level.

Insecticide 1 Insecticide 1 Insecticide 2 Example species Drift rate Field rate Field rate Asaphidion curtum No reduction No reduction Reduction 90% Col; Carabidae Recovery not seen

Bembidion lampros No reduction Reduction 96% Reduction 99% Col: Carabidae Recovery 6 weeks Recovery 6 weeks

Nebria brevicollis Col: Carabidae No reduction No reduction No reduction

Stenus clavicornis Reduction 100% Reduction 100% Col: Staphylinidae No reduction Recovery 8 weeks Recovery 8 weeks

Erigone dentipalpis. Reduction 100% Reduction 100% Male No reduction Recovery 1 year Recovery 1 year Araneae: Linyphiidae

Differences in impact between species of the same family are not uncommon and may be due to exposure of individuals (for example because of different behaviour or prey type), different proportions of the population being exposed at any one time or intrinsically different levels of susceptibility to a particular product. Figures 1 and 2 show trends of pitfall trap catch numbers over time for a staphylinid beetle (Stenus clavicornis) and for females of the Linyphiid spider Oedothorax spp.. In these figures the solid symbols indicate a statistically significant difference from the control at

100 Water control Insecticide 1 Drift rate T1 T2 T3 Mean Insecticide 1 Field rate (n+1) Insecticide 2 Field rate

1 1-Apr 15-Apr 29-Apr 13-May 27-May 10-Jun 24-Jun 8-Jul 22-Jul Date in 2002

Figure 1. Mean number of Stenus clavicornis (Coleoptera: Staphylinidae) collected in six pitfall traps over one week. T1 - T3 indicate treatment dates. Solid symbols indicate statistically significant difference from control in Anova and Tukey (P<0.05).

P<0.05. For S. clavicornis the results show that at their field rates, insecticides 1 and 2 both resulted in a major decline in catch size followed by complete absence from the treated plots. However, by the final sampling date there was no statistically significant difference between treatments and recovery had occurred. The drift rate of insecticide 1 was not statistically different from the control on any date and can be considered to be harmless to S. clavicornis. For Oedothorax spp. females (Fig. 2) a similar response occurred but took place against a backdrop of falling numbers in control plots after treatment followed by increasing numbers towards the end of the study. Whilst the field rates of both insecticides had a major impact on Oedothorax spp. females, numbers had begun to increase over the last three sampling dates. With a significant difference remaining on the final sampling date this can only be considered to be partial recovery. Not all species give clear and easy to interpret results. Fig. 3 shows the pitfall trap catches for the carabid Nebria brevicollis. There were no statistically significant differences between any treatments on any dates suggesting that there were no treatment related effects for this species. However, it is also possible that there was regular movement of individuals from the hedgerow boundary into the plots and that this masked any treatment effects. Such data serve as a reminder that these are the results of pitfall trap catches and not absolute measurements of abundance. It also highlights that recovery for some carabid species can be rapid following treatment.

Water control Insecticide 1 Drift rate Insecticide 1 Field rate Insecticide 2 Field rate 100 T1 T2

Mean (n+1) T3

1 1-Apr 15-Apr 29-Apr 13-May 27-May 10-Jun 24-Jun 8-Jul 22-Jul Date in 2002

Figure 2. Mean number of Oedothorax spp. females (Araneae: Linyphiidae) collected in six pitfall traps over one week. T1 - T3 indicate treatment dates. Solid symbols indicate statistically significant difference from control in Anova and Tukey (P<0.05).

In this regulatory study with 146 taxonomic units it was not meaningful to interpret the results for each one, many were present in low numbers or only occurred on one or two time points in the study. Results for some species, which occurred in low numbers, showed big changes over time, which are as likely to be due to seasonal life cycle variation as well as due to treatment. What constitutes the cut off for low numbers, i.e. the threshold below which the data for a given taxa are not to be interpreted? In arable field studies of the kind described in this paper a mean of 1 individual per sample appears to be appropriate. In this study only 17 of the 34 carabid species were present at more than 1 per sample. Of the 35 staphylinid species 14 exceeded the threshold and of 20 spider taxa 8 exceeded the threshold. In the whole study 46 of the 146 taxa were collected with mean numbers of more than one per sample. This means that 100 of the taxa identified did not generate data that were used in the interpretation of the results. Clearly if these taxa could have been identified at the start of the study it would have been possible to save a great deal of resource. Principal Response Curves (PRC) analysis enables quantitative assessment of effects using the results for every taxonomic unit identified. The PRC analysis creates the response of a virtual species that can be considered to represent the behaviour of the community. In PRC analysis treatment variates are compared with control variates and species scores and the percentage of variance explained are tabulated for each taxon. A species score of 1.0 would mean that the response of that taxon mirrored that of the virtual species. The significance of the first ordination axis was determined using a Monte Carlo permutation test.

100 T1 T2 T3 Water control Insecticide 1 Drift rate Insecticide 1 Field rate Insecticide 2 Field rate Mean (n+1)

1 1-Apr 15-Apr 29-Apr 13-May 27-May 10-Jun 24-Jun 8-Jul 22-Jul

Date in 2002 Figure 3. Mean number of Nebria brevicollis (Coleoptera: Carabidae) collected in six pitfall traps over one week. T1 - T3 indicate treatment dates. There were no statistically significant differences from control in Anova and Tukey (P<0.05) on any sampling dates.

The effect of treatment was significant on the total arthropod community (p = 0.026). Table 3 shows that 48.60% of the total variance can be attributed to time and is thus (implicitly) displayed on the horizontal axis of the PRC whereas, 20.60% can be attributed to treatment (including interaction with time). The variance explained by the first PRC is 68.10% of that accounted for by the treatment time interaction. The second PRC accounts for only 10.20% of the variance indicating that the first PRC best describes the shape of the community response (Fig. 4).

Table 3. Percentages of the total variance that can be attributed to time and treatment regime for the whole dataset on all species/taxa for all treatments. % Variance accounted % Variance explained by treatment regime for by captured by Time Treatment First PRC P-value of Second PRC first PRC 48.60 20.60 68.10 0.026 10.20

PRC: Principal Response Curve

Fig. 4 shows the First Principal Response for the community of arthropods sampled in pitfall traps in this study. The control values have been taken to represent zero and the plot has been inverted to show a reduction in catch size as a decline over time. Whilst insecticides 1 and 2 both had a significant impact there was recovery by the final sampling occasion. Throughout the sampling period no statistically significant differences were noted between the control and the drift rate treated plots at the whole community level.

Treatment dates 0.2 T1 T2 T3

0 25-Mar-02 14-Apr-02 4-May-02 24-May-02 13-Jun-02 3-Jul-02 23-Jul-02 12-Aug-02

-0.2

-0.4

-0.6

-0.8 Water control Insecticide 1 Drift rate -1 Insecticide 1 Field rate Insecticide 2 Field rate -1.2 Sampling date

Figure 4. Principal response curves (PRC) for all 146 species/taxa from pitfall catches.

Species scores are presented in Table 4 for species where at least 30% of the variance could be explained by the first PRC axis. Species scores in Table 4 ranged from 3.397 (Stenus clavicornis, Coleoptera, Staphylinidae) to -0.015 (Pardosa palustris, Araneae, Lycosidae). Sixteen of these 36 taxa were staphylinid beetles, 10 were spiders and 7 were carabid beetles. The remaining three taxa were fungus beetles (Ptomophagus spp., Coleoptera, Leiodidae), soil mites (Acari) and aphids (Aphidoidea). Species with the highest scores more closely follow the shape of the PRC than those with low scores. Species with negative scores tend to show a response opposite to that of the PRC. Given the findings from both the univariate and multivariate (PRC) analyses what can we learn about the level of taxonomy required in regulatory studies and which species or taxonomic units were critical in interpreting the effects of the two insecticides under field conditions? Fig. 5 shows a plot of the numbers of a taxon caught in water treated control plots against its species score. Several species, shown to the bottom right of Figure 5, have relatively high catch size and low species score. Interestingly there is a group of species where the species score is higher than their catch size would suggest should a linear relationship exist. Those with a species score of greater than 2 would fall into this category.

From Table 4 it can be seen that those taxa with a species score of greater than 2.0 comprise 5 staphylinid species, staphylinid larvae, 3 small carabid species and 3 linyphiid species. In this study these species were possibly the best indicators of treatment effect for the products tested. In this study, the catch size of these species in control samples was between 100 and 2000 individuals, i.e. those of moderate to high catch size but not of low or very high catch size. Based on the 1 individual per sample threshold, species level taxonomy was necessary on a range of taxa notably Staphylinidae, Carabidae and Linyphiidae. Grouping species would have masked effects and hidden the impact on these most sensitive species. Species with the highest scores tended to belong to the Staphylinidae which suggested that this group is an important indicator for effects (Table 4). Stenus clavicornis, Philonthus cognatus, staphylinid larvae, Tachyporus hypnorum and Tachinus signatus had high species scores (between 3.397 and 2.749), which indicated good agreement with the community level responses for each treatment in the study. The majority of the spiders taxa affected by the field rate treatments were small web building types belonging to the Linyphiidae. Oedothorax spp. (female), Savignya frontata (male) and Erigone atra (male) showed the highest correlation to the observed PRCs with species scores of 2.455, 2.289 and 2.016 respectively.

Z = Logten(X) Y = -1.9E-01 + 0.526540Z R-Sq = 43.7 %

2 Regression

1 95% CI

0 Species score (AX1 PRC) score (AX1 Species

1.0 3.0 10 30 100 300 1000 3000 10000 30000 100000 Pitfall catch in controls (n+1)

Figure 5. Plot of the abundance of a taxon in samples from water treated control plots against its species score.

These species or taxa could be vulnerable to exposure to direct spray and to the upper parts of the barley plant, which would receive pesticide treatment. Spiders of low sensitivity and hence species score close to zero included larger ground dwelling taxa such as Alopecosa sp. (0.034) and Pardosa palustris (-0.015). Carabids with the highest species scores included the common species Bembidion lampros (2.648), Loricera pilicornis (2.420), Agonum dorsale (2.399) and Bembidion guttula (1.996). These are all relativity small to medium sized species.

Table 4. Species scores for the first axis of the PRC (Species score AX1) and the percentage of the total variance of the species that can be explained by the first axis. Only species for which 30% or more of the total variance can be explained by the first axis are shown (Exp % Var.). Species Order / score Species / taxon Group Stage (AX1) Exp % Var. Stenus clavicornis Staphylinidae adult 3.397 77.31 Philonthus cognatus Staphylinidae adult 3.094 44.15 Staphylinid larva Staphylinidae larva 2.902 58.28 Tachyporus hypnorum Staphylinidae adult 2.894 55.49 Tachinus signatus Staphylinidae adult 2.749 67.42 Bembidion lampros Carabidae adult 2.648 39.27 Oedothorax sp. female Araneae adult 2.455 40.80 Loricera pilicornis Carabidae adult 2.420 35.33 Agonum dorsale Carabidae adult 2.399 50.26 Aloconota gregaria Staphylinidae adult 2.337 38.58 Savignya frontata male Araneae adult 2.289 46.43 Ptomaphagus spp. Leiodidae adult 2.132 48.64 Erigone atra male Araneae adult 2.016 33.50 Bembidion guttula Carabidae adult 1.996 33.76 Gabrius sp. Staphylinidae adult 1.978 58.02 Anotylus rugosus Staphylinidae adult 1.923 53.05 Amischa sp. Staphylinidae adult 1.801 56.07 Tachyporinae (other) Staphylinidae adult 1.706 55.19 Acari other Acari adult 1.528 37.24 Philonthus carbonarius (varius) Staphylinidae adult 1.448 36.00 Aleocharinae other Staphylinidae adult 1.371 40.05 Oxyopoda tarda Staphylinidae adult 1.339 36.90 Linyphiid juvenile Araneae juvenile 1.302 41.48 Xantholinus longiventris Staphylinidae adult 1.292 35.17 Linyphiidae male other Araneae adult 1.241 35.44 Sunius propinquus Staphylinidae adult 1.175 45.43 Pterostichus strenuus Carabidae adult 1.140 41.96 Demetrias atricapillus Carabidae adult 0.734 38.51 Aphidoidea Aphidoidea adult 0.618 32.93 Bathyphantes gracilis Araneae adult 0.574 44.59 Oedothorax retusus male Araneae adult 0.563 36.64 Stenus nanus Staphylinidae adult 0.489 38.64 Agonum assimile Carabidae adult 0.049 42.23 Alopecosa sp. female Araneae adult 0.034 32.57 Tetragnathidae (other) Araneae adult 0.016 32.57 Pardosa palustris female Araneae adult -0.015 36.45 PRC: Principal Response Curve

None of the larger common species (e.g. Nebria brevicollis, Pterostichus melanarius and Pterostichus madidus) featured in Table 4 as their % explained variance was low (5.49, 4.83 and 3.53% respectively). This indicated that the variation in population numbers for these

species was unrelated to insecticide treatment or that effects could not be detected in the study. This can be illustrated by the response observed for N. brevicollis collected per plot shown in Figure 3, which is unrelated to the shape of the curve of the first PRC and where all treatments follow closely the response of the control. If family level taxonomy had included members of the small to medium sized carabids with the larger common species, significant effects of the insecticides would have been obscured. Tables 1 and 2 clearly demonstrate this. A good quality regulatory field study depends on a number of factors including site selection, reproducible sampling with appropriate methodology, highly skilled operators and also good taxonomic determination of a wide range of arthropods to a suitable level (where possible to genus and species). Although determination of all individuals to this level is rarely practical, determination to genus or species level for most arthropods of medium to high catch size seems desirable for a full and correct interpretation of the data. Some species (n = 36) identified in this paper) were shown to be good indicators of effect. Although these cannot be determined a priori, intelligent and expert taxonomy and judgement is necessary to achieve interpretable results based on the individuals sampled. These, together with the judicial and appropriate use of data analysis employing where necessary both univariate and multivariate techniques will allow state of the art interpretation of the complex datasets generated from a properly conducted regulatory field study with non-target arthropods.

Conclusions In conclusion, Principal Response Curves were a useful tool for examining the effects of treatments on the community as a whole. With the benefit of hindsight most information was gained from a small proportion of the data set. In the study analysed here certain staphylinid species appeared to be the most reliable indicator of treatment effects together with smaller Carabidae and some Linyphiidae. PRC analysis allowed the use of all species data so no information was lost in analysis. PRC analysis can be used to give a balanced assessment of the level and duration of impact and subsequent recovery. Overall, regulatory studies need species level precision to be clearly interpretable for both species and communities. However, carefully targeted taxonomy could prove a valuable tool to save time on studies without a loss in precision. In practical terms this means that species of very low or high catch size may not necessarily need to be determined to a detailed taxonomic level.

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticide. – J. Econ. Entomol. 18, 265-267 Van den Brink, P.J, Van Wijngaarden, R.P.A., Lucassen, W.G.H., Brock, T.C.M. & Leeuwangh, P. 1996: Effects of the insecticide Dursban 4 (active ingredient chlorpyrifos) in outdoor experimental ditches: II. Invertebrate community responses. – Environ. Toxicol. Chem. 15: 1143-1153. Van den Brink, P.J. & Ter Braak, C.J.F. 1999: Principal response curves: analysis of time- dependent multivariate responses of biological community to stress. – Enviro. Toxicol. Chem. 18: 138-148. Zar, J.H. 1999: Biostatistical Analysis. – Prentice Hall, New Jersey, USA.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 53-59

The effects of Spinosad on beneficial insects and mites used in integrated pest management systems in greenhouses

M. Miles Dow AgroSciences, European Development Centre, 2nd Floor, 3 Milton Park, Abingdon, OX14 4RN, UK

Abstract: When used according to good horticultural practice, spinosad was shown to be compatible with the use of predatory mites (Phytoseiulus persimilis, Amblyseius californicus, Amblyseius cucumeris, Hypoaspis aculeifer and Hypoaspis miles), predatory Heteroptera (Orius laevigatus and Macrolophus caliginosus), Coccinellidae (Hippodamia convergens and Coccinella septempunctata), Neuroptera (Chrysoperla carnea and Chrysoperla rufibularis) and Diptera (Aphidoletes aphidimyza). Parasitic Hymenoptera were sensitive to spinosad; however toxic effects were short lived due to the low persistence of spinosad but species such as Aphidius colemani, Encarsia formosa and Trichogramma brassicae can be introduced to protected crops within 2 weeks after application. The findings showed that spinosad is highly selective to beneficials and pollinators making it an ideal insect pest control product for use within greenhouse IPM programmes.

Keywords: Insecticide, Spinosad, predatory arthropods, parasitic insects, side-effects, glasshouse

Introduction

Spinosad is the active ingredient in Conserve, Success, SpinTor, Laser and Tracer insecticides (Trademarks of Dow AgroSciences LLC) and can be used to control pests in crops where the conservation of predatory insects and mites is an important component of Integrated Pest Management (IPM). This has considerable importance when growers actively make introductions of commercially available beneficial species for pest control, typically under conditions of protected cultivation. Spinosad is an insect control agent derived by fermentation of the Actinomycete bacterium, Saccharopolyspora spinosa. The active ingredient is composed of two metabolites, spinosyn A and spinosyn D (Thompson et al., 1997). Spinosad controls many caterpillar pests in vines, pome fruit and vegetables (including tomatoes and peppers) and thrips and dipterous leafminers in vegetables and ornamentals. Application rates vary between 25 to 150 g of active ingredient per hectare (g a.i./ha) and 4.8 to 36 g of active ingredient per hectolitre (g a.i./hL) depending on the crop and target pest. The mode of action of spinosad is novel, making it a useful resistance management tool. A novel mechanism of activity on the nicotinic acetylcholine receptors was identified as the primary cause of insect death (Salgado, 1997). Spinosad has additional effects on gamma-aminobutyric acid or GABA receptors, although it has not been shown that these effects contribute to insecticidal activity. The action of spinosad on nicotinic receptors is unique and is different from the action of nicotine and imidacloprid. Studies so far have found no cross-resistance with a variety of resistance mechanisms, making spinosad an excellent fit in resistance management programs.

53 54

Materials and methods

All studies were carried out using spinosad formulated as either a 120 or a 480 g a.i./L SC formulation. A wide range of beneficial species was investigated. All studies were replicated and included appropriate toxic and control references treatments. All studies were conducted to IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) principles and standard characteristics (Hassan 1992). In order to test effects on Amblyseius californicus, young bean plants infested with two spotted spider mite (Tetranychus urticae) were sprayed to run-off with a 19.2 g a.i./hL solution of spinosad. The plants were trimmed to one leaf and infested with five adult female predatory mites. The experiment was conducted under greenhouse conditions following the semi-field initial toxicity method prescribed for Phytoseiulus persimilis (Hassan, 1985). Live predatory mites were counted six days after infestation. A direct spray semi-field test was conducted on P. persimilis where mites were present on the plants at the time of application following EPPO guideline 151 (Anon 1990). Spinosad was applied to the point of run-off at 9.6 and 36 g a.i./hL. Each treatment was replicated four times. One week after application 10 leaves were sampled from each replicate and the number of live predatory mites and eggs counted. To investigate the compatibility of spinosad with Amblyseius cucumeris, Hypoaspis aculiefer and Hypoaspis miles commercial samples of these mites were obtained from a commercial supplier. The predatory mites are packed and applied in a carrier (bran for A. cucumeris and peat / vermiculite for H. aculeifer and H. miles). In tests with A. cucumeris and H. aculeifer, 4g of product was placed in 9 cm diameter plastic Petri dishes, and for H. miles 2 g of product was placed in 5 cm diameter tight fit Petri dishes. Prior to application the number of mites per dish (replicate) was counted. Each dish was sprayed with test solutions of spinosad at either 144 or 540 g a.i./ha. The number of live mites per replicate was counted three days after application using a binocular microscope. Two species of predatory bug were investigated; O. laevigatus and Macrolophus caliginosus. For O. laevigatus, bean plants, approximately 20 cm tall, were sprayed to run-off at 19.2 g a.i/hL employing the semi-field initial toxicity method for Phytoseiulus persimilis (Hassan, 1985) adapted for O. laevigatus. When dry, each plant was infested with five first or second instar nymphs and fed on pollen. The studies were performed in a greenhouse. The number of live nymphs was counted 4 DAA. Two studies were performed on M. caliginosus where spinosad was evaluated at two rates, 9.6 and 36 g a.i./hL. In the first study mature pepper plants were sprayed to run-off and the plants were aged under greenhouse conditions. Leaves were removed for bioassay with adult bugs on the day of application and 2 and 8 DAA. The bugs were fed on pollen and mortality was assessed after two days exposure. In the second study, pepper plants (approximately 30-40 cm tall) were infested with a mixed population of adults and nymphs under greenhouse conditions. The plants were sprayed to run-off and the bugs were fed with pollen. The number of bugs per plant was counted one, three, seven and 14 DAA. Spinosad was evaluated against larvae of two lacewing species (Chrysoperla carnea and C. rufilabris) under laboratory conditions. C. carnea was exposed to pepper leaves treated with spinosad at 36 g a.i./hL and larval mortality and fecundity of the surviving adults was measured following the method of Vogt et al. (2000). C. rufilabris larvae were exposed to glass plates treated with spinosad at 20 g a.i./hL and mortality assessed after 3 days using a shortened adaptation of the method by Bigler (1988). The ladybird Coccinella septempunctata was evaluated following the method of Schmuck et al (2000) with treated leaves used in place of glass plates. Hippodamia convergens was evaluated in the same manner as C. rufilabris.

Pepper plants (20 cm tall) were sprayed with solutions of spinosad at either 9.6 or 36 g a.i./hL to evaluate the effects of spinosad to the aphidophagous gall midge Aphidoletes aphidimyza. When dry, plants were trimmed to one leaf and infested with peach potato aphids (Myzus persiciae) as prey. Five newly hatched A. aphidimyza larvae were placed on each leaf and fresh aphids were supplied daily. Four days after application the number of live pre-pupal larvae on the plants was counted. The aphid parasitoid (Aphidius colemani) was investigated under laboratory conditions by direct application of spinosad to mummified aphids on pepper plants. Wasp emergence and fecundity was measured. A semi-field test on pepper plants under greenhouse conditions investigated the introduction times for A. colemani before and after applications of spinosad. Wasps were released to forage on plants infested with aphids two days before application and 1, 7 and 14 DAA. The performance of the parasitoids was measured by counting the number of parasitised aphids per plant seven to twelve days after wasp release. The effects on pupae protected within mummified aphids were investigated in an extended laboratory test. Plants on which developing 'mummies' were present were sprayed and the number of parasitoid wasps, which successfully emerged, counted. A semi-field test on tomato plant under greenhouse conditions was employed for the white fly parasitoid Encarsia formosa. Plants infested with white-fly were sprayed with spinosad and wasps released on the day of application and after one week. Two weeks after the release of the wasps the number of parasitised white fly per plant was counted. The effects on E. formosa pupae protected within whitefly 'black scales' was investigated in a semi-field test. Tomato plants on which developing black scales were present were sprayed and the number of parasitoid wasps which successfully emerged counted. Effects on the lepidopteran egg paratisoid, Trichogramma brassicae were measured under greenhouse semi-field conditions. Pepper plants were sprayed and wasps were released on the day of application and after 5 and 10 days ageing under greenhouse conditions. Small cards containing Lepidopera eggs were placed three per plant at each timing for the wasps to parasitise. Two to four days after wasp release the egg cards were collected and held under controlled environment conditions and the number of black parasitised eggs was counted. Spinosad was evaluated at 9.6 and 36 g a.i./hL in all studies with parasitoids. The effect of spinosad to each organism was categorised according to the IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classifications (Hassan 1992) described in Table 1.

Table 1. IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classification system for side effects of plant protection products to beneficial and non-target arthropods (Hassan 1992).

Classification % Effect observed Laboratory studies All other studies * Class 1 Harmless <30% <25% Class 2 Slightly Harmful 30 – 79% 25 – 50% Class 3 Moderately Harmful 80 – 99% 51 – 75% Class 4 Harmful >99% >75% Note: *Study types are extended laboratory, semi-field and field tests.

Results and discussion

The effects of spinosad on a wide range of beneficial arthropods is summarised in tables 2 and 3. Spinosad was shown to have no detrimental effect on any predatory mite species tested. Low toxicity was demonstrated to A. californicus (19.2 g a.i./hL) and P. persimilis (36 g a.i./hL) in semi-field tests. No effects were seen on A. cucumeris, H. aculiefer or H. miles at test rates of 144 or 540 g a.i./ha (equivalent to 9.6 g a.i./hL or 36 g a.i./hL applied at 1500L/ha water volume). Spinosad at 19.2 g a.i./hL was harmless to the predatory bug O. laevigatus. Spinosad was harmless to M. caliginosus at 9.6 g a.i./hL and only slightly harmful at 36 g a.i./hL. Where spinosad was applied at 36 g a.i./hL directly to populations of M. caliginosus recovery took place within 14 days. Spinosad was harmless to the ladybird species C. septempunctata and H. convergens at rates of 36 and 20 g a.i./hL respectively. Lacewings were similarly unharmed, where 36 g a.i./hL was harmless to C. carnea and 20 g a.i./hL was harmless to C. rufilabris. Spinosad at 9.6 and 36 g a.i./hL caused no harmful effects on A. aphidimyza larvae. Direct application of spinosad at either 9.6 or 36 g a.i./hL was harmful to adult A. colemani foraging in a greenhouse crop as were one day old residues. However when these residues had been allowed to age under greenhouse conditions one and two weeks after application, spinosad was only slightly harmful at the same rates. However direct application of spinosad at 36 g a.i./hL was harmful to pupal wasps within mummified aphids. Spinosad at either 9.6 or 36 g a.i./hL was harmful / moderately harmful to adult E. formosa and T. brassicae foraging on treated plants on the day of application. One-week-old residues of spinosad at the lower rate were harmless to forging adult E. formosa whereas the higher rate was only slightly harmful. The pattern was repeated for T. brassicae when exposed to ten day old residues. Direct spray at 36 g a.i./hL to pupal wasps (protected within mummified aphids) for A. rhopalosiphi resulted in only 15% wasp emergence (moderately harmful). Spinosad was classified as IOBC class 1 (harmless) to E. formosa pupae, when sprayed directly at 9.6 g a.i./hL and slightly harmful at 36 g a.i./hL. A low level of impact was observed for spinosad on a wide range of commercially available natural enemy species, including predatory mites, bugs, lacewings, lady birds and predatory gall midges. Parasitoids appeared to be at risk from applications of spinosad. However by careful use of introduction periods both spinosad and parasitoids may be used in the same IPM programme successfully. This level of selectivity makes spinosad an ideal product for use within greenhouse IPM programmes where the product can be used to control damaging pests such as caterpillars, thrips and dipterous leafminers, and still allowing the grower to use biological control methods and pollination by bumble bees. The exact mechanism for the selectivity has not been fully investigated, but reasons could include leaf penetration, intrinsic lack of toxicity to a particular species or group, behaviour, exposure, and uptake. Spinosad is highly active against pest such as the thrips species Frankliniella occidentalis (Drinkall and Boogaard, 2001). Where a predator feeds primarily on thrips an application of spinosad will remove much of the available prey. In such cases a reduction in prey may lead to an apparent reduction in predator numbers.

Conclusions

Two key attributes of spinosad include a) exceptional selectivity to plants and b) low toxicity to many commercially important beneficials, natural predators and pollinators. This makes spinosad ideal for use in Integrated Pest Management (IPM) systems within greenhouses.

Table 2. The effects of spinosad to predatory mites and insect species (laboratory, extended laboratory and semi-field tests)

Species Test Conditions Rate / Conc. *Effect (%) Classification Phytoseiulus persimilis Semi-field 9.6 g a.i./hL E = 0.26 Harmless class 1 All stages 36 g a.i./hL E = 13.5 Harmless class 1 Amblyseius californicus Semi-field Adults 19.2 g a.i./hL E = 6.0 Harmless class 1 Amblyseius cucumeris Extended laboratory All stages 144 g a.i./ha M = 0.0 Harmless class 1 540 g a.i./ha M = 0.0 Harmless class 1 Hypoaspis aculiefer Extended laboratory All stages 144 g a.i./ha M = 0.0 Harmless class 1 540 g a.i./ha M = 0.0 Harmless class 1 Hypoaspis miles Extended laboratory All stages 144 g a.i./ha M = 0.0 Harmless class 1 540 g a.i./ha M = 15.8 Harmless class 1 Orius laevigatus Semi-field L1/L2 19.2 g a.i./hL M = 0.0 Harmless class 1 Macrolophus Extended laboratory Adults 9.6 g a.i./hL M = 4.0 (2DAA) Harmless class 1 caliginosus 36 g a.i./hL M = 36.0 (0DAA) Slightly harmful class 2 Semi-field Adults and 9.6 g a.i./hL E = 22.5 (7DAA) Harmless class 1 nymphs 36 g a.i./hL E = 63.6 (7DAA) Recovery in 14 days Chrysoperla carnea Extended laboratory L2 36 g a.i./hL M = 28.8 Harmless class 1 Chrysoperla rufilabris Laboratory bioassay Larvae 20 g a.i./hL M = 0.0 Harmless class 1 Hippodamia convergens Laboratory bioassay Larvae 20 g a.i./hL M = 0.0 Harmless class 1 Coccinella 7-punctata Extended laboratory L2 36 g a.i./hL M = 16.0 Harmless class 1 Aphidoletes aphidimyza Semi-field L1 9.6 g a.i./hL M = 0.0 Harmless class 1 36 g a.i./hL M = 12.5 Harmless class 1 Note: * E = overall effect, M = mortality, DAA = Days After Application 57

58 58 Table 3. The effects of spinosad to parasitic hymenoptera species (extended laboratory and semi-field tests)

Species Test Conditions Conc. *Effect (%) Classification Aphidius colemani Semi-field Adults exposed to 9.6 g a.i./hL P = 69.5 (2DBA) Harmful class 4 treated pepper plants P = 97.8 (1DAA) Harmful class 4 P = 41.2 (7DAA) Slightly harmful class 2 P = 45.6 (14DAA) Slightly harmful class 2 36 g a.i./hL P = 87.7 (2DBA) Harmful class 4 P = 98.1 (1DAA) Harmful class 4 P = 36.0 (7DAA) Slightly harmful class 2 P = 27.1 (14DAA) Harmless class 1 Extended Mummified aphids 36 g a.i./hL M = 85.0 Moderately harmful class 3 laboratory Encarsia formosa Semi-field Adults exposed to 9.6 g a.i./hL P = 73.0 (0DAA) Harmful class 4 treated tomato plants P = 35.0 (7DAA) Slightly harmful class 2 P = 19.0 (14DAA) Harmless class 1 36 g a.i./hL P = 83.0 (0DAA) Harmful class 4 P = 33.0 (7DAA) Slightly harmful class 2 P = 38.3 (14DAA) Slightly harmful class 2 Semi-field Parasitised 9.6 g a.i./hL M = 9.7 Harmless class 1 black scales 36 g a.i.hL M = 39.3 Slightly harmful class 2 Trichogramma Semi-field Adults exposed to 9.6 g a.i./hL P = 63.3 (0DAA) Moderately harmful class 3 brassicae treated pepper plants P = 51.8 (5DAA) Moderately harmful class 3 P = 12.7 (10DAA) Harmless class 1 36 g a.i./hL P = 81.8 (0DAA) Harmful class 4 P = 37.5 (5DAA) Slightly harmful class 2 P = 33.6 (10DAA) Slightly harmful class 2 Note: * P = reduction in parasitism, M = mortality, DBA = days before application, DAA = days after application. Days refer to wasp release timings in relation to spray application.

References

Anon. 1990: Guideline for the evaluation of side-effects of plant protection products, Phytoseiulus persimilis. – EPPO Bulletin 20: 531-550. Bigler, F. 1988: A laboratory method for testing side-effects of pesticides on larvae of the green lacewing, Chrysoperla carnea Steph. (Neuroptera, Chrysopidae). – IOBC/WPRS Bulletin (11) 4: 71-77. Drinkall, M.J. & Boogaard, M. 2001: The development of spinosad for the control of Frankliniella occidentalis in protected ornamentals. – Med. Fac. Landbouww. Univ. Gent 66/2a: 387-393. Hassan, S.A. 1985: Standard methods to test the side-effects of pesticides on ntural enemies of insects and mites developed by the IOBC/WPRS working group ‘Pesticides and Beneficial organisms.’ – EPPO Bulletin 15: 214-225. Hassan, S.A. 1992:Guidelines for testing the effects of pesticides on beneficial organisms. – IOBC/WPRS Bulletin 15 (3): 186 pp. Salgado, V.L. 1997: The modes of action of spinosad and other insect control products. – Down to Earth [Dow AgroSciences] 52 (1): 35-43. Schmuck, R., Candolfi, M.P., Kleiner, R., Mead-Briggs, M., Kemmeter, F., Jans, D., Waltersdorfer, A. & Wilhelmy, H. 2000: Laboratory test system for assessing the effects of plant protection products on the plant dwelling insect Coccinella septempunctata (Coleoptera: Coccinellidae). – In: Candolfi, M.P., et al. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 45-70. Thompson, G.D., Michel, K.H., Yao, R.C., Mynderse, J.S., Mosburg, C.T., Worden T.V., Chio, E.H., Sparks T.C. & Hutchins, S.H. 1997: The discovery of Saccharopolyspora spinosa and new class of insect control products. – Down to Earth [Dow AgroSciences] 52 (1): 1-5. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Viñuela, E., Waldburger, M. & Waltersdorfer, A. 2000: Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). In: Candolfi, M.P., et al. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 27-44.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 61-65

A method to prove long term effects of neonicotinoids on whitefly parasitoids

Ellen Richter Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Horticulture, Messeweg 11/12, D-38104 Braunschweig, Germany, e-mail: [email protected]

Abstract: It is well known that many insecticides have a repellent impact on E. formosa. Currently, there is little information on the persistence of this effect. Although it is known that a spray application of the neonicotinoid imidacloprid is detrimental to E. formosa, soil applications were thought to have little impact. It is believed that soil applications resulted in a minimal exposure. But it is also assumed that either the active ingredient and/or metabolites remain in the plant for a long time. This is evi- denced by the long term effects that this systemically active substance has on insect pests in many crops. An accurate evaluation for the persistent effect has not been previously made, as up till now, no adequate testing method has been available. That is why the long term influence of neonicotionid insecticides (imidacloprid, acetamiprid, thiacloprid) on parasitoid behaviour was examined in extended laboratory tests. This type of investigation became possible due to an imidacloprid resistent line of Bemisia tabaci becoming available. The results show that imidacloprid in particular, frequently used in poinsettia stock plants, has a long lasting repelling and lethal effect on E. formosa, whereas acetamiprid had a minor effect and thiacloprid showed no persistent effect. Apart from this, none of the substances were efficient against the used B. tabaci line.

Key words: Encarsia formosa, test method, pesticid, persistence, side effect, imidacloprid, acetamiprid, thiacloprid

Introduction

Controlling whiteflies with the parasitoid wasp Encarsia formosa in poinsettia (Euphorbia pulcherrima Willd. ex Klotsch) has been a common system for more than three decades. To that time poinsettia were usually infested with the white fly species Trialeurodes vaporari- orum (Westwood). In the middle of the 1980‘s the species Bemisia tabaci (Gennadius), which is more difficult to control with biological or chemical means entered European greenhouses. Subsequently, the use of chemical pesticides increased. Since that time a decline in the efficacy of Encarsia formosa in German poinsettia production has been noted. The objective of our work was to find reasons for this reduced efficacy. One reason for a decrease in efficacy of beneficials can be side effects of pesticides. Hence, one part of our work was to examine the influence of neonicotinoids, especially the active ingredients imidacloprid, acetamiprid and thiacloprid, on parasitation behaviour of the parasitoid. Many insecticides have a repellent impact on E. formosa. The parasitoids do not approach treated plants. Particularly the systemic substance imidacloprid is well known to have a long-term effect on pests in many crops and even on trees. This leads to the assumption that either the active ingredient and/or metabolites remain in the plant for a long time. The producing company describes imidacloprid to be easily uptaken and translocated within the plant. If drenched there would be a supply within the plant that is replenished from the soil reservoir over an extended period of time. Also the active ingredient slowly metabolizes within the plant.

61 62

Although it is known that spraying imidacloprid has a negative effect on E. formosa, drenching plants was supposed to be harmless, because beneficial insects are not exposed directly to the substance. For an accurate evaluation of the persistent effects of pesticide treatments no adaequate test method has existed until now. EPPO recommends a sequential testing scheme with three laboratory tests for direct and indirect toxicity and if the results are doubt- ful a field tests for final rating (Oomen, 1989). But until now it was not possible to expose the wasps to contaminated white fly larvae as food for host feeding and for parasitation.

Material and methods

The influence of three insecticides (imidacloprid, acetamiprid, thiacloprid) on the parasitation behaviour of the parasitoid E. formosa was examined. For these investigations, fortunately, an imidacloprid resistent line of Bemisia tabaci was available. Four infested poinsettia plants were each put into insect safe cages in a climatic chamber. They were about the size of small semi-finished plants infested with B. tabaci two weeks before the insecticide was applied. In the first cage plants were sprayed with a hand sprayer to the point of run-off with the active ingredient, in the second they were drenched and in the third cage they were left untreated (concentration see Table 1). From the time of treatment one infested leaf with 80 to 100 larvae at minimum was taken from each cage weekly. It’s stalk was put into a test tube with water and then the leaf was put together with a commer cially available „Encarsia-card“ into a transparent dish (ca. 20 cm x 20 cm x 8 cm, Figure 1). Each following week, leaves were controlled for larvae of B. tabaci, parasitised larvae, empty cases of parasitised larvae, adult white flies and the condition of the wasps. All insecticides were tested twice.

Table 1. Tested plant protection products

active ingredient trade name concentration imidacloprid Confidor® WG 70 (a.i. 700 g/kg) 0,035 % acetamiprid Mospilan (a.i. 200 g/kg) 0,05 % thiacloprid Calypso (a.i. 480 g/l) 0,025 %

Results and discussion

The system worked well. Most of the leaves could be kept in the dishes for five to six weeks. Depending on the treatment, white flies and wasps could develop well during this time. Only in some cases the leaves wilted or showed growth of fungal myzelia after one to two weeks. On those leaves the parasitation rate was certainly lower because of the short time for development and they had to be removed. With imidacloprid the effects were observed for more than thirty weeks, but from week 18 it became more and more difficult because the plants suffered from white fly infestation and were covered with honeydew. The results also show that there are clear differences in persistence between the three pesticides.

Influence of imidacloprid on parasitation behaviour of Encarsia formosa Figure 2 presents the results on the influence of imidacloprid on the parasitation behaviour of E. formosa. In the first week E. formosa still caused a high parasitation rate on all leaves, treated or not. Further on, on untreated leaves parasitised white fly larvae could be found after 63

two to three weeks. This was clearly visible when pupae of B. tabaci changed their colour from yellow to brown from the second week after being parasitised by E. formosa. Hence, parasitation rate was also depending on the condition of the leaves in the dishes. Low parasitation rates in weeks 6, 7, 10, 11, 18 und 21 resulted from leaves which perished after two or three weeks. In dishes with untreated leaves after a short time all larvae were parasitised or killed by host feeding. No white fly larvae could develop to an adult state. Additionally, live wasps could always be found. Also, from parasitised larvae a new generation of wasps emerged after three to four weeks.

Figure 1. Transparent dish with poinsettia leaf in test tube and „Encarsia-card“

The reason for the delay in the effect of imidacloprid on E. formosa in the first week may be that the active ingredient had still not reached all parts of the plant. Although separated from the plant on treated leaves white fly larvae developed well, adults emerged and repro- duced, while the parasitoids did not. The parasitation rate went to zero. After spraying the plants, the repellent effect of imidacloprid lasted approx. 16 weeks. After drenching the effect lasted even longer. After spraying the leaves with imidacloprid it also took 16 weeks until living wasps could again be found in the dishes. Though some parasitised pupae were found in between, wasps generally died during emergence or afterwards. Surprisingly, in week 16 after treatment parasitation rates rose on sprayed leaves. From these results, it can be concluded that it will take four month until E. formosa works well again. When the plants were drenched, which means the amount of the pesticide was higher and with a better distribution in the plan, this period lasted even longer. Some living wasps were found from week 23 after treatment, but parasitation rate was satisfactory only from week 33 after treatment. This result means that imidacloprid, frequently used in stock plants, has a long lasting repellent and lethal effect on the beneficial wasp E. formosa (Richter et al., 2003).

100 .

40 parasitation rate in % 20

0 1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526 week

"untreated" imidacloprid sprayed imidacloprid drenched

Figure 2. Parasitation rate of Encarsia formosa on poinsettia leaves infested with Bemisia tabaci after treatment with imidacloprid

Parasitation rate of E. formosa on poinsettia leafs infested with B. tabaci

100

. 80

20 Parasitation rate in % 0 1234567891011121314151617 week

untreated acetamiprid sprayed acetamiprid drenched

Figure 3. Parasitation rate of Encarsia formosa on poinsettia leaves infested with Bemisia tabaci after treatment with acetamiprid

Influence of acetamiprid on parasitation behaviour of Encarsia formosa After treatment with acetamiprid the white fly population was reduced but showed a quick recovery, also called resurgence (Birnie & Denholm, 1992), which is probably due to a cross resistance to the active ingredient. A study from Almeria (Spain) revealed that 65

neonicotinoid-resistant Q-type strains were often more than 100-fold less susceptible to thiamethoxam, acetamiprid and imidacloprid when tested in laboratory bioassays (Nauen et al., 2002). Figure 3 presents the results of the influence of acetamiprid on the parasitation behaviour of E. formosa. In the first week E. formosa still achieved a high pest parasitation rate on leaves from untreated and drenched plants. Furthermore, wasps on untreated leaves showed the same reaction as in the previous trial. The reason for the delay in the effect of acetamiprid on E. formosa in the first week again may be that the active ingredient had still not reached all parts of the plant when drenched. The effect after drenching with acetamiprid was clearly different from the effect of imidacloprid. During the four weeks after treatment parasitation rates slowly went to zero and remained there until week 11. Uptake or translocation of acetamiprid within the plants seems to be slower. On sprayed leaves there was no parasitation of E. formosa. This effect lasted also approx. 11 weeks. After 11 weeks in both treatments the parasitation rate of E. formosa was similar to the untreated control. In dishes with untreated leaves living wasps could always be found. After treatment with acetamiprid living wasps could also be found in the dishes but only in small numbers until week 12.

Influence of thiacloprid on parasitation behaviour of Encarsia formosa After treatment with thiacloprid the white fly population was also reduced but showed a quick recovery, which is probably due to a cross resistance to the active ingredient. E. formosa caused a high parasitation rate on leaves from untreated plants. On leaves from treated plants there was a reaction of E. formosa only in the first week. Afterwards the parasitation rates in all treatments adapted. In week 7 the trial was finished because all plants were sticky from honeydew and somehow some individuals of E. formosa had entered all cages and parasitised the white flies.

Acknowledgements

Many thanks to the company Sautter & Stepper for providing the beneficials and the helpful discussions and to all my assistants for helping to count thousands of Bemisia tabaci larvae.

References

Birnie, L.C. & Denholm, I. 1992: Use of field simulators to investigate integrated chemical and biological control tactics against the cotton whitefly, Bemisia tabaci. – Brighton Crop Protection Conference – Pests and Diseases 3: 1003-1008. Richter, E., Albert, R., Jaeckel, B. & Leopold, D. 2003: Encarsia formosa – Eine Erzwespe für den biologischen Pflanzenschutz unter dem Einfluss von Insektiziden und wechseln- den Wirten. – Nachrichtenblatt deut. Pflanzenschutzd. 55(8): 161-172. Nauen, R., Stumpf, N. & Elbert, A. 2002. Toxicological and mechanistic studies on neonicotinoid cross resistance in Q-type Bemisia tabaci (Hemiptera: Aleyrodidae). – Pest Manag. Sci. 58(9): 868-875. Oomen, P. 1989: Guideline for the evaluation of side-effects of plant protection products – Encarsia formosa. – EPPO Bulletin 19: 355-372.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 67-79

Mancozeb: A profile of effects on beneficial and non-target arthropods

M. Miles Dow AgroSciences, European Development Centre, 2nd Floor, 3 Milton Park, Abingdon, OX14 4RN, UK

Abstract: Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide with multi-site modes of action against economically important fungal diseases and is the active substance in Dithane1 fungicides. It is a broad spectrum contact fungicide with high protectant activity. To date there are no recorded incidences of resistance, despite many years of use on high resistance risk diseases. Due to this, mancozeb is a key strategic fungicide in resistance management programmes and is registered for use in a wide range of crops globally. Mancozeb is well known for its side-effects on certain phytoseiid mites; however during the use of the product a wide range of other important beneficials may also be exposed. This paper reviews a wide range of studies on the effects of mancozeb on predatory and parasitic arthropods and provides new information from studies with soil mites, spiders and predatory Heteroptera. Overall mancozeb was shown to have low toxicity to parasitic Hymenoptera, Coccinellidae, Chrysopidae, Syrphidae, Carabidae, Aranea and Laelapidae. Effects were seen on certain species of predatory mite (Phytoseiidae) however, use patterns compatible with Integrated Pest Management programmes and the conservation of predatory mites have been developed which cause minimal impact on naturally occurring populations of predatory mites.

Keywords: Fungicide, Mancozeb, predatory mite, beneficial arthropod, side-effects, field, laboratory

Introduction

Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide with contact activity against a wide range of economically important fungal diseases and is sold and marketed under the trademark of Dithane1 fungicides. Its multi-site mode of action means that to date there have been no recorded incidences of resistance developing despite many years of use on high risk diseases (Anon., 2002). For example, in vines Grape downy mildew (Plasmopara viticola) has developed resistance or reduced sensitivity to a number of important oomycete specific fungicides following their introduction onto the market (Bulletins de Stations d’Avertissements Agricoles, 2003). The role of Mancozeb either as a mixing or alternation partner in helping to manage resistance situations remains critically important (Hutson & Miyamoto, 1998). Mancozeb is an important disease management tool with registrations on over 70 crops for over 400 diseases world wide. Major crops for the use of mancozeb include potatoes, vines, pome fruit, citrus, bananas, cucurbits, corn, lettuce, beets, peas, ornamentals crops and cereals. In Europe the majority of uses are on potato and vine crops. The effects of mancozeb on beneficial arthropods have been investigated with most researchers focusing their activities on the well known potential impact to predatory mites (e.g. Mathys, 1958, Bostanian et al. 1998, Sentenac et al. 2002). A more general range of effects was reported in the third joint pesticide testing programme of the IOBC/wprs where the effects of mancozeb as Dithane Ultra 80% was evaluated according to established IOBC guidelines at the rate of 480 g a.s./ha. This scheme of independent testing established that

1 Trademark of Dow AgroSciences LLC

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mancozeb was of low toxicity (IOBC class 1) to a wide range of predatory and parasitic insects in laboratory and semi-field tests. Toxicity was noted in the laboratory to several phytoseiid mites although repeated applications were necessary to bring about harmful effects in the field. The glasshouse predatory mite Phytoseiulus persimilis was unaffected with an IOBC class 1 category reported for both laboratory and semi-field testing. Mancozeb has successfully undergone the re-registration process conducted in the USA by the USEPA and also recently achieved Annex I listing for an active substance within the European Union (SANCO/4058/2001 - rev. 4.3). These achievements demonstrate the continued importance of mancozeb in crop disease management along with high levels of environmental and human safety. As part of the Annex I listing process and to be in line with current horticultural and agricultural practice the use pattern of mancozeb has been modernised. It is currently proposed that in vines that mancozeb will be used 2 to 4 times per season at an application rate of 1.6 g a.s./ha with a spray interval of 7 to 14 days. In potatoes, the application rate will also be 1.6 g a.s./ha with up to 8 sprays per season applied weekly. Mancozeb, either alone or in mixtures, is used to control fungal diseases in crops where the conservation of predatory insects and mites is an important component of Integrated Pest Management (IPM). A range of laboratory and field tests were conducted with mancozeb on beneficial insects and mites. The objective of this paper was to review the effects of mancozeb to a range of predatory insect and mite species in light of the proposed use patterns in vines and potatoes to give an accurate and up to date profile of this important disease management tool.

Materials and methods

Experimental test systems (study type and species) are outlined in Table 1. All studies were carried out using formulated mancozeb. Although a range of different products were tested, all are considered representative of the toxicity and effects of mancozeb. In all tests a suitable toxic reference and water treated control treatment were also included.

Table 1. Study type and beneficial species tested in side-effects studies with mancozeb formulations.

Study type Test item Species Laboratory (tier I) Manex2 II SC Aphidius rhopalosiphi, Typhlodromus Dithane M45 pyri, Chrysoperla carnea, Poecilus cupreus Extended laboratory Dithane Ultra 75WG T. pyri, Orius laevigatus, Dithane M45 Pardosa spp., Hypoaspis aculeifer, A. rhopalosiphi, C. carnea Field tests Dithane NeoTec 75WG, T. pyri, Euseius stipulatus, Dithane 75WG, Kampimodromus aberans Dithane M45

2 Trade mark of Griffin

Laboratory (tier I) studies Laboratory tier I studies were conducted to investigate the intrinsic toxicity of mancozeb to a range of representative beneficial arthropod species (see Table 1). In all tests a suitable toxic reference and water treated control treatment were also included. Sensitive life stages were exposed to artificial substrates (glass plate, quartz sand) according to internationally recognised guidelines. A study with A. rhopalosiphi followed the method of Poglar (1988) with modifications by Mead-Briggs (1992). Adult wasps were exposed to glass plates treated with mancozeb (as Manex II SC) at 2.6 kg a.s./ha for 48 hours. The parasitism rate of surviving female wasps was assessed using cereal seedlings infested with bird cherry aphids (Rhopalosiphum padi) as a suitable host. In the mortality phase each treatment was replicated three times with ten wasps per test unit and in the parasitism phase, ten individually confined wasps were used per treatment. For the predatory mite T. pyri, a glass, coffin cell method was used (Blümel et al, 2000a). Each test unit consisted of two glass plates which form the top and base of the test units, held together by a middle unit of inert material (Polytetrafluorethylene PTFE). Holes are present in the base glass plate to allow for ventilation, water supply and feeding. All parts of the test unit were treated with mancozeb (as Dithane M-45) in a rate response test to estimate the rate corresponding to 50% mortality (LR50). Twenty protonymph mites were placed in each test unit and fed with untreated pollen. Each mancozeb treatment was replicated four times, the control was replicated five times and the toxic reference (dimethoate) three times. Mortality was assessed after seven days exposure and the fecundity of the surviving female mites was assessed between days seven and fourteen. A total of forty larval C. carnea less than 24 hours old were exposed individually to glass plates treated with mancozeb (as Manex II SC) at 2.4 kg a.s./ha using the method of Bigler (1988). Forty larvae were also exposed to a water treated control and a toxic reference item (pyrazophos). Larval mortality and fecundity of the surviving adults was measured. The effect on adult carabid beetles (P. cupreus) was investigated using the method of Heimbach (1992). Test units containing quartz sand, holding three male and three female beetles were prepared with fly pupae as food. Mancozeb (as Manex II SC) was applied at 2.4 kg a.s./ha. Each treatment was replicated six times and mortality and food consumption assessed up to fourteen days after treatment. Extended laboratory studies These tests are characterised by the inclusion of a natural substrate in the test system. All studies included appropriate control and toxic reference treatments. The effect of mancozeb on the predatory mite T. pyri was investigated using the laboratory tier I open design of Blümel et al (2000a). Leaf discs were substituted for glass plates and a rate response test was conducted to estimate the rate corresponding to 50% mortality. Six rates of mancozeb (as Dithane M-45) were tested and each was replicated three times with each test unit housing 10 mites. To investigate the effect of repeated applications, aged residue extended laboratory tests were conducted on A. rhopalosiphi and C. carnea using a multiple application factor (MAF) to calculate doses. Rates equivalent to 3.9x and 4.8x the single maximum field application rate of mancozeb as Dithane NeoTech 75WG (6.29 and 7.69 kg a.s./ha) were tested to simulate extreme worse case exposure arising from season-long spray programmes. Bioassays were conducted on the day of application (when spray deposits had dried) and after one week of ageing following the methods of Mead-Briggs and Longley (2000) for A. rhopalosiphi and Vogt et al (2000), modified for bean leaf substrate, for C. carnea. For A. rhopalosiphi pots of barley seedlings were sprayed and six replicates of five adult female wasps were used in each bioassay. The fecundity of surviving wasps was assessed. Lacewing larvae two to three days

old were used for the bioassay with forty exposed to each treatment. Fecundity of the surviving adults was assessed. Appropriate toxic reference and control treatments were included for both species. An extended laboratory test was conducted to evaluate the effect of mancozeb as Dithane M-45 to the predatory bug Orius laevigatus following the method of Bakker et al (2000). Detached cow pea leaves were substituted for glass plates using a modified Munger cell design. Mancozeb was tested at three rates of 0.8, 1.6 and 3.2 kg a.s./ha (equivalent to 0.5x, 1x and 2x the application rate). Seven second instar nymphs were placed into each replicate; bugs were fed with untreated pollen and had access to clean water. Mancozeb treatments were replicated nine times; other treatments were replicated seven times. Mortality was observed over a nine day exposure period and egg production was assessed for surviving females. Following the method of Heimbach et al. (2000), adult Pardosa spp. were exposed individually in test units containing LUFA 2.2 soil to mancozeb as Dithane M-45 applied at 1.6 and 3.2 kg a.s./ha. For each treatment, fifteen male and fifteen female spiders were exposed for fourteen days and mortality and food consumption were measured throughout. Soil dwelling mites (Hypoaspis aculeifer) were exposed to mancozeb as Dithane M-45 in artificial OECD soil with a pH of 6.5 containing 10% sphagnum, 20% kaolin clay and 70% sand. Test solutions were homogenously mixed to give three exposure concentrations of 1.1, 2.1 and 4.3 mg mancozeb/kg dry soil (equivalent to spray application rates of 0.8, 1.6 and 3.2 kg a.s./ha applied to bare soil). Treated soil was placed into modified Munger cells and twenty H. aculeifer protonymphs were introduced. Each treatment was replicated five times (except the toxic reference where n = 3) and the predators were fed the mite Tyrophagus putrescentiae as prey. Mortality was assessed 21 days after initiation. Surviving mites were transferred to untreated units and fecundity was assessed twice over a seven day period. The soil mite test followed the method of Bakker et al (2003) which has the benefit of separate mortality and fecundity end points. Field studies A series of field studies in vines was conducted in 2004 to investigate the effect of number and timing of applications of mancozeb to predatory mites. Studies were conducted in Portugal, Germany, France and Italy reflecting a range of mite species and growing conditions. Two studies were undertaken in each country, giving a total of eight trials. The species of mite present in each trial was determined. All studies followed the guideline of Blümel et al. (2000b) and a summary of test information and mite species present is given in Table 2. The impact of two applications of mancozeb applied before flowering was compared to two applied during / post flowering as well as the effects of four sprays over the same pre- to post flowering period. Each mancozeb application was standardized to a rate of 1.6 kg a.s./ha per spray. Methidathion or deltamethrin were applied six times as a toxic reference and a water control or soft reference was included. Details of all treatments are given in Table 3. Treatments were applied to experimental plots using a backpack sprayer at water volumes appropriate to the growth stage of the vines and local practice. Leaves were sampled (minimum of 25 / plot) at intervals and the number of motile stages of mites determined using a washing technique. Leaves were sampled before application, approximately seven days after application timing B (7DAAB), seven days after application timing D (7DAAD), twenty-one days after application timing D (21DAAD), and end of the trial (35 – 45 days after application timing D). The number of mites at each post-treatment assessment time was corrected by control population levels according the method of Abbott (1925).

Table 2. Summary of test information for field trials conducted in 2004 on vines with mancozeb on predatory mites.

Country Test substance Target Trial code Portugal Dithane M45 Typhlodromus pyri POR 1 (800 g a.s./kg WP) POR 2 Germany Dithane NeoTec Typhlodromus pyri GER 1 (750 g a.s./kg WG) GER 2 France Dithane NeoTec Kampimodromus aberrans FRA 1 (750 g a.s./kg WG) Typhlodromus pyri FRA 2 Italy Dithane DG Kampimodromus aberrans ITA 1 (750 g a.s./kg WG) ITA 2

Table 3. Summary of treatment timings for field trials conducted in 2004 on vines with mancozeb on predatory mites.

Treatment Application Growth stage Growth stage description timing code (BBCH code) Mancozeb AB 53 to 57 Pre-flowering 2 x 1.6 Kg a.s./ha Mancozeb CD 60 to 70 Flowering to fruit set 2 x 1.6 Kg a.s./ha Mancozeb ABCD 53 to 70 Pre-flowering to fruit set 4 x 1.6 Kg a.s./ha Soft reference ABCDEF 53 to 75 Pre flowering to pea size (dimethomorph/folpet/water) berries Toxic reference ABCDEF 53 to 75 Pre-flowering to pea size (deltamethrin/methidathion) berries

Table 4. IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classification system for side effects of plant protection products to beneficial and non-target arthropods (Hassan 1992).

Classification of effects The effect of mancozeb was categorised according to the IOBC (International Organisation for Biological and Integrated Control of Noxious Animal and Plants) classifications (Hassan 1992) described in Table 4.

Results and discussion

The results from the laboratory tier I studies with mancozeb conducted with representative beneficial arthropod species are presented in Table 5. Both lethal and sub-lethal effects are listed along with the performance of the untreated and toxic reference treatments. The performance of the control and toxic reference treatments indicated valid studies according to the guidelines in force at the time of conduct. When A. rhopalosiphi adults were exposed to 2.6 kg a.s./ha on glass plates there was no mortality observed above that of the control treatment; however a minor reduction in parasitism rate for those wasps exposed to mancozeb was observed (IOBC class 2). Due to known toxicity of mancozeb to Phytoseiid mites a dose response test was conducted and an LR50 of 26.67 g a.s./ha with 95% confidence intervals (C.I.) of 22.9 - 31.63 g a.s./ha) was estimated for T. pyri. This value is considerably lower than the application rate of 1.6 kg a.s./ha, indicating intrinsic toxicity to this species under worst case tier I conditions. Mancozeb tested at 2.4 kg a.s./ha caused no adverse effects to C. carnea or P. cupreus under tier I laboratory conditions. A range of studies were conducted under more realistic but still highly stringent extended laboratory conditions (Table 6). When O. laevigatus was exposed to treated leaves with rates of 0.8, 1.6 and 3.2 kg a.s./ha a rate related response was noted for mortality with 26, 36 and 48% corrected mortality observed respectively; no effect was noted on fecundity at 0.8 and 1.6 kg a.s./ha but a 32% reduction was observed at the top rate. Consequently mancozeb was classified as IOBC class 2 to O. laevigatus. A. rhopalosiphi and C. carnea were exposed to extreme rates of mancozeb which would be far in excess of those that would be normally encountered in the field (6.29 and 7.69 kg a.s./ha). Both rates had no effect on the survivorship of adult A. rhopalosiphi wasps (Figure 1). A minor effect on parasitism rate compared to the control was noted at the lower rate of 6.29 kg a.s./ha but not at the higher rate. However when wasps were exposed to treated barley which had been aged outside for 7 days both treatments were classified as harmless (less than 25% effect), IOBC class 1. Initially (bioassay on day of application) in the C. carnea study a low level of larval mortality was observed (Figure 2), however the higher rate caused greater than 25% mortality (actual = 36%) indicating IOBC class 2 at this rate. Neither treatment affected reproductive parameters of the surviving insects. When larvae were exposed to leaves which had been allowed to aged for 7 days, mortality was below 25% for both treatments and egg production and hatch was unaffected. Overall these high rates of 6.29 and 7.69 kg a.s./ha had very limited impact on either species tested. A dose-response study with T. pyri conducted on bean leaf discs under extended laboratory conditions gave an LR50 of 104.4 g a.s./ha (95% C.I.48.6 - 213.4 g a.s./ha). At the test rate of 20 g a.s./ha a 53% reduction in egg production was also noted. Mortality and feeding rates for Pardosa spp. were unaffected by mancozeb applied at 1.6 and 3.2 kg a.s./ha (IOBC 1). This is an interesting result as it demonstrates that spiders are not affected in the same way as Phytoseiid mites, indicating a specificity of effects. This was confirmed by a study on the highly sensitive soil mite H. aculeifer. Rates in soil equivalent to 0.8, 1.6 and 3.2 kg a.s./ha had limited effects on exposed mites (IOBC class 2). Although exposure in soil is different to that on leaves this finding does suggest that the effect of mancozeb to certain types of mites could be very specific.

Laboratory (tier I and extended) tests have robustly confirmed that mancozeb is of low toxicity to wide range of beneficial insect, mite and spider species and that the most sensitive group are the leaf dwelling Phytoseiid mites such as T. pyri. The effects of mancozeb to natural populations of the predatory Phytoseiid mites were investigated in vines in a series of field studies conducted in 2004. In order to gain a thorough understanding of the impact to mites new studies were conducted following the revised use pattern and rates for vines (2 – 4 applications at 1.6 kg a.s./ha). The findings at key assessment times are summarized in Table 7. An acceptable level of effect on the mite populations can be defined as 50% or less impact equivalent to IOBC class 1 or 2. This level of impact was deemed acceptable as mite populations would be expected to rapidly recover from this level of reduction with limited impact on pest regulation provided by the predators. This level of effect would be acceptable to most growers and advisors.

Table 5. Effects of mancozeb to a range of beneficial arthropod species in tier I laboratory tests.

Test species Treatment M (%) Corr M P/R/F IOBC (%) (% reduction) Class Aphidius mancozeb 2.6 kg/ha 13.3 -0.4 P = 9.0 (36.2%) 2 rhopalosiphi pyrazophos 100* 100 N/A Control 13.7 --- P = 14.1

Typhlodromus mancozeb LR50 = 26.67 g/ha N/A pyri 0.1 – 31 g/ha (95% C.I. 22.9 – 31.63 g/ha) dimethoate 100* 100 N/A Control 20 --- R = 6.2 Chrysoperla mancozeb 2.4 kg/ha 6.7 -0.3 R = 910 (12.2%) 1 carnea pyrazophos 100* 100 N/A Control 7.0 --- R = 1037 Poecilus mancozeb 2.4 kg/ha 3.3 0.0 F = 4.29 (8.6%) 1 cupreus pyrazophos 86.7* 86.2 2.50* (46.7%) Control 2.5 --- F = 4.69

Notes: * Indicates significant difference from control (P = 0.05). M=Mortality, Corr. M=Corrected mortality, P=parasitism as mummies/female, R= fecundity as eggs/female, F=food consumption No. prey/individual/day.

From the studies (Table 7) it can be seen that there is a range of effects of mancozeb to predatory mites. Less than 50% impact was observed in 6 out of the eight trials when assessed one week after two applications of mancozeb at 1.6 kg a.s./ha applied before flowering. In fact in four of the trials less than 25%impact was noted indicating IOBC class 1, two trials were in IOBC class 2 and two trials in IOBC class 3. No findings were in the harmful

category (IOBC class 4). By the end of the study all observations for this treatment regime were at IOBC class 1 showing full recovery where effects had been seen. Two applications applied at a later timing (during flowering) were seen to have similar limited effects. Seven days after the second application no observation was above 50% effect with three of the observations in IOBC class 1 and five in IOBC class 2. By the end of the season in five out of eight trials mites had returned to control levels (IOBC class 1), one to IOBC class 2 and two at IOBC class 3. Seven days after a series of four applications at 1.6 kg a.s./ha applied from pre-flowering to end of flowering 50% or less effect was noted in half of the studies. IOBC

Table 6. Effects of mancozeb to a range of beneficial arthropod species in tier II extended laboratory tests.

Test species Treatment M (%) Corr M P/R/F IOBC (%) (% reduction) Class

Typhlodromus mancozeb LR50 = 104.4 g/ha N/A pyri 20 – 640 g/ha (95% C.I.48.6 – 213.4 g/ha) R = 53% effect at 20 g/ha dimethoate 100* 100 N/A Control 11.3 --- R = 5.4 Orius laevigatus mancozeb 0.8 kg/ha 41 26 R = 14.6 (11%) 2 mancozeb 1.6 kg/ha 49 36 R = 19.2 (-17%) 2 mancozeb 3.2 kg/ha 59 48 R = 11.1 (32%) 2 dimethoate 100* 100 N/A Control 21 --- R = 16.4 Pardosa spp. mancozeb 1.6 kg/ha 3.0 --- F = 3.8 (5%) 1 mancozeb 3.2 kg/ha 7 --- F = 3.7 (7.5%) 1 lambda-cyhalothrin 43* --- F = 3.5 (12.5%) Control 0.0 --- F = 4.0 Hypoaspis mancozeb 0.8 kg/ha 17 3.0 R = 13.9 (23%) 1 aculeifer mancozeb 1.6 kg/ha 20 7.0 R = 13.8 (23%) 1 mancozeb 3.2 kg/ha 27* 15 R = 13.5 (25%) 2 dimethoate 72* 67 N/A Control 14 --- R = 17.9

a) Bioassay on day of application

100 * 90 mancozeb 6.29 kg a.s./ha 80 mancozeb 7.69 kg a.s./ha 70 control

60 dimethoate

0 %Mortality No. mummies/female %Reduction in parasitism

b) Bioassay 7 days after application

100

90 mancozeb 6.29 kg a.s./ha

80 mancozeb 7.69 kg a.s./ha

70 control

60 dimethoate 50

0 %Mortality No. mummies/female %Reduction in parasitism -10

-20

Figure 1. Effects of mancozeb on Aphidius rhopalosiphi in a tier II aged residue extended laboratory test. * Denotes significant difference from the control at P = 0.05.

a) Bioassay on day of application

100

90 Mancozeb 6.29 kg a.s./ha * 80 Mancozeb 7.69 kg a.s./ha Control 70 Dimethoate 60

50 * 40

30 * 20

0 %Mortality No. eggs/female/day %Egg viability

b) Bioassay 7 days after application

100

90 Mancozeb 6.29 kg a.s./ha 80 Mancozeb 7.69 kg a.s./ha 70

60 Control

0 %Mortality No. eggs/female/day %Egg viability

Figure 2. Effects of mancozeb on Chrysoperla carnea in a tier II aged residue extended laboratory test.* Denotes Significant difference from the control at P = 0.05.

class 1 was recorded in two trials, class 2 in two trials and class 3 in four trials. Good levels of recovery were observed by most populations as in six out of eight trials the final assessment indicated IOBC class 2 or 1. In two trials mite numbers had increased in effect from 7DAAD to Class 3. ANOVA of the data set indicated a highly significant effect of treatment on the mite populations (P < 0.001) and allowed the treatments to be ranked in order of effect). The least harmful treatment was two applications of mancozeb at 1.6 kg a.s./ha applied pre-flowering, followed by two applications during flowering. Four application of mancozeb at 1.6 kg a.s./ha caused a higher level effect than either of the other two application scenarios. The toxic reference insecticide treatments were by far the most harmful to predatory mites in all of the trials. Overall 2 – 4 applications per season of mancozeb at 1.6 kg a.s./ha (3.2 to 6.4 kg a.s./ha in total) was shown to be generally compatible with predatory mites in most vine growing situations. This observation is in good agreement with an analysis conducted by Miles and Green (2002). The impact at the end of each season on residual populations of mites from a wide range of studies in vines and apples was analysed in relationship to the total annual loading of mancozeb to the mites. It was found that in the majority of trials (over 96%) rates up to 8 kg a.s./ha/year of mancozeb was shown to preserve populations of predatory mites. In rare cases where reductions above the 50% levels are observed at the end of a season no between season effects are anticipated. Findings from multi-year field trials in vines have shown that reductions in mite numbers due to applications of mancozeb do not lead to significantly reduced numbers of predators compared to the control in the following year (Mattoida, 1999). Therefore, in the unlikely case that mite numbers would be depressed below the 50% level at the end of a growing season no between years effects are anticipated.

Table 7. Summary of field trial results as % reduction compared to control at key assessment times in field trials in Europe in 2004 with naturally occurring populations of predatory mites.

Trial code %Reduction / Abbott (time) Mancozeb AB Mancozeb CD Mancozeb ABCD Toxic reference 7DAAB End 7DAAD End 7DAAD End Max. End POR 1 34 5 22 22 36 34 98 97 POR 2 21 -19 8 -34 4 17 97 97 GER 1 25 12 40 38 56 44 60 44 GER 2 -28 17 45 62 54 65 67 67 FRA 1 -6 1 -22 -34 -18 -6 91 72 FRA 2 58 16 34 53 49 70 73 78 ITA 1 67 24 46 18 65 14 99 85 ITA 2 -8 18 50 18 56 46 93 81 Note: Figures in bold indicate mancozeb treatments which exceeded 50% impact on mite populations.

Conclusions

It was concluded that mancozeb was highly selective to the majority of beneficial insect, mite and spider species tested in tier I laboratory and extended laboratory studies. Exceptions to this are the findings from studies on the predatory mite T. pyri under tier I and II conditions. Under field conditions the effects of 2 – 4 applications of mancozeb at 1.6 kg a.s./ha on

predatory mites (T. pyri and K. aberrans) in vines was studied. These studies conducted under different climatic and growing conditions in Europe confirmed that two to four applications per season of mancozeb were suitably selective. It was concluded that this use pattern for mancozeb could be integrated into disease control programmes in vines at a variety of spray timings giving the grower excellent flexibility, disease control and selectivity to predatory mites and other beneficial arthropods. The low impact on soil mites and other arthropods confirmed the acceptability of the use programme in potatoes (8 x 1.6 kg a.s./ha). Overall, the proposed use patterns for mancozeb in vines and potatoes are compatible with Integrated Pest Management programmes and the conservation of predatory insects and mites.

References

Anon. (2002). An introduction to Dithane. – Dow AgroSciences technical bulletin Y45-169-001 (07/02) DAS. Abbott, W.S. (1925). A method of computing the effectiveness of an insecticide. – J. Econ. Entomol., 18, 265 – 267. Bakker, F.M., Aldershof, S.A., Vieire, M. v.d., Candolfi, M. P., Izquierdo, J.I., Kleiner, R., Neumann, Ch., Nienstedt, K.M. & Walker, H., (2000). A laboratory test for evaluating the effects of plant protection products on the predatory bug, Orius laevigatus (Fieber) (Heteroptera: Anthocoridae). – In: Candolfi, M.P. et al. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 57-70. Bakker, F.M., Feije, R., Grove, A.J., Hoogendoorn, G., Jacobs, G., Loose, E.D. & Stratum, P. van (2003). A laboratory test protocol to evaluate the effects of plant protection products on mortality and reproduction of the predatory mite Hypoaspis aculeifer Canestrini (Acari: Laelapidae) in standard soil. – Journal of soils and sediments 3 (2): 73-77. Blümel, S; Baier, B; Bakker, F; Brown, K; Candolfi, M; Goßmann, A; Grimm, C; Jäckel, B.; Nienstedt, K; Schirra, K.J; Ufer, A. & Waltersdorfer, A. (2000a). Laboratory residual contact test with the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae) for regulatory testing of plant protection products. – In: Candolfi, M.P. et al. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 121-143. Blümel, S., Aldershof, S., Bakker, F., Baier, B., Boller, E, Brown, K, Bylemans, D., Candolfi, M.P., Huber, B., Linder, C. Louis, F. Müther, J., Nienstedt, K.M., Oberwalder, C., Reber, B., Schirra, K.J., Ufer, A. & Vogt, H. (2000b). Guidance document to detect side effects of plant protection products on predatory mites (Acari: Phytoseiidae) under field conditions: vineyards and orchards. – In: Candolfi, M.P. et al. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS; Gent, Belgium: 145-158. Bigler, F. 1988: A laboratory method for testing side-effects of pesticides on larvae of the green lacewing, Chrysoperla carnea Steph. (Neuroptera, Chrysopidae). – IOBC/wprs Bulletin 11 (4): 71-77. Bostanian, N.J., Thistlewood, H. & Racette, G. (1998). Effect of five fungicides used in Quebec apple orchards on Amblyseius fallacis (Garman) (Phytoseiidae: Acari). – Journal of Horticultural Science 73: 527-530. Bulletins de Stations d’avertissements agricoles (2003). Note National mildiou de la Vigne 2004. Bulletin no. 28 du 19 November 2003 – 3 pages.

Hassan, S.A., 1992: Guidelines for testing the effects of pesticides on beneficial organisms: Description of test methods. – Pesticides and Beneficial Organisms IOBC/wprs Bulletin 1992/XV/3 15: 1-3. Heimbach, U., (1992). Laboratory method to test effects of pesticides on Poecilus cupreus (Coleoptera, Carabidae). – Pesticides and Beneficial Organisms IOBC/wprs Bulletin 1992/XV/3 15: 103-109. Heimbach, F. Wehling, A., Barrett, K. L., Candolfi, M. P., Jäckel, B., Kennedy, P. J., Mead-Briggs, M., Nienstedt, K. M., Römbke, J., Schmitzer, S., Schmuck, R., Ufer, A. and Wilhelmy, H. (2000). A method for testing effects of plant protection products on spiders of the genus Pardosa (Araneae: Lycosidae) under laboratory conditions. – In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, p. 71-86. IOBC/WPRS; Gent, Belgium, ISBN 92-9067-129-7. Mathys G. (1958). The control of phytophagous mites in Swiss vineyard by Typhlodromus species. – 10th International Congress of Entomology 4: 607-610. Mattoida, H. (1999). Effects of mancozeb on T.pyri. Multiyear trial results, – Phytoma, 513; 44-47. Mead-Briggs, M., (1992). A laboratory method for evaluating the side-effects of pesticides on the cereal aphid parasitoid Aphidius rhopalosiphi (DeStephani-Perez). – Aspects of Applied Biology 31: 179-189. Mead-Briggs, M., Longley, M., (2000). An extended laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (DeStephani- Perez) (Hymenoptera: Braconidae). – Unpublished draft method, 12th January 2000. Miles, M. & Green, E. (2002). Field studies to determine the effects of the fungicides Mancozeb and Dinocap on predatory mites in orchards and vineyards in Europe. – BCPC Conf. Pests Dis. (2002), 1, 297-302. Polgar, L., (1988). Guideline for testing the effects of pesticides on Aphidius matricariae Hal. Hym., Aphidiidae: Laboratory contact tests: 1-on adults, 2-on aphid mummies, semi-field test on adults. – Pesticides and Beneficial Organisms IOBC/wprs Bulletin 1988/XI/4: 29- 34. SANCO/4058/2001 - rev. 4.3 (2005) Review report for the active substance mancozeb Finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 3 June 2005 in view of the inclusion of mancozeb in Annex I of Directive 91/414/EEC. Sentenac G., Bonafos R., Ruelle B., Coulon T., Escaffre P., Auger P. & Kreiter S. (2002). Effets non intentionnels de certains produits phytopharmaceutiques sur Typhlodromus pyri, Kampimodromus aberrans et Phytoseius plumifer. – Phytoma 555: 50-55. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Viñuela, E., Waldburger, M. and Waltersdorfer, A. (2000). Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). – In: Guidelines to evaluate side-effects of plant protection products to non-target arthropods, p. 27-44. IOBC/WPRS; Gent, Belgium, ISBN 92- 9067-129-7.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 81-84

Side effects of insecticides used in cotton and vineyard areas of Aegean Region of Turkey on the green lacewing, Chrysoperla carnea (Steph.) (Neuroptera: Chrysopidae) under semi field conditions

Bilgin Güven, M. Ali Göven Bornova Plant Protection Research Institute, Gençlik Street 6, 35040, Izmir, Turkey

Abstract: The side-effects of insecticides used in cotton and vineyards areas on predator Chrysoperla carnea (Steph.) were tested under semi field conditions. The tests were performed according to the standard semi field test method of the IOBC/WPRS working group “Pesticides and Beneficial Organisms”. As a result of these tests Ekalux (a.i. quinalphos), Korvin (a.i. carbaryl), Deltanet (a.i. furathiocarb), Flashed (a.i. profenofos+cypermethrin) were classified as high toxic and Cascade (a.i. flufenuxuron) as moderately toxic products. Dimethoate (reference item) showed high toxicity resulting in a death rate above 75%.

Key words: Chrysoperla carnea, pesticides, side-effects, semi-field

Introduction

Chrysoperla species are important predators in many agricultural systems worldwide. They are marked by their longevity, high fecundity and fast developmental rates (Pfeiffer and Hogmore ,1995). The aim of this study was to determine the side-effects of insecticides used in vineyards and cotton crops on the susceptible life stage of Chrysoperla carnea. The findings are to be used to select the most suitable pesticides for use in areas under Integrated Pest Management (IPM) programs. Insecticides tested in this study revealed high toxicity in the laboratory tier tests.(Güven and Göven, 2003). Therefore, according to the test decision scheme these products need futher testing in higher testing tiers, e.g. under semi-field conditions (Hassan, 1985).

Material and methods

The tests were performed under outdoor conditions under shelter in an experimental field of BPPR Institute during 2002-2004 The side-effects of 5 insecticides used in cotton and vineyards areas were tested on the predator C. carnea (Steph.) under semi-field conditions (see Table 1). The side-effects tests were performed according to the standard semi-field test method (Bigler &Waldburger 1988) and revised by Vogt et al. (2001). The IOBC classification is based on the mortality values of Hassan (1994). Insect rearing In order to establish the culture, adults of C. carnea were collected from vineyard and cotton areas of Manisa(the nearest) and Denizli provinces of Aegean Region. The adults were reared in the laboratory on an artificial diet consisting of 7 parts of honey, 4 parts of brewer’s yeast and 4 parts of water (simple diet) Bigler (1988). Larvae of C. carnea were fed with fresh Ephestia küehniella Zell. eggs and Acyrthosyphon pisum in respect to Kaya & Öncüer (1988). E. küehniella were reared under the same conditions on an artificial diet consisting of ¼ corn flour, 1/2 wheat flour and ¼ beaten pistachio nut in plexiglass cages after Tunçyürek (1972). 81 82

Climatic conditions for the rearing of insects were 25±2°C, 70% rel. humidity and 16 h light, 8 h dark. Test units and number of replications Test units consisted of a plastic container (43 x 29 x 8cm) filled with garden soil. Twenty broad bean (Vicia faba) plants were grown in the inner half of the container at 22°±2°C, 60- 80% RH conditions in the greenhouse. Plants were used for the experiment when they reached 15-20 cm height. The plants were held together with a wooden screen with rubber bands around to prevent possibly escaping of test larvae from the unit. In order to keep the larvae of C. carnea within the test unit, a plastic barrier treated with fluon was set along the edge of the container. In order to keep the adults of C. carnea within the test unit after the application of the PPP the top of each container was covered with fine gauze in a frame. Soil moisture was regulated by wicks pulled through holes at the bottom of each container placed above a water filled tray. Corrugated cardboard strips were placed on the soil for pupation. The test units were placed outside on a table under a waterproof transparent roof with a layer of fine mesh gauze beneath it. In order to protect the pupae from ants, the legs of the table were smeared with tanglefoot glue. The experimental design included 3 treatments: the test substance, a reference substance (Dimethoate (Poligor) 400g a.i./l EC) and a water treated control. Each treatment consisted of 4 units (= 4 replicates) with 20 (2-3 day old) larvae of C. carnea per unit. Application of pesticides The plants were sprayed by using a hand held sprayer with adjustable angle full cone spray type (0.2 mm nozzle orifice, at a pressure of 1.5 atm.) at the highest recommended rate per hectare of the test substance in a water volume of 400 l (Vogt et al. 2001). The water amount needed for test unit and duration of application of this amount were determined before application of PPP The pesticides were tested with the rates as indicated in Table 1.

Table 1. Pesticides tested on C.carnea under semi-field conditions during 2002-2004.

Pesticide amount per ha Active ingredient Trade name Mode of action (formulated product) Vineyard flufenuxuron, 50 g/l Cascade 50 DC 2000 ml Chitin inhibitor quinalphos, 250 g/l Ekalux 1250 ml Cholinesterase inhibitor Cotton carbaryl, 85% Korvin 85% 2000 g Cholinesterase inhibitor dimethoate, 400 g/l Poligor 1000 ml Cholinesterase inhibitor furathiocarb, 400 g/l Deltanet 750 ml Cholinesterase inhibitor profenofos 40 g/l Flashed 2500 ml Cholinesterase inhibitor + cypermethrin 40 g/l

Observations Mortality assessment Larval and pupal mortality was assessed by collecting and counting emerged adults 3 times a week. The experiment was terminated when no adults emerged for a period of 1 week. Reproduction assessment Reproduction was not assessed because of the low number of females.

Results According to the effects on pre-adult stages of C. carnea all four insecticides showed high mortalities (Table 2). Furthermore, as to validity criteria, in all tests, the mortality in the control was below the agreed maximum acceptable value of 30% and the level of mortality in the reference item treatment was above 50 % (Vogt et al. 2001). Therefore, it can be concluded that the effects are treatment related.

Table 2. Pesticides revealed a high toxicity on C. carnea

Active ingredient Treatments IOBC class

Control Test Item Dimethoate Mortality Corrected (reference item) (%) Mortality Mortality (%) (Abbott%) flufenuxuron, 50 g/l 25.0 66.6 85.0 3 profenofos, 40g/l 27.5 75.8 81.0 4 + cypermethrin, 40g/l quinalphos, 245g/l 28.8 86.0 77.5 4 carbaryl, 85% 25.0 84.0 85.0 4 furathiocarb, 400 g/l 26.0 79.7 81.0 4

Conclusion As a result, 5 products were tested, four were class 4 and one, was Class 3. According to the IOBC testing scheme, the pesticides classified as moderately toxic and high toxic need futher testing in higher testing tiers, e.g. in the field (Hassan 1985).

References

Abbott, W.S. 1925: A method of computing the effectiveness of an insecticides. – J. Econ. Entomology 18: 265-267. Bigler, F. 1988: A laboratory method for testing side-effects of pesticides on larvae of the green lacewing, Chrysoperla carnea (Steph) (Neuroptera: Chrysopidae). – IOBC/WPRS- Bulletin 11(4): 127-134. Bigler, F. & Waldburger, M. 1994: Effects of pesticides on Chrysoperla carnea (Steph) (Neuroptera: Chrysopidae) ın the laboratory and semi-field. – IOBC/WPRS-Bulletin 17(10): 55-69. 84

Güven, B. & Göven, M.A. 2003: Side effects of pesticides used in cotton and vineyard areas of Aegean Region on the green lacewing, Chrysoperla carnea (Steth.) (Neuroptera: Chrysopidae) in the laboratory. – IOBC/WPRS-Bulletin 26 (5): 21-24. Hassan, S.A. 1985: Standard methods to test side-effects of pesticides on natural enemies of insects and mites developed by the IOBC/WPRS Working Group ”Pesticides and Beneficial Organisms” – Bulletin OEPP / EPPO 15: 214-255. Hassan, S.A. 1994: Activities of the IOBC/WPRS Working Group ”Pesticides and Beneficial Organisms”. – IOBC/WPRS-Bulletin 17 (10): 1-5. Kaya, Ü. & Öncüer, C. 1988: Laboratuvarda üretilen Chrysoperla carnea (Steph) (Neuroptera: Chrysopidae)’nın biyolojisine farklı iki besinin etkisi üzerinde bir araştırma. – Türkiye Entomoloji Dergisi 12 (3): 151-159. Kişmir, A. & Ç. Şengonca 1981: Anisochrysa carnea (Stephens) (Neuroptera: Chrysop- idae)’nın kitle üretim yönteminin geliştirilmesi üzerinde araştırmalar. – Türk. Bit. Kor. Derg. 5 (1): 36-41. Pfeiffer, D.G. & Hogmire, H.W. 1995: Aphid predators.– In: H.W. Hogmire (ed.). Mid- Atlantic Orchard Monitoring Guide. – Northeast Regional Agric. Engineer. Serv. Publ. 75, Ithaca: 75-80. Stabler, P. 1997: Effects of different NeemAzal-formulations on larvae of the green lacewing Chrysoperla carnea Steph. (Neuroptera: Chrysopidae) in the laboratory and semi-field. – Proceedings of 5th Workshop Wetzlar: 183-188. Tauber, J.M., Tauber, C.A., Daane, K.M. &. Hagen, K.S. 2000: Commercialization of predators: Introduction, systematics and mass production. - Recent lessons from green lacewing. – American Entomologist 46 (1): 26-38. Tunçyürek, C.M. 1972: Bracon hebetor Say (Hym.: Braconidae) ile Cadra cautella (Walk.) ve Anagasta kuehniella (Zeller) (Lep.: Pyralidae)’ya karşı biyolojik savaş imkanları üzerinde araştırmalar. – Tar.Bak.Zir.Müc. ve Zir. Kar.Gn. Md. Araş. Es.Serisi. Teknik Bülten No. 20. Zir. Müc.Arşt.Enst. İzmir: 78 pp. Vogt, H. 1992: Investigation on the side effects of insecticides and acaricides on Chrysopa carnea Steph. (Neuroptera, Chrysopidae). – Mededelingen van de Faculteit Landbouw- wetenschapen, Rijksuniversiteit Gent 57 (2b): 559-567. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Blümel, S., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Vinuela, E., Waldburger, M. & Waltersdorfer, A. 2000: Laboratory method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). – In: Candolfi, M. P., Blümel, S., Forster, R., Bakker, F.M., Grimm, C., Hassan, S.A., Heimbach, U., Mead-Briggs, M.A., Reber, B., Schmuck, R. & Vogt, H. (eds.): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/WPRS, Gent: 27-44. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Blümel, S., Kemmeter, F., Kühner, Ch., Moll, M., Travis, A., Ufer, A., Vinuela, E., Waldburger, M. & Waltersdorfer, A. 2001: Semi-field method to detect residual toxicity of pesticides on larvae of Chrysoperla carnea (Steph.) (Neuroptera: Chrysopidae). – Unpublished, based on a meeting at FAL Zürich-Reckenholz, last revision. Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 85-93

Effects of botanical insecticides on two natural enemies of importance in Spain: Chrysoperla carnea (Stephens) and Psyttalia concolor (Szépligeti)

Pilar Medina, Flor Budia, Manuel González, Benjamín Rodríguez, Aurelio Díaz, Arturo Huerta, Nelson Zapata, Elisa Viñuela Unidad de Protección de Cultivos. Escuela Técnica Superior de Ingenieros Agrónomos. Ciudad Universitaria, s/n. 28040. Madrid. Spain. e-mail: [email protected]

Abstract: Insecticidal properties of some plant extracts are known from ancient times. Despite, their use has been basically limited to subsistence crops in underdeveloped countries. Adverse environmental effects on nontarget organisms and the build up of resistance caused by the abuse of pesticides in developed countries, have contributed to the increase of research on plant-derived pesticides. Nowadays, among plants with a higher potential to be used for the development of active products against insects are an increasing number of species from families Meliaceae and Lamiaceae, rich in secondary metabolites. The bioassay-guided fractionation of Trichilia havanensis (Meliaceae) extracts led to the purification of two limonoids: azadirone (F12) and 1,3+1,7-di-O-acetylhavanensin(4:1) (F18) whereas two compounds (M1 and M9) were hemisynthesized from Teucrium viscidum (Lamiaceae). All these compounds have antifeedant properties against some important pests. In the current study, effects of these compounds have been evaluated on two natural enemies, the generalist predator Chrysoperla carnea (Neuroptera: Chrysopidae) and the olive fruit fly parasitoid, Psyttalia concolor (Hymenoptera: Braconidae). Ingestion bioassays at concentrations of 1000 mg a.i./l were carried out with adults of both beneficials to study potential antifeedant effects as it was observed on phytophagous pests. Results have demonstrated that these bioinsecticides are nearly innocuous for both natural insects at the conditions tested.

Key words: Trichilia havanensis, Teucrium viscidum, Chrysoperla carnea, Psyttalia concolor, plant extracts.

Introduction

The registration of new insecticides in the EU demands more strict requests on selectivity and environmental impacts. The search of new compounds that fulfil the requirements is not an easy task. The presence of a large number of plant secondary metabolites offers excellent perspectives for their extractions, structural identification and evaluation as pesticides (Mc Laren, 1986). Insecticidal properties of some plant extracts are known from ancient times. Their use, however, has been basically limited to subsistence crops in underdeveloped countries. These compounds originate from renewable resources, accessible and cheaper than synthetic insecticides. Adverse environmental effects on nontarget organisms and the build up of resistance caused by the abuse of pesticide use in developed countries, have contributed to the increase of research on plant-derived pesticides. The increase in Organic Farming in industrialized countries has also reemerged the economic interest and search for plants with new insecticidal properties. Structural identification and evaluation of these substances is one of the main goals of natural products chemistry and an important step to develop more rational methods to control pests (McLaren, 1986; Coll, 1988). Moreover, these compounds

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might delay the resistance outbreak due to the mixture of several compounds with different mode of action (Völlinger, 1995). The marketing of insecticides with botanical origin has increased in the last decade, representing currently one per cent of the world trade of insecticides. It is considered that around 5 to 15% of plants have been studied in relation to their active compounds and, commonly, only one type of biological activity (Balandrin et al., 1985). Among plants commercially used as source of active compounds for pest control, stand out those containing pyrethrins, rotenoids, alkaloids and limonoids. They are members of the first generation of insecticides, pyrethrins being the model for synthetic pyrethroids (Casida & Quistad, 1998). Nowadays, there is an increasing number of species from families Meliaceae and Lamiaceae plants with a higher potential to be used for the development of active products against insects (Jacobson, 1989). Meliaceae is a family of tropical plants able to biosynthesize highly oxidized triterpenoids known as limonoids. These compounds possess a high structural diversity and a broad range of biological activity. Leaves, bark and specially seed extracts have been reported as toxic against insects. The more known secondary metabolite is azadirachtin, that acts as insect growth regulator and antifeedant. Trichilia havanensis Jacq. is a Meliaceae widespread in America, also under focus because among the limonoids characterised from this tree, azadirone (F12) and the mixture 1,7 and 3,7-di-O- acetylhavanensin (F18) have shown a strong antifeedant activity against some important pests: Spodoptera littoralis (Boisduval) (López-Olguín et al., 1997); Helicoverpa armígera (Hübner) (López-Olguín et al., 1998), Ceratitis capitata (Wied.) (López-Olguín et al., 2002) or Leptinotarsa decemlineata (Say), (Ortego et al., 1998); Epilachna varivestis Mulsant (Schwinger et al., 1984). On the other hand, Labiatae is a plant family with a high variety of secondary metabolites such as monoterpenoids, sesquiterpenoids, deterpenoids, glycosides and flavonoids (Simmonds & Blaney, 1992). Some genus as Teucrium are a rich source of neo-clerodanic diterpenoids. Yet, none has been commercially used for insecticidal purposes, though some of them have a broad potential. Neoclerodanes isolated from Teucrium had shown toxicity against Spodoptera littoralis, Locusta migratoria L., Prodenia litura F., Helicoverpa armigera and Leptinotarsa decemlineata (Say) (Simmonds et al., 1989; Ortego et al., 1995). To determine if limonoids and neoclerodanes can be useful in IPM programmes, it is necessary to increase our knowledge on the specificity and mode of action, emphasizing at molecular level, and to estimate the purification degree needed to act effectively against pests. Finally, studies on persistance and biodegradation, and toxicity against non-target organisms are required (Rodriguez, 1997) In this context, we have evaluated effects of ingestion of the extracts from T. havanensis and T. viscidum on adults of a generalist predator, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) and a parasitoid, Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) in comparison with two reference botanical insecticides, that are currently on the markets: the non-oil Align®, a commercial of azadirachtin, and Pelitre Hort®, a new formulation of natural pyrethrins synergised with piperonyl butoxide (NP+PBO).

Material and methods

Extracts The limonoids F12 (azadirone) and F18 (1,7+3,7-di-O-acetilhavanensin (4:1)) were isolated from the seed kernels of T. havanensis as described by López-Olguín (1998) with a purity above 95%. M1 and M9 are two compounds chemically transformed (hemisynthesis) from teucjaponin B and teucvin, respectively, extracted from Teucrium viscidum var. miquelanum

(Rodriguez et al., 1994). All botanical compounds were diluted in distilled water using 1% acetonitrile, at a selected concentration of 1000 mg a.i./l. 0.1% Tween 20 was used as emulsifier. Acetonitrile and Tween 20 are useful to solve the compounds and were previously chosen among several solvents and emulsifiers with assurance that they were harmless to both beneficials. Control AC contained water + acetonitrile (1%) + Tween 20 (0.1%). Align® (3.17% azadirachtin, EC, Sypcam Inagra, Spain) and Pelitre Hort® (4% natural pyrethrins+16% piperonyl butoxide, EC, CQ Massó, Spain) were used at the maximum recommended rate (Liñán, 2004): 150 ml/hl and 200 ml/hl, respectively, diluted on distilled water only (Control). Insects Adults of both beneficials were routinely reared in our Crop Protection laboratory, at 25 ± 2ºC temperature, 75 ± 5 R.H. and a photoperiod of 16 h light. C. carnea adults were fed the artificial diet described by Vogt et al., (2000). Females of P. concolor were reared on their substitution host C. capitata as described by Jacas & Viñuela, (1994). Bioassays Botanical compounds were offered to adults ad libitum in the drinking places. During the experiments, adults were maintained in ventilated plastic cages (11 cm in diameter, 5 cm high) under the same environmental conditions described above for the rearing. Artificial diet used for feeding C. carnea was brushed on the wall of the cages (Medina et al., 2001) and 4:1 w/w sugar: brewer´s yeast was provided to P. concolor. C. carnea adults: Experiments consisted of 4 to 6 replicates with 3 pairs of newly emerged adults per compound or control. They ingested F12 and F18 during five days (the preoviposition period) and M1, M9 and reference products continuously until the end of the experiments. The studied parameters were: mortality, fecundity and fertility. Mortality was monitored after seven days. The mean number of eggs per female laid in a 7-days period from the start of egg-laying was used to compare fecundity among treatments. To evaluate fertility, oviposition gauzes with eggs were collected at day 5 after the beginning of oviposition. Gauzes were placed in plastic boxes with Ephestia kuehniella eggs to prevent cannibalism among neonate larvae before being counted. Percentages of hatched eggs were calculated 5-6 days later. P. concolor adults: Fifteen newly emerged females per replicate were exposed continuously to the test products via treated drinking water during seven days. Each experiment was designed with four replicates per compound and control. Mortality was measured as the percentage of dead adults on the 7th day. To assess possible effects on reproduction, two females per replicate and control were individually isolated three days after the beginning of the experiment. During the following three days, 20 fully-grown C. capitata larvae were offered daily to each female for parasitization. After two hours of exposure to parasitization, C. capitata larvae were placed onto Petri dishes to let them pupate. Beneficial capacity was measured as the percentage of attacked host (percentage of puparia without medfly emergence) and progeny size (percentage of parasitoids emerged from parasitized medfly puparia). Statistical analysis One-way analysis of variance (ANOVA) and LSD multiple range test if the F was significant, were performed on the data to determine significant (P<0.05) compound differences, using Statgraphic (STSC, 1987). Experiments using F12 and F18 fractions were carried out in 2003, whereas the rest of compounds, including references were tested during 2005. Therefore, results have been analyzed separately.

Results

Results on survival and reproduction of C. carnea are presented in Table 1, those for P. concolor in Table 2. Azadirachtin and natural pyrethrins+piperonyl butoxide (NP+PBO) killed about 20 and 64% of C. carnea adults, respectively, seven days after continuous ingestion on drinking throughs. On the contrary, the botanical compounds F12 and F18 from T. havanensis and M1 and M9 from T. viscidum were innocuous. Azadirachtin completely inhibited the egg-laying and, therefore, fertility could not be tested. The effects of natural pyrethrins+piperonyl butoxide on C. carnea reproductive parameters could not be measured due to the high mortality on adults. Both fractions from T. havanensis and M1 from T. viscidum affected neither fecundity nor fertility, whereas M9 slightly reduced only fecundity, being classified as 2, according to IOBC standards. Natural pyrethrins+piperonyl butoxide caused a drastic reduction (91.3%, compared to control) on P. concolor survival after seven days of continuous ingestion, whereas azadirachtin, the other botanical reference compound was not toxic. Concerning mortality, not any effects were found for the non-commercial botanicals. Beneficial capacity did not significantly differ from those of controls. However, progeny size was slightly reduced after treatments with azadirachtin. Due to the high mortality of females, no data on beneficial capacity were assessed from natural pyrethrins+piperonyl butoxide.

Discussion

The use of botanical insecticides might be one alternative to synthetized insecticides. In this paper we studied the effects of antifeedant compounds extracted from plants on natural enemies. None of the natural compounds affected significantly neither survival nor reproductive parameters of both beneficials in comparison with the reference products: commercial formulation of azadirachtin and natural pyrethrins+piperonyl butoxide. The behaviour of reference compounds on these insects has been partly studied by Medina et al. (2004) (azadirachtin – C. carnea); Huerta et al. (2004) (natural pyrethrins + piperonyl butoxide – C. carnea); González & Viñuela (1997) (azadirachtin – P. concolor) and Zapata et al. (2005) (natural pyrethrins + piperonyl butoxide – P. concolor). Azadirachtin reduced the egg-laying of C. carnea females as shown by previous studies from Medina et al. (2004). These authors reported a clear reduction in fecundity of egg-laying females when azadirachtin was ingested with the drinking water, once the oviposition had started, or a delay of oviposition for as much days as the females had been exposed to the compound from adult emergence onward. Time of exposure to azadirachtin is the critical point to explain these differences. Medina et al. (2004) only exposed females for four days to azadirachtin ingestion. After that, females drank water and restored their fecundity potential. In this work, exposure, being continuous from emergence until the end of the experiment, totally prevented females from egg-laying. All these effects are due to the interference of azadirachtin with the vitellogenin synthesis and/or its uptake by the oocytes. Continuous feeding on azadirachtin treated water at the higher recommended concentration caused a significant decrease, not only in longevity, but also in the beneficial capacity of the wasp as was reported by González & Viñuela (1997). C. carnea adults seem to be more resistant than the parasitoid concerning survival after being treated with azadirachtin, even though they are much more sensitive taking into account the effects on reproduction.

Table 1. Effects of continuous ingestion of extracts from T. havanensis (F12 and F18) and T. viscidum (M1 and M9), compared to two commercial botanical compounds used as references (Align® (azadiracthin 3.17%) and Pelitre Hort® (natural pyrethrins 4% + piperonyl butoxide 16%)), on survival and reproduction of Chrysoperla carnea adults.

Compound Conc. Mortality after 7 days Fecundity Fertility IOBC (mg a.i./l) (%) Corrected Eggs/female/day Reduction4/ Egg hatch (%) Reduction4/ class5 mortality3/ IOBC IOBC class IOBC class Control 0±0a - 28.4±3.3a - 87.4±5.3 - Azadirachtin 48 19.4±7.9b 19.4 / 1 0 100 / 4 Not measured - 4 NP+PBO1 80+320 63.9±12.5c 63.9 /2 Not measured Not measured Not measured - 2

Control AC2 0±0a - 40.7±1.8a - 82.2±3.7a - M1 1000 0±0a 0 /1 33.1±1.7b 18.6 / 1 85.2±5.2a -3.6 / 1 1 M9 1000 5.5±5.5a 5.5 /1 26.3±3.0b 35.3 / 2 91.3±1.8a -11 / 1 2

Control AC2 0±0a - 26.3±1.9a - 69.8±2.6a - F12 1000 0±0a 0 / 1 25.4±2.7a 3.4 / 1 71.4±5.6a -2.3 / 1 1 F18 1000 0±0a 0 / 1 25.3±2.7a 3.8 / 1 67.7±3.7a 3.0 / 1 1 Data followed by the same letter are not significantly different (P>0.05). ANOVA and LSD mean separation.

1 Natural pyrethrins+piperonyl butoxide (NP+PBO). 2 Distilled water+acetonitrile (1%)+Tween 20 (0.1%). 3 Corrected mortality by Schneider-Orelli :[(Mtreated-Mcontrol)/(100-Mcontrol)]*100. 4 Reduction (%)=[(1– (Parameter value (treated)/ Parameter value (Control)]*100 5 Highest value among the three IOBC categories calculated previously (mortality, fecundity and fertility)

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Table 2. Effects of continuous ingestion of extracts from T. havanensis (F12 and F18) and T. viscidum (M1 and M9), compared to two commercial botanical compounds used as references (Align® (azadiracthin 3.17%) and Pelitre Hort® (natural pyrethrins 4% + piperonyl butoxide 16%)) on survival and reproduction of Psyttalia concolor adults.

Compound Conc. (mg Mortality after 7 days Attacked host Progeny size IOBC a.i./l) (%) Corrected Eggs/female/day Reduction4/ Egg hatch (%) Reduction4/ class5 mortality3/ IOBC class IOBC class IOBC class Control 5.0±3.2a - 78.0±3.5a - 59.2±3.1a - Azadirachtin 48 10.0±4.3a 5.2 / 1 61.6±5.9a 21.1 / 1 32.5±6.2b 45.1 / 2 2 NP+PBO1 80+320 91.7±5.0b 91.3 / 3 Not measured - Not measured - 3

Control AC2 6.7±2.7a - 74.0±3.9a - 65.0±7.9a - M1 1000 11.7±1.7a 5.3 / 1 64.9±5.2a 12.3 / 1 49.4±4.4a 24 / 1 1 M9 1000 6.7±3.8a 0 / 1 66.9±6.0a 9.6 / 2 51.2±3.8a 21.2 / 1 1

Control AC2 8.4±1.7a - 78.1±5.9a - 62.0±6.0a - F12 1000 10.0±3.3a 1.7 / 1 74.2±3.5a 4.9 / 1 63.6±3.4a -2.5 / 1 1 F18 1000 11.7±3.2a 3.6 / 1 70.6±5.5a 9.6 / 1 57.5±6.9a 7.2 / 1 1 Data followed by the same letter are not significantly different (P>0.05). ANOVA and LSD mean separation.

Natural pyrethrins + piperonyl butoxide had a higher knock-down effect on P. concolor in agreement to data reported by Zapara et al. (in press) who found LC50=63.4 mg a.i./l after three days of treatment. Huerta et al. (2004) did not find effects on C. carnea survival after four days of ingestion, which demonstrated that this compound has to be ingested during a longer time to be effective on the predator. Therefore, we believe NP+PBO would not have any effect on C. carnea after outdoor treatments. The absence of toxicity after continuous ingestion of non-commercial botanical compounds (fractions from T. havanensis and T. viscidum) can be explained by the amount of insecticide that target the insect. Effects on phytophagous were detected after the ingestion of this compounds mixed into the artificial diet or supplied on leaf discs, sometimes only with doses larger than 1000 mg a.i./l. On the other hand, the commercial botanical compounds used as references, are prepared with synergist or coadyuvants that help to increase their toxicity, whereas the non-commercial compounds did not have these substances. Effects on mortality are not so common, being often the delay on the development caused by a deficient feeding. Many botanical extracts are remarkably benign to spiders, butterflies, honey bees and many other natural enemies. This is because these products must be ingested to be effective. Thus, insects that feed on plant tissues will be affected by the extracts, while those that feed on nectar or other insects rarely contact lethal concentrations (Williams & Mansingh, 1996). We forced our natural enemies to ingest the products, without obtaining nearly any effect in the laboratory. Therefore, our work has confirmed that fractions F12 and F18 from T. havanensis and M1 and M9 from T. viscidum would not cause any deleterious effects on C. carnea and P. concolor if they were marketed to control pests.

Acknowledgements

This work was supported by the Spanish Ministry of Education and Culture (project AGL 2001-1652-C02-02 to E. Viñuela).

References

Balandrín, M.F., Klocke, J.A., Wartele, E.S. & Bollinger, W.H. 1985. Natural plant chemicals: sources of industrial and medicinal materials. – Science. 228: 1154-1160. Casida, J.E. & Quistad, G.B. 1998. Golden age of insecticide research: Past, Present, or Future? – Annu. Rev. Entomol. 43: 1-16. Coll, J. 1988. Hormonas juveniles, juvenoides y juvenógenos. – In: Insecticidas biorracio- nales. CSIC. Coord. Bellés: 87-112. González, M. & Viñuela, E. 1997. Effects of two modern pesticides: azadirachtin and tebufenozide on the parasitoid Opius concolor (Szépligeti). – IOBC/wprs Bull. 20 (8): 233-240. Huerta, A., Medina, P., Budia, F., Del Estal, P. & Viñuela, E. 2004. Evaluación de la toxicidad de cuatro insecticidas y el colorante Floxín-B en larvas y adultos de Chryso- perla carnea Stephens (Neuroptera: Chrysopidae). – Bol. San. Veg. Plagas. 30: 721-732. Jacas, J. & Viñuela, E. 1994. Analysis of a lab method to test the effects of pesticides on adult females of Opius concolor, a parasitoid of the olive fruit fly Bactrocera oleae. – Biocontrol Science & Technology 4: 147-154. Jacobson, M. 1989. Botanical pesticides: past, present and future. – In: Arnason, Philogene and Moran (eds.): Insecticides of plant origin. American Chemical Society Symposium Series 387: 1-10.

Liñán, 2004. Vademecum de productos sanitarios y nutricionales 2005. – Ediciones Agro- técnicas, S.L. López-Olguín, J.F. 1998: Actividad y modo de acción de productos de Trichilia havanensis Jacq. y Scutellaria alpina subsp. javalambrensis (Pau), sobre Leptinotarsa decemlineata (Say) y Spodoptera exigua (Hübner). – Tesis Doctoral. UPM. Madrid. López-Olguín, J.F., Adán, A., Ould-Abdallahi, E., Budia, F., Del Estal, P. & Viñuela, E. 2002: Actividad de Trichilia havanensis Jacq. (Meliaceae) en la mosca mediterránea de la fruta Ceratitis capitata (Wied.). – Bol. San. Veg. Plagas 28: 301-308. López-Olguín, J.F., Budia, F., Castañera, P. & Viñuela, E. 1997: Actividad de Trichilia havanensis Jacq. (Meliaceae) sobre larvas de Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). – Bol. San. Veg. Plagas 23: 3-10. López-Olguín, J.F., De La Torre, M.C., Viñuela E. & Castañera, P. 1998: Actividad de extractos de semillas de Trichilia havanensis Jacq., sobre larvas de Helicoverpa armigera (Hübner). – Bol. San. Veg. Plagas 24: 629-636. McLaren, J.S. 1986. Biologically active substances from higher plants: status and future potential. – Pestic. Sci. 17: 559-578. Medina, P., Budia, F., Del Estal, P. & Viñuela, E. 2004. Influence of azadirachtin, a botanical insecticide on Chrysoperla carnea (Stephens) reproduction: Toxicity and Ultrastructural Approach. – J. Econ. Entomol. 97: 43-50. Medina, P., Budia, F., Smagghe, G. & Viñuela, E. 2001: Activity of spinosad, tebufenozide and azadirachtin on eggs and pupae of the predator Chrysoperla carnea (Stephens) under laboratory conditions. – Biocontrol Sci. & Technol. 11:597-610. Ortego, F., López-Olguín, J.F., Ruíz, M. & Castañera, P. 1998: Effects of toxic and deterrent terpenoids on digestive protease and detoxication enzyme activities of colorado potato beetle larvae. – Pestic. Biochem. Physiol. 63: 76-84. Ortego, F., Rodríguez, B. & Castañera, P. 1995. Effects of neoclerodane diterpenes from Teucrium on feeding behaviour of Colorado potato beetle larvae. – J. Chem. Ecol. 21: 1375-1386. Rodríguez, B., de la Torre, M.C., Perales, A., Malakov, Y., Papanov, G.Y., Simmonds, M.S.J. & Blaney, W.M. 1994. Oxirane-opening reactions of some 6,19-oxygenated 4α, 18- epoxy-neo-clerodanes isolated from Teucrium. Biogenesis and antifeedant activity of their derivatives. – Tetrahedron 50: 5451-5468. Rodríguez, B. 1997. Diterpenos de tipo clerodano y su actividad antialimentaria. – In: Insecticidas de origen natural y protección integrada y ecológica en agricultura. Consejería de Medio Ambiente, Agricultura y Agua. Serie Congresos 10: 67-74. Schwinger, M., Ehhammer, B., & Kraus, W. 1984. Methodology of the Epilachna varivestis bioassay of antifeedants demonstrated with some compounds from Azadirachta indica and Melia azedarach. – In: Natural Pesticides from the Neem Tree and Other Tropical Plants. Rauischholzhausen: 181-198. Simmonds, M.S.J., Blaney, W.M., Ley, S.V., Savona, G., Bruno, M. & Rodriguez, B. 1989. The antifeedant activity of clerodane diterpenoids from Teucrium. – Phytochemistry 28: 1069-1071. Simmonds, M.S.J. & Blaney, W.M. 1992. Labiatae–insect interactions: Effects of Labiatae- derived compounds on insect behaviour. – In: Advances in Labiatae science. Eds. Harley & Reinolds: 375-392. STSC, 1987: Statgraphics user’s guide, versión 5.0. – Graphic Software System. Stsc. Rockville. USA. Vogt, H., Bigler, F., Brown, K., Candolfi, M.P., Kemmeter, F., Kühner, C.H., Moll, M., Travis, A., Ufer, A., Viñuela, E., Waldburger, M. & Waltersdorfer, A. 2000: Laboratory

method to test effects of plant protection products on larvae of Chrysoperla carnea (Neuroptera: Chrysopidae). – In: Candolfi, M.P., Blümel, S., Forster, R., Bakker, F., Grimm, C., Hassan, S.A., Heimbach, U, Mead-Briggs, B., Reber, R., Schmuck, R., Vogt, H. (eds): Guidelines to evaluate side-effects of plant protection products to non-target arthropods. IOBC/wprs, Gent: 27-44. Völlinger, M. 1995. Studies on the probability of development of resistance of Plutella xylostella to neem products. – In: Schmutterer, H. (ed.): The neem tree: source of unique natural products for integrated pest management, medicine, industry and other purposes: 477-483. Williams, L.A.D. & Mansingh, A. 1996. The insecticidal and acaricidal actions of compounds from Azadirachta indica (A. Juss.) and their use in tropical pest management. – Integrated Pest Management Reviews 1: 133-145. Zapata, N., Medina, P., Viñuela, E. & Budia, F. 2005. Toxicidad de malation, pimetrocina, piretrinas naturales+PBO y triflumuron en adultos del parasitoide Psyttalia concolor (Szèpligeti) (Hym.: Braconidae). – Bol. San. Veg. Plagas 31: 111-118.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 95-103

Comparative sensitivity of four ladybird species to five pesticides

Jansen, J-P. & Hautier, H. Department of Biological control and Plant genetic resources, Agricultural Research Centre, Chemin de Liroux, 5030 Gembloux – Belgium, [email protected]

Abstract: Since 2003, four ladybird species have been found in open fields in Belgium: the three native species Coccinella septempunctata (L.), Adalia bipunctata (L.), Propylea quatuordecimpunctata (L.) and the invasive species Harmonia axyridis (Pallas). As this last species could be a problem in the future for native species, experiments were carried out to assess its sensitivity to pesticides compared to native species, to determine whether the use of pesticides in agricultural ecosystems could give an advantage to H. axyridis. The results also provide information on the sensitivity of the four species and whether it is possible to extrapolate data obtained for one species to another. The LR50 of 5 pesticides (three insecticides: imidacloprid, zeta-cypermethrin, triazamate and two fungicides: spiroxamine and metalaxyl-M + fluazinam) was assessed on glass plates. Products were tested on basis of a range-finder test (5 doses in a dilution range 5-10x + control, 10 larvae per test unit) and a definitive test (5 doses in an adapted range to ideally cover 0-100 % mortality + control, 20 larvae per unit). The larvae used for the tests were 2-3 day old and were confined for 7 days on glass plates. Mortalities were recorded daily and final assessment was made after 7 days of exposure.

The LR50 results (ml of formulated product/ha ± sd) after 7 days of exposure can be summarised as follows: Impulse Epok Aztec Fury Confidor C. 7-punctata 1155.5 ± 79.0 681.2 ± 158.8 54.1 ± 8.7 0.4766 ± 0.064 300.5 ± 56.7 P. 14-punctata 810.0 ± 54.8 211.6 ± 51.2 75.7 ± 8.5 0.0279 ± 0.0032 1.26 ± 0.27 A. bipunctata 1123.4 ± 74.5 36.1 ± 9.9 21.8 ± 2.9 0.0118 ± 0.0014 0.54 ± 0.11 H. axyridis 1584.4 ± 111.6 68.3 ± 17.7 143.4 ± 18.4 0.0437 ± 0.0058 0.49 ± 0.11

Comparison of LR50 values show no clear relationship between species tested and sensitivity to the different pesticides. A. bipunctata was most of the time the most sensitive species, but there were exceptions, for example in the case of Impulse (P. 14-punctata) and Confidor (H. axyridis). However, if the results obtained with C. septempunctata are omitted, the sensitivity of the three other species is more or less comparable, with LR50 ratio from the least sensitive to the most sensitive in a range of 1– 6fold. C. septempunctata was most of the time the least sensitive species. For Confidor, the LR50 of C. 7-punctata was up to 600 times higher than that of H. axyridis, the most sensitive species. These results suggest a possible resistance mechanism for this species.

Keywords: ladybirds, Coccinella septempunctata, Adalia bipunctata, Propylea quatuordecimpunctata, Harmonia axyridis, LR50, triazamate, imidacloprid, fluazinam + metalaxyl-M, zeta- cypermethrin, spiroxamine, pesticides, plant protection products.

Introduction

Ladybirds are important aphid predators in agro-ecosystems. Their actions limit the development of several aphids known to be serious pest problems in many crops and reduce the need for insecticide treatments (Hodek, 1973; Rautapää, 1976; Chambers et al., 1983; Latteur & Oger, 1987; Poehling & Borgemeister, 1989). In open fields, Coccinella

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septempunctata, Propylea quatuordecimpunctata and Adalia bipunctata were the the most common species in several crops (Dedrijver et al., 1985; Honek, 1995; Jansen, 2000). As for other beneficial insects encountered in agriculture, ladybirds are exposed to pesticides. An intensive work has been undertaken to assess possible side-effects of these compounds on ladybirds, generating a sum of data. However, the data are most of the time limited to one or a few species, because of standardization of test methods and species. This is particularly the case with studies realized in the context of product registration at the European level, where C. septempunctata is the recommended ladybird species (Anonymous, 1994, 2000). Even in an IPM context, data are only generated on a limited number of species. By example, in the different Joint Pesticide Testing Programs organized by the IOBC, a sum of results were obtained on C. septempunctata (Hassan et al., 1987) or Semiadalia undecim- punctata (Col.; Coccinellidae) (Hassan et al., 1988, 1991, 1994; Sterk et al. 1999) or H. axyridis (Hassan et al., 1988). The problem with this standardization is that there is actually very little information on comparative sensitivity of the different ladybird species and the possibility to extrapolate the results obtained on one specific species to another one is limited. In 2003, the multicolored Asian ladybeetle H. axyridis was found for the first time in Belgium in potato crops (Jansen et al., 2004). The presence of this species was confirmed in 2004 and 2005 (Jansen, unpublished). Several studies have shown that the Asian ladybeetle was very aggressive and able to dominate C. septempunctata and A. bipunctata, both by competition for exploitation of food resources and by direct predation (Yasuda et al., 2001; Kajita et al., 2000; Hautier, 2003; Hokkanen et al., 2003). The Asian ladybeetle is well known in the USA to be a problematic invasive species, with problems on indigenous ladybird community (Lamana & Miller, 1996; Colunga-Garcia & Gage, 1998; Michaud, 2002; Brown, 2003; Alyokhin & Sewell, 2004). Depending on the competitive advantage of H. axyridis compared to native species and the presence of all these ladybirds in open fields where pesticides are regularly sprayed, the possible differences in sensitivity of these species to various pesticides can play a role in the establishment and development of the Asian multicolored ladybird. In order to determine whether pesticide side-effect data can be extrapolated from one ladybird species to another and to determine if the use of pesticides in the field, where the multicolored Asian ladybeetle is found, could give a competitive advantage to this species, a specific research program was initiated to compare the sensitivity of different pesticides to four ladybird beetle species, including the invasive species H. axyridis and the native species C. 7-punctata, P. 14-punctata and A. bipunctata.

Material and methods

Plant protection products Table 1 lists the five pesticides selected for testing. These products are all known to be toxic to ladybirds, at least on glass plates, and are used in crops where ladybirds may be important aphid predators. They all belong to a different chemical class. Mass rearing of the ladybirds and toxicity tests The four ladybird species were reared in the laboratory in a same way. Adult ladybirds were kept in plastic cages (40 cm x 30 cm x 20 cm) with 20-40 adults per cage. The bottom of the cages was protected by a piece of filter paper. Pea aphids (Acyrtosiphon pisum) on cut french bean plants, honeybee pollen and cut blackwheat (Fagopyrum esculentum) flowers were offered ad libitum as food. A piece of paper was put into the cages for egg laying. Three times per week beetles were transferred into new cages and their food renewed, except the blackwheat flowers which were only changed when needed. Patches of eggs were harvested 97

and kept on petri dishes for hatching. Young larvae were kept on petri dishes and fed with pea aphids till the pupal stage when they were then used for the mass rearing or used for the tests. All the rearings were done in climatic chambers at 20°C ± 2°C and 60-90%RH, with sodium lamp lightning on a basis of 16h light/8h dark.

Table 1: List of plant protection products used in this study

Commercial active(s) a.s./l or kg Formulation Chemical class name ingredient(s) Aztek * triazamate 140 g EW carbamate Confidor imidacloprid 200 g SL neonicotinoid Epok fluazinam + 400 g + 200 g EC dinitroaniline + metalaxyl-M acylalanine Fury 10EW zeta-cypermethrin 100 g EW pyrethroid Impulse spiroxamine 500 g EC strobilurin * spelt Aztec in summary

Mass rearings of ladybirds were initiated with adults sampled in field hedges and fallow lands in agricultural ecosystems. A. bipunctata rearing started in 1995, C. 7-punctata in 1998 and H. axyridis and P. 14-punctata in 2003. New adults (10-20) were introduced each year to renew the rearing. About 50-100 adults of each species were kept in a same time to produce a sufficient number of larvae for the tests. For the toxicity test, products were applied with a Burgerjon spray tower (Burgerjon, 1956) onto small glass discs (∅ 5cm) at a rate of 200 l ± 10% of spray mixture/ha. Spray deposit was assessed by gravimetry. One to two hours after application, when pesticide residues were dry, the glass plates were put in small plastic dishes and covered with a plastic ring coated with Fluon GP1 to prevent the beetle from escaping. One 2-3 day old ladybird larvae was then released into each unit. The larvae were kept for 7 days in these units. Pea aphids were added daily and larval mortality was checked every day. The products were tested first in a range-finder test with 5 doses in a 5-10x dilution rate and a control, with 10 larvae per rate. According to the results obtained in the range-finder, a definitive test was conducted with a narrower dilution range, generally 2x-5x dilution rate according to product, and 20 larva per rate. Mortality obtained for these 5 doses was corrected for control mortality (Abbott, 1926) and used to calculate LR50 values using Probit analysis (Minitab 13.20) and fitting a log 10 normal distribution model.

Results and discussions

The LR50 values of the five tested products for the 4 ladybird species are illustrated by Figures 1-5. LR50 values are expressed in ml formulated product/ha and given with 95% confidence interval. Numbers followed by the same letter are not significantly different (P<0.05). Control mortality reached a mean of 4.5% for all final tests (species x product), with a maximum of 15.0% in one test out of 20.

2000 1584 c

1600 1156 b 1123 b 1200 810 a 800 ml product/ha 400

0 C. 7-punctata P. 14-punctata A. bipunctata H. axyridis

Figure 1: LR50 of Impulse (spiroxamine) for 4 ladybird species

1200 681,2d 1000

800

600 211,6c 400 ml product/ha 36,1a 68,3b 200

0 C. 7-punctata P. 14-punctata A. bipunctata H. axyridis

FigureFigure 2: 2:LR50 LR50 of of Epok (fluazinam (fluazinam + metalaxyl-M) + metalaxyl-M) for 4 ladybird for 4 ladybird species species

200,0 143,4c 160,0

120,0 75,7b 80,0 54,1b

ml product/ha 21,8a 40,0

0,0 C. 7-punctata P. 14-punctata A. bipunctata H. axyridis

FigureFigure 3: 3:LR50 LR50 of of AztecAztec (triazam (triazamate)ate) for 4for ladybird 4 ladybird species species

0,7 0,477d 0,6 0,5

0,4

0,3

ml product/ha 0,2 0,028b 0,012a 0,044c 0,1

0 C. 7-punctata P. 14-punctata A. bipunctata H. axyridis FigureFigure 4: LR50 4: LR50 of of Fury Fury 10EW10EW (zeta-cypermethrin) (zeta-cypermethrin) for 4 forladybird 4 ladybird species species

500 300,5c 400

300

200 ml product/ha 100 1,26b 0,54a 0,49a 0 C. 7-punctata P. 14-punctata A. bipunctata H. axyridis

FigureFigure 5: LR505: LR50 of of Confidor Confidor (imidacloprid) (imidacloprid) for 4for ladybird 4 ladybird species species

The response of different ladybird species to each product was in some cases very similar, as for Impulse, but in most cases there were great differences between species for the same product, for example for Fury 10EW and Confidor. A comparison of the LR50 values (most sensitive species LR50=1), given in Table 2, showed that the LR50 range reached a factor of 40 and 600 for these two compounds, respectively, while the range was limited to 2 for Impulse. If C. 7-punctata results are omitted, the range was more limited, between 1-2 (Impulse, Confidor) to 1-6 (Fury, Aztec, Epok).

Table 2: Comparative ratio of LR50 values of five products for 4 ladybird species. Most sensitive species, LR50=1. Glass plate test with larvae.

Impulse Epok Aztec Fury Confidor C. 7-punctata 1.43 19.17 2.49 40.39 611.79 P. 14-punctata 1 5.87 3.48 2.37 2.57 A. bipunctata 1.39 1 1 1 1.09 H. axyridis 1.96 1.89 6.59 3.70 1

100

A sensitivity classification of the ladybird species based on LR50 (Table 3) showed that generally A. bipunctata was the most sensitive species and C. 7-punctata the least. P. 14- punctata and H. axyridis sensitivity was intermediate compared to the two other species. However, this standing only gave an indication and clearly showed that it is impossible to generalize.

Table 3: Sensitivity classification of 4 ladybird species to 5 products. 1=most sentitive to 4= least sensitive. Glass plate test with larvae.

Impulse Epok Aztec Fury Confidor Mean A. bipunctata 2 1 1 1 2 1,4 P. 14-punctata 1 3 3 2 3 2,4 H. axyridis 4 2 4 3 1 2,8 C. 7-punctata 3 4 2 4 4 3,4

Discussion

Comparison of the LR50 results shows that the sensitivity of H. axyridis to pesticides seems to be similar to that of A. bipunctata and P. 14-punctata on glass plates. Comparative LR50 values were in a range of 1-6 and even for some products, H. axyridis was more sensitive than P. 14-punctata and A. bipunctata. In comparison with C. 7-punctata, H. axyridis was clearly more sensitive, but several questions must be answered concerning results obtained with the seven-spot ladybird. These results are different than those obtained in the context of the 4th Joint Pesticide Testing Program, where H. axyridis appeared to be less sensitive to several pesticides than S. 11-notata, a European native species (Hassan et al., 1988). However, although the products and the doses tested were similar, the methods differed, making it difficult to make a direct comparison of the results. These results suggest that the use of pesticides in the field is unlikely to confer a competitive advantage on H. axyridis. However, under field conditions the phenology of insects directly influences possible exposure to pesticide. Thus, treatment positioning and presence of H. axyridis in the field, (that arrive a little later than other species), can modify this trend. Importance of searching activity of the larvae and aphid ingestion can also modify pesticide uptake, both by contact and by ingestion of contaminated food. Higher activity and voracity of H. axyridis compared to other species would be expected to increase pesticide toxicity. The results of this study suggest that extrapolation of results obtained for one species to another one can be complicated. As a general trend A. bipunctata appears to be the most sensitive species and products that are harmless for this species could be expected to be harmless for the other species. C. 7-punctata was the least sensitive species and products that were harmful for this ladybird will probably be harmful for the other species. The LR50 values were variable in range according to the product tested, with sometimes a very good correlation between species, by example for Impulse, with LR50 ratio 1-2 or to a lesser extend for Aztec, with LR50 ratio 1-6. For other products, correlation was not good, with LR50 ratio up to 600, making extrapolation between species hazardous. However, most of high LR50 ratios came from C. 7-punctata results. If these results are omitted, correlation of results and extrapolation seems to be better, with LR50 ratios of 1-2 to 1-6 at maximum. If an indicator species for standard tests must be selected, A. bipunctata and H. axyridis are the best candidates: A. bipunctata because of its sensitivity and H. axyridis because of its handling and ease of rearing and the possible extrapolation of results to the other species. P. 14-punctata, 101

which was not the least sensitive species and is not so easy to produce in laboratory, is not a candidate for selection as an indicator species. Results obtained for C. 7-punctata suggest that this species is not an useful indicator species. With LR50 ratio of up to 600 on glass plates, there is a high risk to rate harmless for ladybirds a product that is harmless for C. 7-punctata alone. The results obtained with Fury and Confidor, with high differences between the seven spot ladybird and the other ones, while these differences do not appear so clearly with other products, are the indication of a possible resistance mechanism developed by the strain used for the test. The C. 7-punctata rearing of the laboratory was started and renewed with adults sampled in field margins, hedges and plants in an agroecosystem and the ladybird population was probably exposed to a range of pesticides over many years, including imidacloprid and pyrethroid insecticides. But it was strange that only the C. 7-punctata strain could develop resistance while the other species, that were sampled more or less in the same places, could not. A set of experiments with C. 7- punctata strains from other origins are needed to discover whether the results of this study apply to the species or just to the particular strain tested. According to these results the usefulness of C. 7-punctata as an indicator species for side effects should be reconsidered.

References

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Vanwetswinkel, G. 1987. Results of the third joint pesticide testing programme of the IOBC/WPRS-Working Group "Pesticides and Beneficial Organisms". – Journal of Applied Entomology 103: 92-107 Hassan, S.A.; Bigler, F.; Bogenschütz, H.; Boller, E.; Brun, J.; Chiverton, P.; Edwards, P.; Mansour, F.; Naton, E.; Oomen, P.A.; Overmeer, W.P.J.; Polgar, L.; Rieckmann, W.; Samsøe-Petersen, L.; Stäubli, A.; Sterk, G; Tavares, K.; Tuset, J.J.; Viggiani, G. & Vivas, A.G. 1988: Results of the fourth joint pesticide testing programme by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". – Journal of Applied Entomology 105: 321-329. Hassan, S.A.; Bigler, F.; Bogenschütz, H; Boller, E.; Brun, J.; Calis, J. N. M.; Chiverton, P.; Coremans-Pelseneer, J.; Duso, C.; Lewis, G.B.; Mansour, F.; Moreth, L.; Oomen, P.A.; Overmeer, W.P.J.; Polgar, L.; Rieckmann, W.; Samsøe-Ppetersen, L.; Stäubli, A.; Sterk, G; Tavares, K.; Tuset, J.J. & Viggiani, G. 1991: Results of the fifth joint pesticide testing programme carried out by the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". – Entomophaga 36: 55-67. Hassan, S.A.; Bigler, F.; Bogenschütz, H.; Boller, E.; Brun, J.; Calis, J.N.M.; Coremans- Pelseneer, J.; Duso, C.; Grove, A.; Heimbach, U.; Helyer, N.; Hokkanen, H.; Lewis, G.B.; Mansour, F.; Moreth, L.; Polgar, L.; Samsøe-Petersen, L.; Sauphanor, B.; Stäubli, A.; Sterk, G; Vainio, A.; Van de Veire, M.; Viggiani, G. & Vogt, H. 1994: Results of the sixth joint pesticide testing programme of the IOBC/WPRS-Working Group "Pesticides and Beneficial Arthropods". – Entomophaga 39: 107-119. Hautier, L. 2003: Impact sur l'entomofaune indigène d'une coccinelle exotique utilisée en lutte biologique. – DES en Gestion de l'Environnement, Université Libre de Bruxelles, Belgium. Hodek, I. 1973: Biology of Coccinellidae. – Dr W. Junk, The Hague. Hokkanen, H.M., Babendreier, D. Bigler, F., Burgio, G., Kuske, S. Van Lenteren, J.C., Loomans, A.J., Menzler-Hokkanen, I., Van Rijn, P.C., Thomas, M.B., Tommasini, M.G & Zeng, Q.Q. 2003: Evaluating environmental risks of biological control introductions into Europe (ERBIC), final report. Honek, A. 1995: Habitat preferences of aphidophagous coccinellids. – Entomophaga 30: 253- 264. Jansen, J.-P. 2000: A three-year study on the short-term effects of insecticides used to control cereal aphids on plant dwelling aphid predators in winter wheat. – Pest management Science 56: 533-539. Jansen, J.-P. & Warnier, A.-M. 2004. Aphid specific predators in potato in Belgium. – Comm. Appl. Biol. Sci., Ghent University 69/3. Kajita, Y., Takano, F., Yasuda, H. & Agarwala, B.K. 2000: Effects of indigenous ladybird species on the survival of an exotic species in relation to prey abundance. – Applied Entomology and Zoology 35: 473-479. Lamana, M.L. & Miller, J.C. 1996: Field observations on Harmonia axyridis Pallas (Coleoptera: Coccinellidae) in Oregon. – Biological Control 6: 232-237. Latteur, G. & Oger, R. 1987. Principes de base du système d'avertissement relatif à la lutte contre les pucerons des froments d'hiver en Belgique. – Annales ANPP 6(II-III): 129- 136. Michaud, J.-P. 2002: Invasion of the Florida citrus ecosystem by Harmonia axyridis (Coleoptera: Coccinellidae) and asymetric competition with a native species, Cycloneda sanguinea. – Environmental Entomology 31: 827-835. 103

Poehling, H.-M. & Borgemeister, C. 1989: Abundance of coccinellids and syrphids in relation to cereal aphid density in winter wheat fields in Northern Germany. – IOBC/WPRS Bulletin 12 (1): 99-107. Rautapää, J. 1976: Population dynamics of cereal aphids and method of predicting population trends. – Annals Agricultura Fenniae 15: 272-293. Sterk, G.; Hassan, S.A.; Baillod, M.; Bakker, F.; Bigler, F.; Blümel, S.; Bogenschütz, H.; Boller, E.; Bromand, B.; Brun, J.; Calis, J.N.M.; Coremans-Pelseneer, J.; Duso, C.; Garrido, A.; Grove, A.; Heimbach, U.; Hokkanen, H.; Jacas, J.; Lewis, G.B.; Moreth, L.; Polgar, L.; Rovesti, L.; Samsøe-Petersen, L.; Sauphanor, B.; Schaub, L.; Stäubli, A.; Tuset, J.; Vainio, A.; Van de Veire, M.; Viggiani, G.; Vinuela, E. & Vogt, H. 1999: Results of the seventh joint pesticide testing programme carried out by the IOBC/WPRS- Working Group "Pesticides and Beneficial Organisms". – Biocontrol 44: 99-117. Yasuda, H. & Katsuhiro, S. 1997: Cannibalism and interspecific predation in two predatory ladybirds in relation to prey abundance in the field. – Entomophaga 42: 153-164. Yasuda, H., Kikuchi, T., Kindlmann, P & Sato, S. 2001: Relationships between attack and escape rates, cannibalism and intraguild predation in larvae of two predatory ladybirds. – Journal of Insect Behavior 14: 373-384.

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 pp. 105-109

Natural enemies of plum brown scale Parthenolecanium corni Bouché (Homoptera: Coccidae) in plum orchards in the region of Plovdiv

Vesselin Arnaoudov1, Remigiusz Olszak2, Hristina Kutinkova1 1 Fruit Growing Institute, kv. “Ostromila” 12, 4004 Plovdiv, Bulgaria, e-mail: [email protected] 2 Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100, Skierniewice, Poland

Abstract: Parthenolecanium corni Bouche (Homoptera: Coccidae) is considered as a serious pest of stone fruits and some ornamental plants in Bulgaria. In the years 2002-2004 a survey was conducted in the region of Plovdiv, Bulgaria, aimed at determining the species of parasitoid and predatory insects associated with P. corni and their density. Seven species of hymenopterous parasitoids were found in association with P. corni, including 4 primary parasitoids – Coccophagus lycimnia Walk., Blastothrix confusa Erd., Metaphycus insidiosus Merc., Metaphycus punctipes Palm and 3 secondary – Pachyneuron concolor Först., Pachyneuron solitarium Andre and Marietta picta Andre. Out of predatory insects attacking P. corni, 10 species of predators were found, belonging to 3 orders: Coleoptera, Heteroptera and Neuroptera. In the region of Plovdiv C. lycimnia and B. confusa were parasites of the greatest importance in regulating population density of P. corni. C. lycimnia was more important as a parasite of over-wintering larvae and B. confusa was the most important parasite of adult females.

Key words: plum brown scale, Parthenolecanium corni, predators, parasitoids

Introduction

Plum brown scale Parthenolecanium corni Bouché (Homoptera: Coccidae) is an economically important pest of plum crop in Bulgaria. Along with plum (Prunus sp.) it also attacks black locust (Robinia pseudoacacia L.), hazelnut (Corylus avellana Mill.), grapevine (Vitis vinifera L.) and a number of other fruit and ornamental tree species. The study of natural enemies of the plum brown scale was an object of many investigations. Fulmek (1943) announced 52 species, Borhsenius (1957) – 22, Mitic-Muzina (1964) – 26, Talitskii et al. – 15 (1966), Blahutiak (1977) – 16 species, etc. During the studies a special attention was paid to the problems related to the description of the range of species and the ratio of parasites in the plum brown scale (Mitic-Muzina, 1964; Talitskii et al., 1966; Saakyan-Baranova et al., 1971; Blahutiak, 1977). Sugonyaev and Talitskii (1961) were the first to describe the role of Blastothrix confusa Erd. as a factor regulating population of P. corni in Moldova. Entomophages of P. corni in Bulgaria were first described by Chorbadzhiev (1933), but more profound studies on the range of species and the regulatory role of these entomophages on the pest populations were carried out by Tsalev (1977) and Stancheva (1989). Tsalev (1977) discussed B. confusa as the most efficient parasite of P. corni while Stancheva (1989) considered both C. lycimnia and B. confusa equally efficient in that respect. The present study was aimed at defining the species of entomophages related to Parthenolecanium corni and their possibility to regulate the population density of the pest in plum orchards of the Plovdiv region.

105 106

Material and methods

The studies were carried out in the period 2002-2004 in neglected plum orchards in the surroundings of Plovdiv, Asenovgrad and in the village of Trud. The parasite range of species and the percentage of parasitising on the over-wintering larvae and adults of P. corni were detected by individual cultures of the inhabited host until flying of the parasites. For that purpose, 20 twigs (10 cm long) infested with larvae or adults of P. corni were sampled in spring and summer from a plot of at least 10 trees, randomly selected. The same twigs were inspected in a laboratory, by binocular, and the number of the present living scales was registered. Sampled twigs were put then into a photoeclector (described from Stancheva, 1989) and flying insects were caught for determination of the range of parasite species. The percentage of parasitising on the over-wintering larvae and adults of P. corni was determined by counting the destroyed scales. The range of predatory species was studied by visual observations and by application of the method of shaking. Arthropod predators were assessed bimonthly during the season by visual controls on 20 twigs infested with P. corni colonies in all orchards. The same twigs were selected randomly in the orchards among those, which showed a high infestation of P. corni. Specimens of adult predators were collected bimonthly by shaking of heavily infested trees, randomly selected. All insects described as scale predators in this study were observed as feeding on P. corni.

Results and discussion

As a result of the studies carried out in the fruit-growing region of Plovdiv, 17 species of natural antagonists of the plum brown scale (Parthenolecanium corni Bouche) were detected, including 4 primary parasitoids, 3 secondary parasitoids and 10 species of predators. The useful activity of these entomophages was quite diverse, manifested mainly by destroying particular stages of the host or by restricting its fertility or life activity. The following predatory insects feeding on different developmental stages of Р. corni were detected: Brachytarsus nebulosus Först. (Coleoptera: Anthribidae), Еxochomus qudripustulatus L., Chilocorus bipustulatus L., Coccinella septempunctata L. and Adalia bipunctata L. (Coleoptera: Coccinellidae); Campylomma verbasci Mey-Dur, Pilophorus perplexus Dougl.-Scott and Deraeocoris ruber L. (Heteroptera: Miridae); Sympherobius pygmaeus Ramb. (Neuroptera: Hemerobiidae) and Chrysoperla carnea Steph. (Neuroptera: Chrysopidae). The complex of predatory insects attacking Р. corni at different stages of its development was quite diversified in content and density, but their role in restricting the pest density was comparatively small. Three most abundant predatory families in all orchards were Coccinellidae (39%), Miridae (27%) and Anthribidae (17%). Chrysopidae (15%) and Hemerobiidae (2%) were also observed preying on P. corni larvae (Table 1). The most abundant species was Brachytarsus nebulosus Forst. This predator was destroying more than 50% colonies of P. corni in plum orchards of Asenovgrad and Trud. The coccinellids were the most common and most abundant predatory group in all orchards. Coccinella septempunctata L. and Adalia bipunctata L. were the most abundant during the first part of the survey, when the population of aphids in the tree canopy increased, whereas Exochomus quadripustulatus, Chilocorus bipustulatus were the most common on trees, which had a higher infestation of P. corni. The occurrence of mirids, chrysopids and hemerobiids were related to the density of P. corni and other available host pests in plum orchards.

107

Table 1. The most common predators and their density in plum orchards of Plovdiv region in the period 2002-2004. Region studied Insect group and species Asenovgrad Plovdiv Trud Total No. % No. % No. % No.. % Coleoptera 303 57.7 210 47.3 459 59.6 972 55.9 Anthribidae 120 22.8 0 0 167 26.9 287 16.5 Brachytarsus nebulosus Frost. 120 22.8 0 0 167 26.9 287 16.5 Coccinellidae 183 34.9 210 47.3 292 32.7 685 39.4 Exochomus quadripustulatus L. 35 6,7 52 11.7 67 8.7 154 8.9 Chilocorus bipustulatus L. 32 6.1 37 8.3 58 7.5 127 7.3 Coccinella septempunctata L. 82 15.6 75 16.9 96 12.5 253 14.5 Adalia bipunctata L. 34 6.5 46 10.4 71 9.2 151 8.7 Heteroptera 125 23.8 141 31.8 203 26.4 469 27.0 Campylomma verbasci M.D. 90 17.1 76 17.1 168 21.8 334 19.2 Piloporus perplexus D.S. 35 6.7 20 4.5 35 4.6 90 5.2 Deraecoris ruber L. 0 0 45 10.1 0 0 45 2.6 Neuroptera 97 18.5 93 20.9 108 14.0 298 17.1 Chrysoperla carnea Steph. 74 14.1 87 19.6 108 14.0 269 15.4 Sympherobius pygmaeus 23 4.4 6 1.3 0 0 29 1.7 Ramb. Total effective 525 100 444 100 770 100 1739 100

The following parasites attacking the larvae of Р. corni were detected: Coccophagus lycimnia Walk., Blastothrix confusa Erd., Metaphycus insidiosus Merc. and Metaphycus punctipes Palm. The mean percentage of parasitized overwintering larvae of Р. corni for the whole period was reported to be 52.9 %. (Table 2). The highest percentage of parasitizing was registered for C. lycimnia – 32.9 % on average. B. confusa came the second – 16.7 %, followed by M. insidiosus – 3.1 %. For M. punctipes parasitizing was only 0,2 %; this showed that the latter species did not play any significant role in restricting the population of Р. corni. The following species were detected in the adult females of Р. corni: Coccophagus lycimnia, Blastothrix confusa, Metaphycus insidiosus and Metaphycus punctipes Palm as primary parasites and Pachyneuron concolor Först., Pachyneuron solitarium André and Marietta picta André as secondary parasites. Parasitizing on the adult females of Р. corni in particular regions and years varied within a broad range, from 46 to 67 %, the average percentage for the period being 55.5 % (Table 3). It is worth mentioning that, in contrast to the larvae which were mainly parasitized by C. lycimnia, the adult females were mostly parasitized by B. confusa, amounting up to 40.9 % on average. As for M. insidiosus and C. lycimnia that percentage was comparatively low (8.5 and 6.0, respectively) and varied greatly depending on the region and season, without definite predominance of any species. The role of the primary parasitoid M. punctipes, as well as of the secondary parasitoids P. concolor, P. solitarium and M. picta in reducing the population of Р. corni was insignificant. Summarizing the results of the studies, it may be concluded that in the plum orchards of the region of Plovdiv, in which chemical treatments were not carried out, a great number of entomophagous species was found and they were able to keep the population of the plum 108

brown scale at a comparatively low level. The parasite species C. lycimnia and B. confusa were of the greatest importance as they parasitized successfully both the larvae and the adult females of Р. corni. M. insidiosus made also a significant contribution for decreasing the pest population.

Table 2. Parasitism on the overwintering larvae of Р. corni. Regions studied Parasitoids Asenovgrad Plovdiv Trud 2002 2003 2004 2002 2003 2004 2002 2003 2004 Total number of reported plum brown scales 220 238 216 240 226 248 198 218 204 Out of them parasitized [%] 43.2 49.6 46.8 49.6 55.3 51.2 54.5 66.5 59.8 In % by species: Coccophagus lycimnia 27.3 34.9 26.4 29.2 32.3 30.3 31.3 48.2 35.8 Blastothrix confusa 12.7 12.2 18.5 17.1 19.0 16.9 18.7 15.1 20.1 Metaphycus insidiosus 3.2 2.5 1.9 2.9 4.0 3.6 4.0 2.8 3.4 Metaphycus punctipes 0.0 0.0 0.0 0.4 0.0 0.4 0.5 0,4 0.5

Table 3. Parasitism on the adult females of P. corni. Regions studied Parasitoids Asenovgrad Plovdiv Trud 2002 2003 2004 2002 2003 2004 2002 2003 2004 Total number of reported plum brown scales 268 245 259 248 262 276 254 246 238 Out of them parasitized [%] 38.8 56.3 42.5 48.5 66.8 46.7 59.9 72.3 67.9 In % by species: Blastothrix confusa 26.9 46.1 33.6 32.7 46.2 32.9 42.5 58.1 46.3 Coccophagus lycimnia 6.7 3.2 4.3 7.3 5.3 3.6 6.3 8.1 8.8 Metaphycus insidiosus 5.2 7.0 4.6 8.5 14.1 9.8 9.5 6.1 11.3 Metaphycus punctipes 0.0 0.0 0.0 0.0 1.2 0.4 1.6 0.0 1.3

The results obtained showed a high percentage of parasitizing on the overwintering larvae by C. lycimnia and in the adult females – by B. confusa. To a certain extent they differed from those reported by Tsalev (1977) about the region of Kostinbrod where B. confusa proved to be the most efficient species irrespective of the stage of development during infestation. The change in the dominancy of the species observed in the different regions was apparently due to the disturbance of the interspecific competition resulting from changes occurring in the trophic conditions, which could favour to a greater extent one species or another. A similar opinion was also expressed by other authors (Blahutiak, 1977). 109

Conclusions

• In the agrocenosis of Plovdiv fruit growing region 17 species of natural enemies of the plum brown scale were detected, including 4 primary parasites, 3 secondary ones and 10 species of predatory insects. • Coccophagus lycimnia was the major parasitoid of overwintering larvae of Parthenolecanium corni, with between 26.4% and 48.2% of plum brown scale parasitized by this species in three surveyed regions over a three-year period. Blastotrix confusa parasitized between 12.2% and 20.1% of overwintering larvae. • B. confusa was the major parasitoid of adult females of P. corni, with between 26.9% and 58.1% of scales being attacked over three years in three regions surveyed. • C. lycimnia was of minor importance as a parasite of adult females scales, parasitizing between 3.6% and 8.8% over the three-year period of studies.

References

Blahutiak, A. 1977: Prirodozeni nepriatelia puklice slivkove (Lecanium corni Bouché). (Homoptera: Coccidae), v CSSR. – Polnospodarska veda, Seria A, Bratislava. Borhsenius, N.S. 1957. Homoptera. Subord. Coccids (Coccoidea) Fam. Pseudococcidae and Coccidae. – In: Fauna of USSR. Ed. Academy of Sciences of USSR, Publishing house “Nauka”, Moscow – Leningrad, 9: 493 pp. Chorbadzhiev, P. 1933: Pest insects in the fruit trees in Bulgaria. – Issue of the Ministry of Agriculture Agricultural Library, Sofia, No. 9, 240. Fulmek, L.S. 1943: Wirtsindex der Aleyrodiden und Cocciden Parasiten. – Entomologische Beihefte 10. Saakyan-Baranova, A.A., Sugonyaev, E.S. & Sheldeshova, G.G. 1954: Plum brown scale Parthenolecanium corni Bouché (Homoptera: Coccidae) and its parasitoids. 165, Leningrad. Sugonyaev, E.S. & Talitskii, V.I. 1961: Parasitoids of plum brown scale (Parthenolecanium corni Bouché) in Moldavian SSR. – Works of Moldavian Research Institute of Viticulture and Wine Production 7: 101-117. Stancheva, I.S. 1989: Plum brown scale Parthenolecanium corni Bouché (Homoptera: Coccidae) and its entomophages. – Ph.D thesis, Plovdiv: 192 pp. Talitskii, V.I., Sugonyaev, E.S. & Goanza, I.K. 1966: Parsitoid and predator insects of the plum brown scale (Parthenolecanium corni Bouché) in Moldavian SSR. – Works of Moldavian Research Institute of Viticulture and Wine Production 13: 317-359. Tsalev, M. 1977: The role of the entomophages to reducing the number dynamics of the plum brown scale (Parthenolecanium corni Bouché). – In: Biological and Integrated Control in Plant Protection. Issue of the Ministry of Agriculture and Food Industry, Sofia: 34-40.

ABSTRACTS

Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 113

Consideration of side effects on beneficial organisms during product development in the agrochemicals industry

Hans-Jürgen Schnorbach Bayer CropScience AG, Agronomic Development Insecticides, Monheim, Germany, e-mail: [email protected]

Specificity and selectivity are important prerequisites of a modern, IPM compatible plant protection product (PPP). The identification of a selective compound in the early research/ screening process favours its development. Preliminary tests on the predatory mite Typhlo- dromus pyri and ladybird beetle larvae Coccinella septempunctata are performed for an early differentiation of the spectrum of activity. During the early development process side effects of products on foliar dwelling beneficial arthropods are investigated in semi-field cage tests. Representative beneficial insects like ladybird beetles (C. septempunctata), parasitoids (Aphidius colemani), predatory bugs (Anthocoris nemoralis), predatory midges (Aphidoletes aphidimyza) and hover flies (Episyrphus balteatus) are chosen for these tests. Effects on eggs, larvae, pupae and adults are examined to quantify the possible specificity of a compound with respect to the developmental stage. Insects are either directly treated, exposed to treated surfaces or are fed with contaminated prey. Hatching of parasitoids is investigated after treatment of the mummies. In the course of the main development of a compound a wide range of field tests is conducted with various beneficial organisms in the most relevant crops (e.g. apple, pear, citrus, wine, cereals, rice, vegetables, cotton). Important parameters like prey-predator ratio, parasitisation rate, residual efficacy and time to recovery are determined under practical conditions. Knowledge on the selectivity of a PPP allows to design treatment strategies involving a combination or succession with commercial beneficials. In case any adverse effects are observed, the waiting period until beneficial organisms may be released is investigated. Recommendations with regard to alternative application methods like drench or seed dressing can also contribute to a safe use for beneficial organisms. Trial results from various field research stations throughout the world give a representative overview of local situations with regard to the interaction of climatic conditions, crops, pests and beneficials. All information gathered on the side effects of a PPP on beneficial arthropods contributes to the definition of an use pattern in compliance with national IPM rules.

113 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 114

Effects of Imidacloprid on Poecilus cupreus larvae depending on the mode of application

Barbara Baier, Claudia Norr, Detlef Schenke and Tanja Scharnhorst Institute for Ecotoxicology and Ecochemistry in Plant protection, Federal Research Centre for Agriculture and Forestry, Königin-Luise-Straße 19, D-14195 Berlin, Germany, e-mail: [email protected]

Imidacloprid can be applied as seed treatment (seed dressing and pelleting) or it can be sprayed. The aim of the laboratory investigations was to evaluate the effects of imidacloprid on Poecilus cupreus larvae: experiment 1) applied as seed dressing (winter wheat) and pelleting (sugar beet); and experiment 2) applied as seed dressing compared to spray application. Winter wheat seeds were dressed with Gaucho 350 FS and Gasur (both 350 g imidacloprid/100 kg seeds), respectively. Sugar beets were pelleted with Gaucho WS (91 g imidacloprid/100000 seeds). Confidor 70 W was sprayed (63.5 g imidacloprid/ha). The tests were carried out with glass tubes used for the standard test for soil-surface-applied plant protection products with 5 cm2 surface and larger containers (winter wheat 92 cm2 surface, sugar beet 92, 188 and 384 cm2 surface). Larger containers were selected to achieve a more realistic exposure regarding seed density in the field. Lufa 2.1 was used as substrate. One larva of 24 to 48 hours age was released into each test unit. In addition to the biological investigations, the imidacloprid amount on seeds with mean corn weight and the imidacloprid concentration in the soil were determined. Chemical analyses indicated that only a zone of approximately 2.4 cm in diameter around the coated seed was exposed to higher imidacloprid concentration. The field winter wheat seed density (app. 4.5 million seeds/ha) and the 2fold field seed density with sugar beets (260000 seeds/ha) led to an effect of 58% and 6% (corrected mortality) on Poecilus cupreus larvae. Therefore the effect of Imidacloprid applied as seed treatment on larvae of Poecilus cupreus was dependent on the number of coated seeds/ha primarily. 100% mortality of the Poecilus cupreus larvae was observed when imidacloprid was sprayed.

114 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 115

Side effects of pesticides on Aphelinus mali and other antagonists of the woolly apple aphid

Heidrun Vogt & Pia Ternes Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 10, D-69221 Dossenheim, e-mail: [email protected]

In the last years an increase in infestations of the woolly apple aphid, Eriosoma lanigerum, has been observed in organic as well as in integrated apple production. In order to enhance the biological control of this pest, the safe-guard of its natural antagonists, especially the parasitoid Aphelinus mali, but also earwigs, coccinellids and lacewings is a main objective. For this reason, investigations were carried out on the side effects of pesticides used in organic apple production and trials were started with the neonicotinoids Confidor (a.i. imidacloprid, 700g/kg) and Calypso (a.i. thiacloprid, 480g/l) used in integrated apple production. The tested organic pesticides were Quassia extract and its active ingredients Quassin and Neoquassin, Kumulus WG (sulphur, 800 g/kg), Funguran (756 g cupperoxychloride/kg) und lime sulphur (a.i. Calciumpolysulfid 80%, sulphur 23 %). As the active ingredients of Quassia extracts vary depending on the origin of the Quassia wood, a defined extract, produced by Trifolio-M GmbH (Lahnau, Germany), was used. The parasitoids came from an own rearing. Fresh residues of Quassin, Neoquassin and Quassia – extract in rates as recommended in practice (up to 18 g/ha) were harmless for the parasitoid adults. When applied via the food, i.e. mixed in fructose solution, Quassin and Quassia-extract resulted in dose dependent effects, though not exceeding 30% at the highest rate of 18g/ha. Neoquassin applied via the food was harmless at the highest rate of 18 g/ha. Quassin and Quassia extracts did not harm the parasitoids during their development within the woolly apple aphid mummies and did not affect reproduction of the subsequent generation. Furthermore Quassia was harmless for For- ficularia auricularia, Coccinella septempunctata and Chrysoperla carnea (direct spraying and oral application). Residual contact of fresh residues of Kumulus (0,4 - 2 kg/ha)and Funguran (0,2 – 0,5 kg/ha) resulted in low mortalities (≤ 10%), whereas lime sulphur (6 l and 15 l/ha) caused 80- 100% mortality. In the field, 5 applications of Kumulus (2,5 kg/ha each) and 2 applications of lime sulphur (15 and 20 l/ha) did not cause reductions in parasitization compared to the control. In the lab (residual contact), even very low rates of Confidor resulted in high mortalities of Aphelinus mali adults, whereas Calypso, applied in rates as used in practice caused mortalities between 10 and 40 %. Both neonicotinoids did not affect the protected stage of the parasitoid within the woolly apple aphid mummy, when the mummies were directly sprayed. Further investigations aim to check sublethal effects and potential influence on the behavior of the parasitoid.

115 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 116

Effects of toxins in transgenic crops on natural enemies

Zbigniew T. Dąbrowski and Julia Górecka Department of Applied Entomology, Warsaw Agricultural University, Nowoursynowska 159, 02-787 Warsaw, Polan. E-mail: [email protected]; [email protected]

First successes in introduction of transgenic crops in the USA, Argentina, Canada and other countries brought a hope of their potential beneficial effects as follows: increased flexibility in crop management; decreased dependency on synthetic pesticides and season long protection; enhanced yields and considerable financial savings. First reports in the late 1990- ties showed that there was 30 – 50% reduction of pesticide usage on maize in the USA. At the same time some scientific reports gave evidences of negative side effects of genetically modified plants (GMP). In 2002 Entomological Society of America (ESA) released its position statement on transgenic insect-resistant crops: potential benefits and hazards . The statement empha- sizes that the evaluation of hazards connected with the release of GMP should consider procedures previously developed for pesticides as follows: human health, environmental impacts, insect resistance to transgenic plants, management of resistance. At the same time the wisdom of using a specific-resistant crop should be evaluated relative to the long-term goals of reducing pesticide use and fostering sustainable crop production systems. The challenge facing entomologists and pest managers is to ensure that these crop varieties are used properly and that scientific information remains a cornerstone of debate regarding their deployment. Genetically modified plants (GMP) may soon be commercially cultivated in several countries of the European Union (EU). According to the EU Directive 2001/18/EC, pre- release risk assessment and post-market monitoring for commercial GMP cultivation has to be implemented, which allows for detection and prevention of adverse effects on human health and environment. Currently there is neither EU - or even between scientists - wide consensus on how relevant procedures have to be designed to provide sound scientific data. Already some European authors have carried out large scale field experiments on the effect of transgenic cultivars on natural enemies. The data interpretation should however separate a direct effect of GMP toxins on natural enemies from an indirect effect through reduced abundance of phytophagous prey. The paper reviews methods and techniques used by various authors in studies of GMP effects on natural enemies. The initiatives of members of the IOBC working group “GMO in integrated plant production” in studies on ecological impact of genetically modiefied organisms and their wide differences in opinion on selection of species as bio-indicators in risk assessment and post-release monitoring are presented.

116 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 117

Influence of the treated media on the residual toxicity of several insecticides to Chrysoperla carnea and Chrysoperla externa in laboratory

Gladys Contreras, Pilar Medina, Elisa Viñuela Protección de Cultivos. E.T.S.I.Agrónomos. Polytechnic University of Madrid. E-28040-Madrid. Spain. E-mail: [email protected]

Laboratory and extended laboratory studies were performed to ascertain the susceptibility to pesticides of two predator species of the genus Chrysoperla (Neuroptera, Chrysopidae): the cosmopolitan C. carnea (Stephens) and the widely distributed in America C. externa (Hagen). The residual toxicity of fresh residues of several commercial insecticides applied at the maximum field recommended concentrations registered in Spain or at that recommended by the manufacturer for caolin, were evaluated following IOBC guidelines: Volck Miscible® (83% summer mineral oil, EC, Agrodán, 1.5 l/hl), Surround® (95% caolin, WP, Agrovital, 5 kg/hl), Sistematon 40® (40% dimethoate, EC, Agrodán, 150ml/hl), Karate King® (2.5% lambda-cyhalothrin, WG, Syngenta, 80 g/hl) and Juvinal® (10% pyriproxyfen, EC, Kenogard, 30 ml/hl). Individualised young larvae (L2) of the two predator species were exposed to pesticides residues deposited either on glass or on olive leaves. Glass plates (11.8 x 11.8 cm) or leaves were treated under the Potter precision spray tower at 55 kPa pressure (deposit 1.4 and 1.6 mg/cm2 for C. externa and C. carnea) and seven prism plastic containers (3.5 cm diameter in the bottom and 2.5 cm in the top; 4 cm high) previously coated with talc to prevent larvae from climbing the walls, were placed on each glass plate. The petiole of each leaf was introduced in an eppendorf with a nutritive solution and placed in a ventilated plastic cage (9 cm in diameter, 2 cm high, cover with a 4 cm in diam ventilation hole covered by a mesh). Eggs of Ephestia kuehniella Zeller were always provided ad libitum as food. Larval mortality as well as percentages of pupae and adult emergence were recorded, and insecticides were classified in the four IOBC toxicity ratings for the total effect. Results show that the two Chrysoperla species were equally susceptible to the studied pesticides. Mineral oil and caolin were harmless, dimethoate and lambda-cyhalothrin slightly or moderately harmful and pyriproxyfen harmless on leaves but harmful on glass because totally prevented pupae formation.

117 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 118

Side effects of various pesticides on Feltiella acarisuga

Koen Altena1, Ed Moerman1 1 Koppert Biological Systems, PO Box 155, 2650 AD Berkel en Rodenrijs, the Netherlands, e-mail: [email protected], [email protected]

Biological control of two-spotted spider mite Tetranychus urticae (Koch) in cut roses has largely contributed to the increasing use of the predatory gall midge Feltiella acarisuga (Vallot). This required a better understanding of the impact of the application of various pesticides. Laboratory trials with direct application of these pesticides on F. acarisuga larvae on sweet pepper leaves on agar in Petri dishes, followed by counts and qualitative observations helped to improve the understanding ànd the success rate of commercial introductions.

Key words: Feltiella acarisuga, side effects, abamectine, bifenazate, hexythiazox, imidacloprid, milbemectin, pymetrozine, pyridaben, pyriproxifen, spinosad, tebufenpyrad.

Side effects of various pesticides on Amblyseius swirskii

Pilar Vanaclocha Arocas1, Hans Hoogerbrugge1, Ed Moerman1 1 Koppert Biological Systems, PO Box 155, 2650 AD Berkel en Rodenrijs, the Netherlands, e-mail: [email protected], [email protected]

The unique feature of a beneficial that contributes to the control of both thrips and whitefly has generated a wide interest in the predatory mite Amblyseius swirskii. In the first year of commercial availability, the product SWIRSKI-MITE has been introduced in hundreds of hectares of protected sweet pepper, aubergine and cucumber, first in the Netherlands and then in other European countries. This publication describes the results of laboratory trials with 12 insecticides/acaricides and 6 fungicides as well as the first field experiences with the side effects of some pesticides.

Key words: Amblyseius swirskii, side effects, abamectin, azoxystrobin, Bacillus thuringiensis, bifenazate, bitertanol, boscalid, bupirimate, carbendazim, cyromazin, fenarimol, fenbutatinoxide, imazalil, imidacloprid, kresoximethyl, milbemectin, pirimicarb, potassiumsalts of fatty acids, pymetrozine, pyridaben, spinosad, sulphur, tolylfluanide, triflumizole, vegetable oil, Verticillium lecanii.

118 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 119

Side-effects of IGR on development of an aphid-parasitoid Aphidius colemani (Hymenoptera: Braconidae) Viereck

Delphine Juan and Jean Baptiste Ferré ENIGMA, Hameau de St. Véran, F-84190 Beaumes de Venise

Four insect growth regulators and one neurotoxic insecticide were tested to evaluate their effects on several life history parameters of the aphid parasitoid Aphidius colemani Viereck. The standardised laboratory methods were used. The neurotoxic compound (organophosphate) was toxic on adults. The organophosphate and pymetrozine increased host mortality, reduced mummification and emergence rate when sprayed during all the steps of parasitoid development. All the IGR tested were slightly toxic for adults. However, Fenoxycarb increased host mortality when sprayed on young instars of the parasitoid, and decreased mummification when the application was done when the parasitoid was realising its first or second moulting. Flufenoxuron increased host mortality when it was sprayed on parasitoids during their first moulting, or sprayed when the parasitoid was at its two first larval instars, or when it was doing its nymphosis. Flufenoxuron reduced mummification when it was sprayed on parasitoids before nymphosis. Buprofezin had no effect either on host mortality, mummification or emergence at all the steps of parasitoid development. Consequently in spite of their low toxicity on adults of aphid parasitoids, IGR have to be used carefully in integrated pest management strategies considering susceptible instars of beneficials which depend on their respective modes of action.

Keywords : IGR, aphid parasitoids, host mortality, emergence, mummification, mode of action.

119 Pesticides and Beneficial Organisms IOBC/wprs Bulletin Vol. 29 (10) 2006 p. 120

The loss of earwig populations in Belgian orchards: testing side-effects on orchard management

Bruno Gobin Department of Zoology, Royal Research Station for fruitgrowing at Gorsem, Belgium [email protected] +32 11 67 43 18

Earwigs are key generalist predators to a variety of orchard pests. However, the once held believe that earwigs damage and spoil fruits led to control strategies and eventually the loss of large earwig 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 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, requiring larger sample sizes. 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. In addition to this, transplants of large earwig populations to previously unoccupied orchards are seldom successful, limiting clear-cut experimental design. These issues need to be properly addressed to limit their impact on the outcome of side-effect testing in field tests.

120