Proceedings of the Meeting Comptes Rendus De La Réunion

IOBC / WPRS

Working group „Insect Pathogens and Insect Parasitic Nematodes” Subgroup “Melolontha”

OILB / SROP

Groupe du travail “Entomopathogènes et Nématodes Parasites d’Insectes” Sous-Groupe “Melolontha”

Proceedings of the Meeting

Comptes Rendus de la Réunion

at / à

Innsbruck (Austria)

11-13 October 2004

Edited by Siegfried Keller

IOBC wprs Bulletin Bulletin OILB srop Vol. 28 (2) 2005

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]

ISBN 92-9067-174-0

Preface

The Working Group “Integrated Control of Soil Pests“ subgroup “Melolontha“ held its fourth meeting within the frame of IOBC. The topics were opened to include other soil dwelling pests like wireworms and Diabrotica which resulted in a very attractive programme. 53 participants from 12 countries met from 11-13 October 2004 in Innsbruck, Austria. The local arrangements and a half-day excursion to Melolontha sites in the Oetztal were perfectly organised by Barbara Pernfuss and Hermann Strasser and their team which is greatly acknowledged. The meeting was opened with review presentations on Diabrotica, the scarab situation in Europe and non-target effects of entomopathogenic fungi. 22 oral and 17 poster contributions were presented dealing with the following topics: Melolontha spp. – actual situation, control strategies and risk assessment; “new scarabs” – control strategies and ecology; wireworms – control, ecology, sampling and monitoring. The dissemination of Diabrotica in Europe is of great concern. Control concentrates on curative and preventive insecticide applications and on crop rotation, while no biological control method is available. The increasing problems with wireworms attracted many scientists and were vivaciously discussed. The problems with Melolontha spp. are increasing in central and east Europe with some local exceptions. They are mainly due to M. melolontha L. and concern grassland, orchards and reforestation areas, occasionally vineyards and other crops. Successful control strategies are the use of the entomopathogenic fungus Beauveria brongniartii and the placements of nets to protect expensive crops. From the other scarab species Amphimallon spp. and Phylloperta horticola show increasing populations. Metarhizium anisopliae and nematodes are considered good candidates for their control. A highlight of the meeting was the excursion to the Oetztal. This valley suffered from heavy white grub damages which eventually got under control with the application of the fungus Beauveria brongniartii. In presence of involved farmers the representatives of the local extension service explained the situation before and after the treatment and how the treatment was organised. The scientific part was followed by a visit of the Oetzi-village and a delicious dinner in alpine heights accompanied with local music and dance. Future work of the group will concentrate on improving the existing non-chemical control measures and exploring new ones especially with regard to wireworms and Diabrotica. At the administrative meeting Jürg Enkerli, Agroscope FAL Reckenholz, Zürich, was elected as new convenor of the subgroup. Further, it was decided that the name of the subgroup should be changed to “Soil insect pests” and that future meetings are held every two years alternating with the IOBC/wprs Working Group “Insect pathogens and insect parasitic nematodes”. Therefore, the next meeting will be in autumn 2006 at the Research Centre Laimburg, South Tyrolia, Italy. A press conference concluded the meeting.

Many sponsors supported the working group meeting and contributed substantially to its success. They are greatly acknowledged.

Siegfried Keller Convenor of the subgroup ii

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List of Participants

BASSO Barbara CATE Peter C. National Research Council Österreichische Agentur für Gesundheit University of Milano und Ernährungssicherheit Ges.m.b.H. (AGES) Via Celoria, 26 Spargelfeldstr. 191 20133 Milano 1226 Vienna Italy Austria Tel: + 39 02 50314718 Tel: + 43 (0)1 73216 5223 Fax: + 39 02 50314764 Fax: + 43 (0)1 73216 5216 E-mail: [email protected] E-mail: [email protected]

CLERES Sabine BENKER Ullrich lbu-Labor für Boden- und Umweltanalytik Bayerische Landesanstalt für Landwirtschaft der ERIC SCHWEIZER SAMEN AG Institut für Pflanzenschutz Postfach 150 Lange Point 10 3602 Thun 85354 Freising Switzerland Germany Tel: + 41 (0) 33 227 57 30 Tel: + 49 8161 71 5720 Fax: + 41 (0) 33 227 57 39 Fax: + 49 8161 71 5753 E-mail: [email protected] E-mail: [email protected]

DALLA VIA Josef BLACKSHAW Rod Land- und Forstwirtschaftliches Versuchs- School of Biological Sciences zentrum Laimburg University of Plymouth Laimburg 6 Drake Circus 39040 Auer PL4 8AA Plymouth Italy United Kingdom Tel: + 39 0471 969510 Tel: + 44 1626 325600 Fax: + 39 0471 969599 E-mail: [email protected] E-mail: [email protected]

EILENBERG Jørgen BRENNER Hermann Royal Veterinary and Agricultural University LBBZ Arenenberg Department of Ecology Fachstelle Pflanzenschutz und Ökologie Thorvaldsensvej 40 Arenenberg 1871 Frederiksberg 8268 Salenstein Denmark Switzerland Tel: + 45 35 282692 Tel: + 41 71 663 31 40 Fax: + 45 35 282670 Fax: + 41 71 664 28 67 E-mail: [email protected] E-mail: [email protected] ENKERLI Jürg Swiss Federal Research Station BRUNNER Nina for Agroecology and Agriculture Ludwig Boltzmann Institut Reckenholzstrasse 191 für biologischen Landbau 8046 Zürich Felbigergasse 93 /11 Switzerland 1140 Vienna Tel: + 41 1 377 7206 Austria Fax: + 41 1 377 7201 Tel: + 43 (0) 1 0650 8262545 E-mail: [email protected] E-mail: [email protected] iv

ERICSSON Jerry HADAPAD Ashok University of British Columbia University of Hohenheim Department of Plant Science Institute of Phytomedicine, Dept. of Entomology 301 – 1249 Granville Street Otto-Sander Str. 5 V6Z 1M5 Vancouver 70593 Stuttgart Canada Germany Tel: + 604 694 0791 Tel: + 49 711 459 3220 Fax: + 604 850 2150 Fax: + 49 711 459 2408 E-mail: [email protected] E-mail: [email protected]

HOZZANK Alexandra ESTER Albert InfoXgen Applied Plant Research, Arable farming Königsbrunnerstrasse 8 and field production of vegetables 2202 Enzersfeld Edelhertweg 1 Austria 8219 PH Lelystad Tel: + 43 (0)2262 672214 31 The Netherlands Fax: + 43 (0)2262 672214 33 Tel: + 31 320 291633 E-mail: [email protected] Fax: + 31 320 230479 E-mail: [email protected] JUEN Anita Centre for Mountain Agriculture University of Innsbruck Technikerstrasse13 FRÖSCHLE Manfred 6020 Innsbruck Landesanstalt für Pflanzenschutz Austria Reinsburgstr. 107 Tel: + 43 (0)512 507 5695 70197 Stuttgart E-mail: [email protected] Germany Tel: + 49 711 6642445 KABALUK Todd Fax: + 49 711 6642499 Agriculture and Agri-Food Canada E-mail: [email protected] c/o Pacific Agri-Food Research Centre Box 1000 Agassiz British Columbia FURLAN Lorenzo Canada VOM 1AO Group of Entomolgy, Departement of E-mail: [email protected] Agronomy, University of Padova Via Carozzani 18 KATZUR Katrin 30027 San Donà di Piave Biologische Bundesanstalt für Land- und Italy Forstwirtschaft Tel: + 39 0335 7162739 Messeweg 11-12 Fax: + 39 0421 596659 38104 Braunschweig E-mail: [email protected] Germany Tel: + 49 531 2994576 Fax: + 49 531 2993008 GUENTER Martin E-mail: [email protected] Andermatt Biocontrol AG Stalermatten 6 KELLER Siegfried 6146 Grossdietwil Federal Research Station for Agroecology Switzerland Reckenholzstrasse 191 Tel: + 41 62 917 50 05 8046 Zürich E-mail: [email protected] Switzerland Tel: + 41 1 377 7211 Fax: + 41 1 377 7201 E-mail: [email protected] v

KOLLER Robert LANDL Marion Biologische Bundesanstalt für Land- und Institut für Pflanzenschutz Forstwirtschaft Universität für Bodenkultur Institut für biologischen Pflanzenschutz Peter Jordan Strasse Heinrichstraße 243 1180 Vienna 64287 Darmstadt Austria Germany Tel: + 49 (0) 6151 407 0 Tel: + 43 (0) 1 47654 3355 E-mail: [email protected] E-mail: [email protected]

KOUTNY Andreas MUŠKA František Landeslandwirtschaftskammer Tirol State Phytosanitary Admin. SRS-OPOR Fachabteilung: Pflanzenbau - Landtechnik Zemedelska 1a Brixnerstrasse 1 61300 Brno 6020 Innsbruck Czech Republic Austria Tel: + 420 5 45137057 Tel: + 43 (0) 512 5929 230 Fax: + 420 5 45211078 E-mail: [email protected] E-mail: [email protected]

KRENN Andreas NEUHOFF Daniel F. Joh. Kwizda GmbH Institute of Organic Agriculture Dr. Karl Lueger-Ring 6 1010 Vienna University of Bonn Austria Katzenburgweg 3 Tel: + 43 (0)1 53468 235 53115 Bonn Fax: + 43 (0)1 53468 280 Tel: + 49 228 735616 E-mail: [email protected] Fax: + 49 228 735617 E-mail: [email protected] KROMP Bernhard Ludwig Bolzmann Institute for PAFFRATH Andreas Biological Agriculture & Applied Ecology Landwirtschaftskammer Nordrhein-Westfalen Rinnboeckstr. 15 Endenicher Allee 60 1110 Vienna 53115 Bonn Austria Germany Tel: + 43 1 712 98 99 E-mail: [email protected] Tel: + 49 228 7031537 Fax: + 49 228 7038537 E-mail: [email protected] KRON-MORELLI Roberto Agrifutur srl Via Campagnole 8 PARKER William 25020 Alfianello (BS) ADAS Italy Woodthorne Tel: + 39 030 9934776 WV6 8TQ Wolverhampton Fax: + 39 030 9934777 United Kingdom E-mail: [email protected] Tel: + 44 1746 712815 E-mail: [email protected] LAENGLE Tobias Institut für Mikrobiologie van der PAS Rick Leopold-Franzens-Universität Koppert Biological Systems Technikerstrasse 25 6020 Innsbruck Veilingweg 17 Austria 2651 BE Berkel en Rodenrijs Tel: + 43 (0) 512 507 6010 The Netherlands Fax: + 43 (0) 512 507 2929 Tel: + 31 10 514 0444 E-mail: [email protected] Fax: + 31 10 512 1005 E-mail: [email protected] vi

PÁZMÁNDI Christian RODRIGUES Sonia Centre for Mountain Agriculture Agroscope FAL Reckenholz University of Innsbruck Reckenholzstrasse 191 Technikerstrasse 13 8046 Zürich 6020 Innsbruck Switzerland Austria Tel: + 41 1 377 7467 Tel: + 43 0512 507 5689 E-mail: [email protected] E-mail: [email protected] ROT Mojca PERNFUSS Barbara Kmetijsko gozdarski zavod Nova Gorica Institut für Mikrobiologie Pri hrastu 18 Leopold-Franzens Universität Innsbruck 5000 Nova Goriza Technikerstrasse 15 Slovenia 6020 Innsbruck Tel: + 386 5 3351211 Austria Fax: + 386 5 30 27 312 Tel: + 43 (0)512 507 6012 E-mail: [email protected] Fax: + 43 (0)512 507 2929 E-mail: [email protected] SCHEPL Ute

Landwirtschaftskammer Nordrhein-Westfalen PETERS Arne Endenicher Allee 60 e-nema GmbH 53115 Bonn Klausdorfer Str. 28-36 Germany 24223 Raisdorf Tel: + 49 2287031598 Germany Fax: + 49 2287038598 Tel: + 49 4307 82950 E-mail: [email protected] Fax: + 49 4307 829514 E-mail: [email protected] SCHNETTER Wolfgang PILZ Christina Erlenweg 10 Universität für Bodenkultur 69429 Waldbrunn-Schollbrunn Spittelauerlände 19-21/5/5/16 Germany 1090 Vienna E-mail: [email protected] Austria heidelberg.de Tel: + 43 69911370292 E-mail: [email protected] SCHWEIGKOFLER Wolfgang Research Centre for Agriculture POZENEL Anka and Forestry - Laimburg Kmetijsko gozdarski zavod Nova Gorica 39040 Auer Pri hrastu 18 Italy 5000 Nova Goriza Tel: + 39 0471 969643 Slovenia E-mail: [email protected] Tel: + 386 5 3861787 E-mail: [email protected] SHAH Syed Farooq Abbas School of Biological Sciences RAFFALT Josef University of Wales, Swansea F. Joh. Kwizda GmbH Singleton Park Dr. Karl Lueger-Ring 6 SA2 8PP Swansea 1010 Vienna United Kingdom Austria Tel: + 44 1792 513652 Tel: + 43 (0)1 53468 235 E-mail: [email protected] Fax: + 43 (0)1 53468 280 E-mail: [email protected]

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SIERPINSKA Alicja VERNON Robert S. Forest Research Institute in Warsaw Agriculture and Agri-Food Canada 05-090 Raszyn Pacific Agri-Food Research Center Sekocin-Las PO Box 1000 Poland Agassiz, British Columbia Tel: + 48 22 7150 547 / 544 Canada Fax: + 48 22 7150 557 E-mail: [email protected] E-mail: [email protected]

WEISSTEINER Sonja STRASSER Hermann Institut für Forstzoologie und Waldschutz Institut für Mikrobiologie Buesgenweg 3 Leopold-Franzens-Universität 37077 Göttingen Technikerstrasse 25 Germany 6020 Innsbruck Tel: + 49 551 393609 Austria Fax: + 49 551 392089 Tel: + 43 (0)512 507 6008 E-mail: [email protected] E-mail: [email protected]

ZELGER Roland TOEPFER Stefan Research Center for Agriculture CABI Bioscience CH and Forestry Research, Laimburg c/o Plant Health Service 39040 Auer of Csongrad County in Hungary Italy Rarosi ut 110 PO 99 Tel: + 39 0471 969601 6800 Hodmezovasarhely Fax: + 39 0471 969599 Hungary E-mail: [email protected] Tel: + 36 62 535740 Fax: + 36 62 246036 E-mail: [email protected] ZIMMERMANN Gisbert Biologische Bundesanstalt für Land- und Forstwirtschaft TOTH Miklos Institute for Biological Control Plant protection Institute HAS Heinrichstrasse 243 Herman O. u. 15 64287 Darmstadt 1022 Budapest Germany Hungary Fax: + 49 6151 407 290 Tel: + 361 3918639 E-mail: [email protected] Fax: + 361 3918655 E-mail: [email protected]

TRAUGOTT Michael Centre for Mountain Agriculture University of Innsbruck Technikerstrasse 13 6020 Innsbruck Austria Tel: + 43 (0)512 507 5696 E-mail: [email protected]

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Contents

Preface...... iii

List of participants...... v

Invited Papers

Options for developing integrated control measures against the maize pest Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) in Europe Stefan Toepfer, Jozsef Kiss, Gyorgy Turoczi, Judit Komaromi & Ulrich Kuhlmann ...... 3

Scarabs and other soil pests in Europe: Situation, perspectives and control strategies Siegfried Keller & Gisbert Zimmermann ...... 9-12

Non-target effects of insect pathogenic fungi Nicolai V. Meyling, Jørgen Eilenberg & Charlotte Nielsen ...... 13

Melolontha

Field Experience in the Control of Common Cockchafer in the Bavarian Region Spessart Ullrich Benker & Bernhard Leuprecht ...... 21

Isolation of B. brongniartii from soil: Are the available isolation tools neutral? Jürg Enkerli, Priska Moosbauer, Franco Widmer, Silvia Dorn & Siegfried Keller ...... 25

Development of the Melolontha populations in the canton Thurgau, eastern Switzer- land, over the last 50 years Siegfried Keller & Hermann Brenner...... 31

Biocontrol of the forest cockchafer (Melolontha hippocastani): Experiments on the applicability of the “Catch and Infect”-Technique using a combination of attractant traps with the entomopathogenic fungus Beauveria brongniartii Robert Koller, Kerstin Jung, Stefan Scheu, Gisbert Zimmermann & Joachim Ruther...... 37-44

„New“ white grubs

Control of the garden chafer Phyllopertha horticola with GranMet-P, a new product made of Metarhizium anisopliae Barbara Pernfuss, Roland Zelger, Roberto Kron-Morelli & Hermann Strasser ...... 47

Timing of nematode application to control white grubs (Scarabaeidae) Arne Peters & Henk Vlug ...... 51 x

Metarhizium anisopliae for white grub control in Nepal Yubak Dhoj GC, Siegfried Keller ...... 57

Screening and selection of virulent isolates of the entomopathogenic fungus Beauveria brongniartii (Sacc.) Petch for the control of scarabs A. B. Hadapad, A. Reineke & C. P. W. Zebitz ...... 63

Wireworms

European wireworms (Agriotes spp.) in North America: Distribution, damage, monitoring, and alternative integrated pest management strategies Robert S. Vernon, Wim Van Herk & Jeff Tolman ...... 73

Monitoring and control of Agriotes lineatus and A. obscurus in arable crops in the Netherlands Albert Ester & Klaas van Rozen ...... 81

Practical implementation of a wireworm management strategy – lessons from the UK potato industry William E. Parker...... 87

An IPM approach targeted against wireworms: what has been done and what has to be done Lorenzo Furlan...... 91

Strategies to regulate the infestation of wireworms (Agriotes spp. L.) in organic potato farming: results Ute Schepl & Andreas Paffrath...... 101

Status-Quo-Analysis and development of strategies to regulate the infestation of wireworms (Agriotes spp. L.) in organic potato farming Ute Schepl & Andreas Paffrath...... 105

Metarhizium anisopliae as a biological control for wireworms and a report of some other naturally-occurring parasites Todd Kabaluk, Mark Goettel, Martin Erlandson, Jerry Ericsson, Grant Duke & Bob Vernon...... 109

Evaluation of different sampling techniques for wireworms (Coleoptera, Elateridae) in arable land Nina Brunner, Bernhard Kromp, Peter Meindl, Christian Pázmándi & Michael Traugott...... 117

Bait and pheromone trapping of Agriotes sp. in Lower Austria (first results) Marion Landl, Lorenzo Furlan & Johann Glauninger...... 123

A stable isotope analysis of wireworms puts new light on their dietary choices in arable land Christian Pázmándi & Michael Traugott ...... 127 xi

Pheromone composition of European click beetle pests (Coleoptera, Elateridae): common components – selective lures Miklós Tóth & Lorenzo Furlan ...... 133

Diabrotica

The Monitoring Program for the Western Corn Rootworm (Diabrotica virgifera virgifera Lec.) in Austria 2004 Peter C. Cate...... 145

Trap types for capturing Diabrotica virgifera virgifera (Coleoptera, Chrysomelidae) developed by the Plant Protection Institute, HAS, (Budapest, Hungary): performance characteristics Miklós Tóth...... 147

Miscellaneous

The impact of the fungal BCA Metarhizium anisopliae on soil fungi and animals Martin Kirchmair, Lars Huber, Elke Leither & Hermann Strasser ...... 157

Biocontrol potential of entomopathogenic nematodes against nut and orchard pests Stefan Kuske, Claudia Daniel, Eric Wyss, Jean-Paul Sarraquigne, Mauro Jermini, Marco Conedera & Jürg M. Grunder...... 163

Occurrence and harmfulness of Brachyderes incanus L. (Coleoptera: Curculionidae) to young Scots pine (Pinus sylvestris L.) trees planted on post-fire areas Henryk Malinowski & Alicja Sierpinska...... 169

Is differentiated host plant preference of Agriotes sp. and Melolontha sp. mediated by root volatiles? Sonja Weissteiner & Stefan Schütz ...... 175

Persistence of the insect pathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin on soil surface and on oilseed rape leaves Christina Pilz, Siegfried Keller & Rudolf Wegensteiner ...... 179

The natural distribution of the entomopathogenic soil fungus Metarhizium anisopliae in different regions and habitat types in Switzerland Sónia Rodrigues, Ralf Peveling, Peter Nagel & Siegfried Keller...... 185

What have BIPESCO and RAFBCA achieved that could help with risk assessment and registration? Hermann Strasser & Barbara Pernfuss...... 189 xii xiii

Invited Papers Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 1-7

Options for developing integrated control measures against the maize pest Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) in Europe

Stefan Toepfer1, Jozsef Kiss1, Gyorgy Turoczi1, Judit Komaromi1, Ulrich Kuhlmann2 1 St. Istvan University, Plant Protection Department, Pater K. Street 1, HU - 2100 Gödöllö, mailto: [email protected] 2 CABI Bioscience Switzerland Centre, Rue des Grillons 1, CH-2800 Delémont

Abstract: One of the most important North American maize pests, the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) was accidentally introduced into Serbia in the late 1980s, and is currently invading all European maize production areas. Diabrotica v. virgifera is a univoltine maize herbivore whose eggs overwinter in the soil, three larval instars feed on maize roots, and adults feed on maize leafs, silk and pollen. Yield losses are mainly caused by reduced water and nutrient uptake as well as lodging of plants due to extensive larval feeding. The presence of D. v. virgifera will significantly change European plant protection practises currently applied in maize production. This paper summarises the options that European farmers and researchers have when developing an IPM strategy against D. v. virgifera. First, cultural practises might be changed; here crop rotation techniques similar to those successful in the United States could be applied. Second biological control strategies and products could be developed such as classical biological control, or biological control with indigenous commercially available entomopathogenic nematodes or fungi. Third, the selection for host plant tolerance (such as compensation for larval damage by fast secondary root development), or host plant resistance (mainly the genetic modification of maize with the Bt-toxin gene) could be used. Fourth, selective chemical control measures could be considered such as 'attract and kill' and / or seed coating.

Keywords: Zea mays, Western Corn Rootworm, IPM, crop rotation, classical biological control, host plant tolerance and resistance, GM maize

Introduction

Maize (Zea mays L) is grown in almost all countries in Europe, but it is a particularly important crop in Serbia and Montenegro, Croatia, Romania, Hungary, Slovakia, Ukraine, France, Italy, Germany, and Austria (FAO 2004a). Recently, one of the most important North American maize pests, the Western Corn Rootworm, Diabrotica virgifera virgifera LECONTE (Col.: Chrysomelidae), was accidentally introduced into the Balkan region. Larvae-induced damage was first observed near Belgrade in the former Fed. Rep. of Yugoslavia in 1992 (Baca 1993). Within 10 years, this insect spread over Central Europe and its eradication became impossible. Recently, D. v. virgifera was even detected in Belgium, the Netherlands, England, Czech Republic and Slovenia bringing the total affected area in Europe to 310 000 km2 (Kiss et al. 2004a). This invasive chrysomelid is univoltine with eggs overwintering in the soil, larvae feeding on maize roots, and adults feeding on maize leaves, silk and pollen. The root-feeding larvae cause economic damage due to reduced water and nutrient uptake by the damaged root system and due to plant lodging. Occasionally, the adults cause damage in seed maize production by extensive silk feeding interfering with pollination (Tuska et al. 2001).

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Diabrotica beetles are among the most important insect pests of maize production in the United States, where 10 to 12 million hectares of maize are treated annually with soil insecticides to protect the crop from larval damage. Crop losses and control costs attributed to rootworms approach $1 billion annually (Krysan & Miller 1986). So far, serious yield losses in Europe have been reported from Serbia, Hungary, Croatia, and Romania (Kiss et al. 2004a). It is therefore evident that D. v. virgifera has the potential to significantly change European maize production practices. As many farmers, seed maize companies, customers and environmental bodies are concerned about the spread of D. v. virgifera, sustainable integrated control strategies are urgently needed and those options are summarised.

Material and methods

Four main options for the development of an integrated pest management of D. v. virgifera could be considered: (1) cultural practises, (2) biological control, (3) host plant tolerance and/or host plant resistance, and (4) selective chemical control measures. These options were reviewed in the context of their feasibility and in terms of minimizing hazards to environmental, crop and human health. This was based on a review of North American studies on IPM of Diabrotica spp. (Toepfer & Kuhlmann 2004a), and on information gained from the European research project DIABROTICA QLRT-1999-01110 (Vidal et al. 2004), the FAO activities (Edwards et al. 1999, Kiss 2004c), the Diabrotica subgroup of the IOBC/IWGO (Berger 2001) and the IPM guidelines of the IOBC (Boller et al. 1997).

Results and discussion

Option 1: Cultural control practises Cultural practises such as crop rotation, tillage systems, planting and harvesting date, modifying the soil environment or irrigation might be adopted to support the growing the maize or to prevent/decrease the outbreaks of pest insects (Toepfer & Kuhlmann 2004a). Crop rotation is a major non-chemical control option for preventing population increases of D. v. virgifera (Ostlie & Noetzel 1987, Levine & Oloumi 1991, Nelson et al. 1994), and is required by the IPM guidelines of the IOBC for maize (Boller et al. 1997). Diabrotica v. virgifera is univoltine and its larvae require maize for their development. From experiences in the United States (Levine & Oloumi 1991, Gray et al. 1998), it is expected that crop rotation will be the major method to prevent larval damage in Europe (Kiss et al. 2004b). In principle, all possible crops, fallows or vegetables can be rotated with maize for D. v. virgifera management. However, certain crops might be less promising in long term rotation with Diabrotica- infested maize fields, such as soybean (Glycine max) or monocotyledonous crops. About 21 Poaceae are known to serve to some degree as secondary food plants for D. v. virgifera larvae (Branson & Ortman 1967, Branson & Ortman 1970, Moeser 2003) and adults feed on nearly every pollen source (Levine et al. 2002, Hatvani & Horvath 2002, Moeser 2003). However, larval damage on cultivated Poacean plants other than maize has not yet been recorded. In the common United States rotation system maize-soybean-maize, D. v. virgifera started to develop behavioural resistance by laying eggs in soybean fields, and larval development occurred if maize was planted in the following year (Gray et al. 1998, Levine et al. 2002). In a four-year crop rotation trial in Hungary (Kiss et al. 2004b), the adult emergence in maize after soybean was found to be much lower than in maize-after-soybean fields in Indiana, USA (Barna et al. 1998). And finding few emerging adults in maize after soybean (or other non-maize crops) does not necessarily imply an adaptation of the population to the rotation. A part of the D. v. virgifera population naturally immigrates to non-maize crop 3

stands (Levine et al. 2002) to feed on flowers and pollen, e.g. on sunflower (Hatvani & Horvath 2002), or on volunteer winter wheat in harvested wheat fields. Moreover, a small proportion of 5 to 15% of the D. v. virgifera adult population was found active near the ground surface of non-maize crops probably laying eggs so that larvae develop in the subsequent maize (Komáromi et al. 2002, Kiss et al. 2004b). In conclusion, to reduce selection pressure for developing behavioural resistance, the above-mentioned critical crops should not be long-term rotated with maize over whole agricultural regions. The rotation should be kept as diverse as possible partly including fields with two successive years of maize and other fields with two successive years of non-maize.

Option 2: Biological control A three-year field survey conducted in Hungary, Serbia and Croatia, aimed to determine the occurrence of indigenous natural enemies of D. v. virgifera in Europe (Toepfer & Kuhlmann 2004b). Though Toth et al. (2002) reported on the presence of spiders preying on D. v. virgifera, and FAO (2004b) is currently screening generalist predators of D. v. virgifera, it was concluded from the survey results that host-specific and/or effective indigenous natural enemies are not currently attacking any of the life stages of the alien invasive pest in Europe (Toepfer & Kuhlmann 2004b).

Classical biological control Classical biological control provides an opportunity to reconstruct the natural enemy complex of an invading alien pest and its application to manage D. v. virgifera populations in Europe should be considered (Kuhlmann & Burgt 1998). Therefore, the natural enemy complex of Diabrotica species was surveyed in their area of origin in Central America (Kuhlmann et. al. 2004) and Celatoria compressa (Diptera: Tachinidae) was the only parasitoid found on the target species, D. v. virgifera. Its host range is considered to be restricted to Diabroticite beetles, and thus Celatoria compressa would be safe for introduction because direct and indirect impacts on other organisms would be extremely low (Kuhlmann et al. 2004). Classical biological control is considered as only one element within an IPM strategy that is compatible and applicable with other control measures.

Inundative biological control using entomopathogenic nematodes (EPN) EPNs are known to have great potential as biological control agents of insects (Poinar 1979, Gaugler 2002) and are successfully applied in horticultural and green house pest control. In contrast, the application of EPN at field scale against field pests is still an innovative field of research. EPN species commercially available in Europe have been screened for their efficacy against both the root-feeding larvae and silk-feeding adults of D. v. virgifera in laboratory. Findings suggest the development of a biocontrol product with EPNs against the pest larvae (Rasmann & Turlings 2004)

Inundative biological control using entomopathogenic fungi (EPF) More than 750 fungi species infect insects and mites. Many EPF are known as important natural regulators of pest populations, however, Diabrotica-populations in the United States are usually not regulated by fungi (Maddox & Kinney 1989). Also, in Hungary, the fungi Beauveria bassiana (Bals.) Vuill. (Mitosporic fungi; formerly Deuteromyces) and Metarhizium anisopliae (Metsch.) Sorok (Mitosporic fungi) naturally attack adults of D. virgifera only at a low level < 1% (Toepfer & Kuhlmann 2004b). However, in laboratory bioassays, M. anisopliae (isolated from Melolontha melolontha by E. Dormannsné, Plant Health Service, Hodmezovasarhely, Hungary) appeared to be highly effective in killing 4

second and third instar larvae of D. v. virgifera. Within 14 days, 96% ±5.1 SD of the D. virgifera larvae died after indirect infestation with spores (Petri-dishes with one larva, a maize seedling, and with M. anisopliae spores sprayed on filter paper). This was significantly more than the 44% ± 28 SD natural mortality in larvae in the control dishes (P< 0.005, Nonparametric Chi Square Test, n = 26). Consequently the fungi increased mortality in larvae by about 52%. About 18% ± 2.3 SD) adult D. virgifera emerged from the third instar larvae when the soil with maize plants had been infested with M. anisopliae spores in laboratory (34 soil trays of 190 x 120 x 45 cm with maize roots, sprayed with 3 x 106 spores per cm2; 4 to 5 days after maize germination each tray infested with 6 second or third instar larvae; 38 days experiment in green house, natural light, 23 to 27 °C). In the control, about 38% ± 38 SD adults emerged from larvae when the soil had not been infested with fungi (difference not significant due to high variation in emergence data, P = 0.068, Nonparametric Mann Whitney Test, Z = - 1.8). These promising results, based on the fact that M. anisopliae is widely distributed in arable land (Keller et al. 2003), and that products based on this fungus are registered in several countries (Inglis et al. 2001), suggest further research towards the development of a fungal biocontrol product.

Option 3: Host plant tolerance and host plant resistance A host plant tolerance strategy against D. v. virgifera is an option for IPM in maize. Although no current commercial and non-GM maize hybrids suppress larval populations, hybrids with an extensive root system and an abundant development of secondary roots, as well as a tolerance to drought stress, can compensate for the effect of larval feeding (list of Genotypes in Baca et al. 1995; Croatian hybrids in Ivezic et al. 2001). Genetically modified maize varieties with Bt toxin expressed in its roots, (Cry3Bb1, Cry34Ab1, Cry135Ab1; Pershing 2001, Monsanto 2003) can prevent larval damage. The transgenic maize MON 863 (Cry3Bb1) is currently under review for import in the EU (J. Romeis, pers. comm. 2004). The European Food Safety Agency has recently recommended allowing the Bt-maize to enter the EC market (EFSA 2004). These developments make it likely that MON 863 maize will become available as a tool for D. v. virgifera control in Europe. However, prior to considering the use of GM maize as an IPM strategy, precautionary risk assessments are necessary (Boller et al. 1997). Bt-transgenic maize could affect biological control agents or other non-targets either directly through the Bt-toxin or indirectly due to an altered nutritional quality of the prey or host species (Dutton et al. 2003, Al-Deeb & Wilde 2003).

Option 4: Selective chemical control The last step in an IPM strategy is the possibility of using direct control measures during pest outbreaks. One may apply foliar 'contact kill' insecticides against adults to reduce silk feeding damage and/or reduce egg laying. For this purpose, broad-spectrum pyrethroids and organophosphates are generally used. However, several cases of resistance to insecticides are already known for D. v. virgifera, such as regionally developed resistances to organophosphates, e.g. methyl parathion, or to carbamate insecticides, e.g. carbaryl. Moreover their honeybee toxicity, their re-entry interval, and application difficulties due to maize height are limiting factors for application. These insecticides also endanger the biological control of the European corn borer, Ostrinia nubilalis, with Trichogramma parasitoids (R. Burger, pers. comm. 2004). A recently (2003) registered management option in Hungary is the ‘attract and kill’ method, where a small amount of insecticide (usually 10% of the registered amount) is combined with natural feeding stimulants/arrestants or attractants, e.g. cucurbitacins (INVITE EC). A seed treatment with the systemic clothianidin (neonicotinoid) reduces D. v. virgifera larval density as well as other root feeding pests (Altmann & Springer 2003). 5

In conclusion, the chemical control of D. v. virgifera in an IPM strategy which is knowledge intensive and should be based on proper pest monitoring and economic thresholds.

Conclusion

The invasive insect pest Diabrotica v. virgifera, is rapidly spreading over Europe, and thus ecologically sound and economically feasible control strategies are urgently needed. This paper summarizes the options for developing an integrated management approach against D. v. virgifera in European maize production. The application of one control option alone will probably not control this alien invasive pest in the long term, therefore farmers and researchers face challenges to apply crop rotation, develop a classical biological control strategy for Europe, develop entomopathogenic nematodes and/or fungi as biocontrol products, and to improve compatibility and/or competitiveness of these control options with transgenic Bt maize and chemical insecticides.

Acknowledgements

We would like to thank for useful communications with Dr. J. Romeis (Agroscope, FAL, Reckenholz, Switzerland) and R. Burger (Landi REBA, Switzerland). We like to thank E. Dormannsne (Plant Health Service, Hodmezovasarhely, Hungary) for providing the fungal strain. We are grateful to B. Kiefer (CABI Bioscience Centre, Switzerland) and K. Imre (University of Gödöllö, Hungary) for technical assistance, and Lars Andreassen (University of Manitoba, Winnipeg, Canada) for reviewing the English text.

References

Altmann, R. & Springer, B. 2003: Control of rootworms (Diabrotica spp.) in corn (Zea mays) using seed treatment of Clothianidin. – International Symposium on the ecology and management of Western Corn Rootworm, 19-23 January 2003, University Goettingen, Germany. Al-Deeb, M.A. & Wilde, G.E. 2003: Effect of Bt corn expressing the Cry3Bb1 toxin for corn rootworm control on aboveground non-target arthropods. – Environmental Entomology 32: 1164-1170. Baca, F. 1993: New member of the harmful entomofauna of Yugoslavia Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae). – IWGO Newsletter 13 (1-2): 21-22. Baca, F. & Camprag, D. 1995: Western corn rootworm Diabrotica virgifera virgifera LeConte. – Kukuruzna Zlatica, Drustvo za zasitu bilja Serbije. Beograd, Studio Stanisic: 112 pp. Barna, Gy, Edwards, C.R., Gerber, C., Bledsoe, L.W. & Kiss, J. 1998: Management of Western Corn Rootworm (D. v. virgifera) in corn based on survey information from previous soybean crop. – Acta Phytopathologica et Entomologica Hungarica 33: 173-182. Berger, H.K. 2001: The XXIth IWGO meeting in Venice, Italy. – 8th IWGO Diabrotica Subgroup meeting, 1-3 November 2001, Padova, Italy, Veneto Agricoltura. Boller, E.F., Malavolta. C. & Jörg, E. 1997: Guidelines for integrated production of arable crops in Europe. Technical guideline III. – IOBC/WPRS Bulletin 20(5): 2-10. Branson, T.F. & Ortman, E.E. 1967: Host range of larvae of the western corn rootworm. – Journal of Economical Entomology. 60: 201-203. Branson, T.F. & Ortman, E.E. 1970: The host range of larvae of the western corn rootworm: further studies. – Journal of Economic Entomology. 63: 800-803. 6

Dutton, A., Romeis, J. & Bigler, F. 2003: Assessing the risks of insect resistant transgenic plants for beneficial arthropods: Bt maize expressing Cry1Ab as a case study. – BioControl 48: 611-636. Edwards, C.R., Kiss, J. & Barcic, J.I. 1999: Results of the 1997-1998 multi-country FAO activity on containment and control of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in Central Europe. – Acta Phytopathologica et Entomologica Hungarica. 34(4): 373-386. EFSA 2004: Opinion of the Scientific Panel on genetically Modified Organisms on a request from the Commission related to the safety of foods and food ingredients derived from insect-protected genetically modified maize MON 863 and MON 863 x MON 810, for which a request for placing on the market was submitted under Article 4 of the Novel Food Regulation (EC) No 258/97 by Monsanto. – The EFSA Journal 50: 1-25. FAO 2004a: FAOSTAT. FAO statistical database. – http://apps.fao.org/default.jsp. FAO 2004b: Integrated pest management of western corn rootworm in Central and Eastern Europe. FAO GTFS/RER 017/ITA project. – http://www.mkk.szie.hu/dep/nvtt/. Gaugler, R. 2002: Entomopathogenic Nematology. – CABI Publishing, Wallingford, UK. Gray, M.E. & Levine, E. 1998: Adaptation to crop rotation: Western and northern corn rootworms respond uniquely to a cultural practice. – Recent Research Development in Entomology. 2: 19-31. Hatvani A. & Horvath Z. 2002: Damage of Western Corn Rootworm after sunflower in North Baczka. – Novenyvedelem 38: 513-517. (In Hungarian). Inglis, G.D., Goettel, M.S., Butt, T.M. & Strasser, H. 2001: Use of hyphomyctous fungi for managing insect pests. – In: T.M. Butt & Magan, N. (ed.): Fungi as Biocontrol Agents. CABI Publishing, Wallingford, UK: 23-69. Ivezic, M. & Raspudic, E. 2001: Evaluation of Croatian corn hybrids for tolerance to corn rootworm (Diabrotica v. virgifera) larval feeding. – 8th IWGO Diabrotica Subgroup meeting, 1-3 November 2001, Padova, Italy, Veneto Agricoltura. Keller, S., Kessler, P. & Schweizer, C. 2003: Distribution of insect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metarhizium ansiopliae. – BioControl 48: 307-319. Kiss, J., Edwards, C.R., Berger, H.K. Cate, P., Cheek, S., Derron, J, Festic, K., Furlan, L, Irgc Barcic, J., Ivanova, I. Lammers, W., Princzinger, G., Sivcev, I., Sivicek, P, Rosca, I., Urek, G., & Vahala, O. 2004a: Monitoirng of western corn rootworm D. v. virgifera, in Europe. – In: Vidal, S., Kuhlmann, U. & Edwards, C.R. (ed.): Western Corn Rootworm: Ecology and Management. CABI Publishing, Wallingford, UK. (in press). Kiss, J., Komaromi, J., Bayar, J.K., Edwards, C.R. & Hataláné Zseller, I. 2004b: Western corn rootworm (D. v. virgifera) and the crop rotation system in Europe. – In: Vidal, S., Kuhlmann, U., Edwards, (ed.): Western Corn Rootworm: Ecology and Management. CABI Publishing, Wallingford, UK. (in press.). Kiss, J. 2004c: IPM for Western Corn Rootworm in Central and Eastern Europe: FAO GTFS/RER/017/ITA project. – 10th IWGO Diabrotica Subgroup meeting, 14-16 January 2004, Engelberg, Switzerland: 65. Komaromi, J, Bayar K., Kiss, J. Edwards, C.R. & Szell, E. 2002: Is the development of western corn rootworm possible in corn-soybean and corn-alfalfa rotation systems. – 9th IWGO Diabrotica Subgroup Meeting, 3-5 November 2002, Belgrade, Serbia: 40-41. Krysan, J.L. & Miller, T.A. 1986: Methods for study of pest Diabrotica. – New York, Springer. 7

Kuhlmann, U. & W.A.C.M. Burgt 1998: Possibilities for biological control of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in Central Europe. – Biocontrol News and Information 19(2): 59-68. Kuhlmann, U., Toepfer, S. & Zhang, F. 2004: Is classical biological control against Western Corn Rootworm in Europe a potential sustainable management strategy ? – In: Vidal, S., Kuhlmann, U. & Edwards, C.R. (ed.): Western Corn Rootworm: Ecology and Management. CABI Publishing, Wallingford, UK. (in press.). Levine, E. & Oloumi, S.H. 1991: Management of Diabroticite Rootworms in corn. – Annual Review of Entomology 36: 229-255. Levine, E., Spencer, J.L., Isard, S.A., Onstad, D.W. & Gray, M.E. 2002: Adaptation of western corn rootworm to crop rotation: Evolution of a new strain in response to a management practise. – American Entomologist 48: 94-107. Maddox, J. & Kinney, K. 1989: Biological control agent of the corn rootworm. – Natural History Survey Report 3 (287): 3-4. Moeser, J. 2003: Nutritional ecology of the invasive maize pest Diabrotica v. virgifera LeConte in Europe. PhD thesis, Faculty of Agricultural Sciences, University Goettingen, Germany: 89 pp. Monsanto 2003: Safety Assessment of YieldGard RootwormTM Corn. – Monsanto Company, St. Louis, USA: 44 pp. Nelson, S.D. & Dyryon, L.M. 1994: Effects of a new rootworm infestation on continuous and rotated corn under four tillage systems. – Journal of Sustainable Agriculture 4: 31-37. Ostlie, K. & Noetzel, D. 1987: Managing corn rootworms. – University of Minnesota. Pershing, J.C. 2001: Biotech approach to corn rootworm control: Development status of Monsanto's corn rootworm resistant maize. – IWGO Newsletter 22(1-2): 41-42. Poinar, G.O. Jr. 1979: Nematodes for biological control of insects. – Boca Raton, CRC Press. Rasmann, S. & Turlings, T. 2004: New tools to study below ground tritrophic interactions- The example of Diabrotica virgifera virgifera LeConte. – IWGO Newsletter. 25(1): 35. Toepfer, S. & Kuhlmann, U. 2004a: IPM strategies for the Western Corn Rootworm (Coleoptera: Chrysomelidae): Current status and potential adoptations to the European maize production systems. – Mitteilungen der Deutschen Gesellschaft für allgemeine und angewandte Entomologie. 14 (1-6): 391-398. Toepfer, S. & Kuhlmann, U. 2004b: Survey for natural enemies of the invasive alien chrysomelid, Diabrotica virgifera virgifera, in Central Europe. – BioControl 49(2): 385- 395. Toth, F., Horvath, J., Komaromi, J, Kiss, J. & Szell, E. 2002: Field data on the presence of spiders preying on western corn rootworm (D. v. virgifera) in Szeged region, Hungary. – Acta Phytopathologica et Entomologica Hungarica. 37 (1-3): 163-168. Tuska, T., Kiss, J. Edwards, C.R., Szabo, Z., Ondrusz, I., Miskucza, P. & Garai, A. 2001: Effect of silk feeding by Western Corn Rootworm adults on yield and quality of seed and commercial corn. – 8th IWGO Diabrotica Subgroup meeting, 1-3 November 2001, Padova, Italy, Veneto Agricoltura: 107-113. Vidal, S., Kuhlmann, U. & Edwards, C.R. 2004: Western Corn Rootworm: Ecology and Management. – Wallingford, UK, CABI. (in press.). 8 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 9-12

Scarabs and other soil pests in Europe: Situation, perspectives and control strategies

Siegfried Keller1, Gisbert Zimmermann2 1 Agroscope FAL Reckenholz, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland 2 Federal Biological Research Centre for Agriculture and Forestry, Institute for Biological Control, Heinrichstrasse 243, D-64287 Darmstadt, Germany

Abstract: Scarabs and other soil dwelling pest insects are of increasing importance in Central Europe. In order to get an overview on the present situation, the damages caused, the population development and on the control strategies used, a questionnaire was sent to representatives of several European countries. Answers were obtained from Austria, Belgium, Czech Republic, part of France (Lorraine), Germany, Italy (South Tyrolia, Aosta), Poland and Switzerland. The results show, that both Melolontha species occur on about 200 000 ha (M. melolontha on 155 000 ha, M. hippocastani on 43 000 ha) in Europe, while economic damage is caused on about 80 000 ha. The garden chafer, Phyllopertha horticola, is present on 31 000 ha, mainly in Austria, causing economic damage on nearly the same area. Curculionids are important pests in Polish forests on 27 000 ha, while wireworms are of increasing importance in several other European countries occurring on about 9.600 ha. Generally, chemical insecticides, biological (e.g. Beauveria brongniartii) and mechanical means (e.g. rotary hoes, nets) are used for control. – The information received must be considered as incomplete. The necessity for optimisation and installation of monitoring systems is discussed.

Keywords: Melolontha, white grubs, soil dwelling pest insects, Europe, damage, control strategies

Introduction

The larvae of the two cockchafer species Melolontha melolontha and M. hippocastani, the June beetles (Amphimallon solstitiale und A. majale) and the garden chafer (Phylloperta horticola) are actually considered to be the most damaging white grubs in Central Europe. There is no overview on the present situation, on the damages caused, on the trends of the population development and on the strategies used for their control. Apart from these scarab species there are other soil dwelling pest insects in Europe of regional or general distribution like Curculionidae, Elateridae (wireworms), Noctuidae and Tipulidae whose importance is also not recorded and summarized. This study aims to present data on the occurrence and importance of these pest insects in different European countries. Such informations are needed to set priorities for control strategies, unite research activities, and to justify research projects.

Material and methods

A questionnaire was sent to representatives of the following countries: Austria, Belgium, Czech Republic, Denmark, France, Germany, Italy, The Netherlands, Poland, Slovakia and Switzerland. The questions concerned the area colonised, the area with economic damage worth to be controlled, the financial yield losses, the tendency of population development and methods used to control these pest species. Additional informations were requested on problems with other soil dwelling pests. Answers were obtained from eight countries, i.e.

9 10

Austria, Belgium, Czech Republic, part of France (Lorraine), Germany, Italy (South Tyrolia, Aosta), Poland and Switzerland.

Results

Both Melolontha species colonise about 200 000 ha (M. melolontha 155 000 ha) and cause economic damage on about 80 000 ha (M. melolontha 70 000 ha) (Tab. 1). M. melolontha occurs in all eight European countries. In the alpine region (Austria, northern Italy, Switzerland) the population densities of M. melolontha are considered as stable although in some areas they are decreasing and in others they are increasing. The largest populations exist in France although only the department Lorraine is considered, followed by Austria (Tab. 2). From Bulgaria we have no actual data, however, a paper from 1960 (Popov, 1960) reveals a nearly landwide medium to heavy outbreak of M. melolontha.

Table 1: Summary of the estimated importance of scarabs and other soil dwelling pest insects in 8 European countries (Austria, Belgium, Czech Republic, France, Germany, Italy, Poland and Switzerland).

Pest insect Size of area Size of area Control strategies used colonised (ha) with economic damage (ha)

Melolontha 155 000 70 000 insecticides, Beauveria melolontha brongniartii, mechanical means (nets) M. 42 700 7 950 insecticides, B. brongniartii hippocastani Amphimallon 1 100 45 insecticides solstitiale A. majale 100 60 Metarhizium anisopliae (trials) Phylloperta 31 000 30 000 insecticides, nematodes, horticola mechanical Hoplia spp. 3 3 nematodes wireworms 9 600 2 000 insecticides (Elateridae) Noctuidae 35 25 insecticides Tipulidae 50 50 insecticides Curculionidae 27 125 22 670 insecticides, nematodes, mechanical Others: Anomala dubia 28 28 Polyphylla fullo 2 2

M. hippocastani has pest status in Germany where over 30 000 ha of forests are colonised by this species, mainly in the southern parts, while nearly 5 000 ha are damaged. In 11

the Czech Republic the forest cockchafer occurs on 12 000 ha, causing an estimated amount of damage of 800 000 €. In both countries, an increasing occurrence is noticed (Tab. 2). The second most important soil dwelling pest after the two Melolontha spp. is Phylloperta horticola which is present on 31 000 ha and damaging nearly the same area. Most of the area is in Austria where the populations are strongly increasing. The species is widely distributed in Switzerland but has no pest status, however, the populations are increasing. Other scarab species are of minor importance. Noteworthy are the damages caused by Amphimallon solstitiale and A. majale. The latter species was cryptic until 2003 when heavy damages occurred on golf courses and other sport grounds in alpine regions of Switzerland. Since some years, Hoplia spp., mainly H. philanthus, are new pests in Germany again.

Table 2: Estimated importance of Melolontha melolontha and M. hippocastani in various European countries. (B.br. = Beauveria brongniartii).

Pest insect Country Size of Size of area Estimated Tendency Control area with amount of of popula- strategies colonised economic damage tion deve- (ha) damage (ha) (EURO) lopment

Melolontha Austria >30 000 30 000 20 000 slightly B.br. melolontha increasing Belgium present no data increasing Czech Rep. 500 5 16 000 increasing insecticides Denmark Present no data France 100 000 35 000 no data increasing mechanical Germany ca. 3 000 several increasing insecticides, 100 ha B.br., mechanical Italy 13 000 1 500 no data stable insecticides, B.br., mechanical. Poland 1 012 1 012 – – insecticides, mechanical Switzerland 9 500 1 300 1 000 B.br. Melolontha Czech 12 000 250 800 000 strongly insecticides, hippo- Republic increasing B. bassiana castani Germany 31 000 ca. 5 000 increasing insecticides, B.br.

Curculionidae and wireworms also turned out to be important soil pests. In Poland, several Curculionid species are occurring in forests on about 27 000 ha (Tab. 1), and the species Hylobius abietis, Pissodes notatus and Brachyderes incanus are causing economic damage on about 22 500 ha. In several European countries, wireworms are of increasing importance. They occur on 9 600 ha and cause damages on about 2 000 ha of arable land. 12

The control strategies used against scarabs and other soil dwelling pests are mainly chemical insecticides, biological control (Beauveria brongniartii, B. bassiana, Metarhizium anisopliae and entomoparasitic nematodes), and mechanical means.

Discussion

This presentation on the occurrence and importance of Scarabs and other soil dwelling pest insects in Europe documents, that several species are important pests, most of them have a wide distribution, and damage is caused on thousands of hectares. Although the questionnaire was sent to representatives of all European countries known to have Melolontha populations, the information received and presented here must be considered as incomplete and as a rough estimation of the real situation. This does not refer only to Melolontha spp. and other white grubs, but in particular also for the wireworm situation. For example, we did not have exact data from the UK, although wireworms, especially Agriotes obscurus, A. sputator and A. lineatus, have become increasingly important pests of potato in recent years (Parker, 2005) In farmer’s journals also the wireworm situation is presented as a widely distributed problem. According to these papers the area of damages done by these pests must be much bigger and the financial consequences for the farmers, mainly for potato growers, are enormous and justify the initiation of specific research and control projects. The lack of information on the occurrence and damage of pest insects is considered to be the result of lacking staff charged with monitoring of the pest situation. This is of disadvantage for the farmers who are uninformed and can not present proper data and adequately articulate their problem on the political level. It is also disadvantageous to scientists who are interested to develop control solutions. It is well known, that the development of effective and environmentally safe control methods needs many years of intensive research. For example, in the UK, the insecticide aldrin was the standard product for wireworm control until 1989, when it was withdrawn. That means, no significant research on wireworms was done in the UK between 1960-1988 (Parker, 2005). Exact data on the pest status are needed to plan and develop strategies for environmental friendly pest control methods. This can be best achieved with the installation of an official monitoring system.

Acknowledgements

We thank all colleagues from the plant protection and forest services for sending back the filled in questionnaire and for supporting this presentation.

References

Popov, P.A. 1960: Untersuchungen über die Gattung Melolontha in Bulgarien. – Z. Pflanzenkrankh. Pflanzenschutz 67: 399-407. Parker, W.E. 2005: Practical implementation of a wireworm management strategy – lessons from the UK potato industry. – IOBC/wprs Bull. 28(2): 87-90. Integrated Protection in Oilseed Crops IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 13-18

Non-target effects of insect pathogenic fungi

Nicolai V. Meyling1, Jørgen Eilenberg1, Charlotte Nielsen1, 2 1 Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; 2 Department of Entomology, Cornell University, Ithaca NY14853, USA

Abstract: Evaluations of non-target effects of insect pathogenic fungi for biocontrol are often carried out under laboratory conditions where only the physiological host ranges of the pathogens are investigated. We argue that the ecological host range should be given much more attention and be examined in field trials over medium to relatively long time intervals. The arguments are discussed based on recent Danish studies on 1) soil application of Metarhizium anisopliae in greenery plantations and 2) the genetic diversity of a local indigenous population of Beauveria bassiana.

Keywords: Non-target effects, ecological and physiological host range, habitat and host selection

Introduction

When using insect pathogenic fungi as biological control agents the non-target effects are generally considered to be lethal infections of non-target insects present in the habitat of the target insect. These non-target hosts can be predators, parasitoids or herbivores. Here, we discuss definitions of ecological and physiological host range relevant for the evaluation of non-target effects. We also present some main results of two studies to elucidate potential non-target effects: 1) a field study of effects on non-target arthropods after application of Metarhizium anisopliae; and 2) a study of naturally occurring Beauveria bassiana genotypes in soil and plant habitat as well as different host species on one locality.

Physiological vs. ecological host range

One important aspect of evaluating non-target effects of entomopathogens is to consider whether it is the physiological or the ecological host range that is investigated. Hajek & Butler (2000) suggested that the range of species that a fungus can infect often differs between that found in the laboratory (physiological host range) and that found in nature (ecological host range). Onstad & McManus (1996) did, however, include evolutionary aspects in the definitions: ‘The ecological host range is the current, and evolving, set of species with which a parasite naturally forms symbiosis, resulting in viable parasite offspring. Physiological host range is based solely on laboratory observations of infection and propagule production’. The physiological host range thus gives information about the potential of a given fungus to infect different species under optimal conditions. The ecological host range (with or without the evolutionary aspect) gives information about the expectations of infections in different species in the eco-system under study. We consider the ecological host range as the most appropriate to evaluate non-target effects of insect pathogenic fungi used for biological control. Also, we suggest that the definition by Onstad & McManus (1996) is generally best suited for addressing ecological host range since it includes an evolutionary and/or host adaptation aspect.

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1) Case study: Soil application of Metarhizium anisopliae in a Danish greenery plantation In a field experiment effects on non-target arthropods were surveyed after soil application of a conidial suspension of M. anisopliae for biocontrol of the pest weevil Strophosoma melano- grammum in a Danish greenery plantation. The effects were studied as prevalences in non- target arthropod populations. Relatively abundant arthropods were collected by sweep-netting before application of M. anisopliae as well as 7, 14, 75 and 277 days after application (Table 1) from both untreated and treated plots and subsequently reared in the laboratory in order to identify any infection of the applied fungus. As seen from Table 1, we found infections among non-target species from distantly related taxa. The highest prevalences were found 7- 14 days after application, but infections were still observed in Coccinellidae at a low level after almost a year. In addition, the applied fungus could, by soil plating onto selective media, be documented in the soil after more than a year The question is whether the sampled non-target insects represent the ecological host range of the fungal isolate. The conclusion depends on the aspect of time as well as the origin of the infection. If the prevalence in the non-target hosts relied solely on the initial spraying of the fungal pathogen, the host range investigated can be interpreted as representing the physiological rather than the ecological host range, as the whole study can be viewed as a ‘large laboratory experiment’. However, if the long-term prevalence depended on repeated infections and establishment of the biocontrol agent in the habitat then the ecological host range (as defined by Onstad & McManus, 1996) of the fungal isolate was evaluated. As seen in Table 1, one major constraint in sampling of non-targets over time in an eco-system is that it is not possible to sample sufficient amounts of all non-target species at all times, and each study needs to compromise and obtain data from non-targets which are present at each time of sampling.

Table 1. Prevalence of Metarhizium anisopliae in selected non-target populations after application in a greenery plantation in Denmark. The table shows the number of infected/the number of sampled in the treated plots. ‘–‘ indicates that no specimens were sampled.

Days after treatment Non-target species 7 14 75 277 Psocoptera 0/40 – – – Hemiptera, Cicadellidae 3/4 5/44 0/60 – Hemiptera, Miridae 33/64 9/47 2/82 – Coleoptera, Coccinellidae – – – 3/30 Arachnida, Ixiodae – 38/67 – –

Studies of field trials thus need to take the time scale into account to evaluate ecological host range, and we suggest three time scales: 1) immediate effects (which show, in fact, rather the physiological host range than the ecological), 2) medium time effects (which includes survival and maybe even a limited cycling of the fungus agent in the environment), and 3) long-term establishment in non-target populations (which includes the adaptation to another host species). These time scales should be considered for the evaluation of non-target effects, and we expect only the third scale to evaluate the true ecological host range of the biological control agent. In addition, experiments need to be carried out at several locations and over several seasons since not all arthropods are equally abundant between localities and years.

Figure 1. Cladogram based on Universally Primed PCR profiles of B. bassiana isolates from a single agroecosystem in Denmark. Grey bars to the right represent the isolates in the study. The isolates are divided into six origins of isolation. A: Agricultural soil. B: Hedgerow soil. C: Leaf surfaces in hedgerow. D: Heteropterans. E: Other insects. F: B. brongniartii.

Habitat vs. host selection

The ecological host range includes permanent establishment within the habitat and will thus reflect evolutionary adaptations of the fungal pathogen. The traditional approach to the evolution of host range in Hyphomycete/Deutoromycete entomopathogenic fungi has been to assume the insect host as the prime selective parameter, and this has been explored in numerous studies of associations between insect host taxa and genetic groups of the pathogen. However, limited evidence has been produced for this to be the case. Recently Bidochka et al. (2001) proposed a hypothesis that habitat selection, not host selection, is driving the evolution of these insect pathogenic fungi. This was demonstrated for Canadian isolates of M. anisopliae (Bidochka et al., 2001) and B. bassiana (Bidochka et al., 2002). In both studies, however, mostly isolates obtained from the soil were investigated, so the data cannot be directly related to the insect hosts.

2) Case study: Genetic diversity of B. bassiana in a single agroecosystem To explore the population structure of a local population of B. bassiana within a single agroecosystem we isolated the fungus from soil, different insect groups and leaf surfaces in an organic field and an associated hedgerow in Taastrup, Denmark. Sampling from different spatial compartments as well as host groups in a presumably stable system gives the possibility of examine both the habitat and the host selection hypotheses. The 84 isolates were characterised by Universally Primed PCR and similarities were compared based on the produced banding patterns. Resulting groupings are presented in Figure 1. Overall, a very large diversity was found on the locality and isolates originating from insects were scattered among all derived genetic groups. The only obvious pattern was that soil isolates from the field, but not from the hedgerow, clustered closely together. The clustering of soil isolates from the field is in accordance with the findings of Bidochka et al. (2001; 2002) who suggested that the abiotic factors in the agricultural soil (relatively high UV radiation, temporarily high temperatures, etc.) select for specific genotypes in the specific habitat. However, the large diversity and scattered pattern of hedgerow soil isolates as well as isolates from various insect orders found in the present study suggest that in the more diversified habitat of the hedgerow many niches are available and thus sustain a diverse population of B. bassiana.

Figure 2. Hypothetical dispersal pathways of B. bassiana in the investigated Danish agroecosystem. Insects can be infected by contact with other insects or by inoculum in the soil or on plants. The specific abiotic conditions in the soil and the low diversity of insect hosts in the agricultural field causes the low diversity of B. bassiana in this habitat. Contrarily, high insect host diversity mediated through a diversified plant community in the hedgerow habitat create the foundation for a large diversity of genetic groups of B. bassiana. The soil environment, in which the fungi can survive or probably grow saprophytically, is repeatedly inoculated from above and within by dead infected invertebrates.

Significant habitat selection shapes the B. bassiana community in the agricultural field soil and we suggest that there is no apparent association between insect host taxa and fungus genetic groups, thus host selection in the traditional sense is not significantly shaping the B. bassiana community of the hedgerow. The fungi presumably infect with limited specificity, but the abundance of available hosts in the semi-natural habitat of the hedgerow create basis for spending less time outside hosts and thus less impact of abiotic factors as a selective force as in the agricultural field. Habitat selection can be separated into abiotic and biotic habitat factors indicating which type is most dominating in the specific system. We propose that abiotic factors are most dominant in the agricultural field thus favouring specific trades in the B. bassiana strains that can cope with these. By contrast, the varied composition of the hedgerow habitat and the abundance of potential hosts select less for abilities to withstand specific abiotic conditions and thus allow various genetic groups to coexist. Both scenarios are defined by the habitat, thus we suggest the broader definition of abiotic and biotic habitat selection. Sampling of B. bassiana in the hedgerow habitat will thus reveal a sample of the entire population that is present at the time. Based on this study evaluation of non-target effects depends on the specific habitat under investigation. In an extreme habitat like the agricultural field few genotypes would probably survive over time, but if they entered the diversified habitat of the hedgerow the possibility of survival and long-term establishment would increase. Recovery of an augmented genotype in this habitat would, however, become difficult if it establishes at low frequencies among the very diverse indigenous population. Figure 2 summarises the hypothesis of diversity and dispersal pathways of B. bassiana in the investigated agroecosystem.

Conclusions

The natural populations of hyphomycete insect pathogenic fungi are very complex and structured by the specific habitat (defined broadly as both abiotic and biotic conditions) they encounter, and studies of the effects of fungal biocontrol agents on indigenous populations are thus challenging. To investigate non-target effects in the field it is for each study necessary to define ecological host range with or without the evolutionary aspect and to sample several taxa and different spatial habitats of the field site. Also, considerations have to be made of the time scale of the effects, either as immediate effects, medium time effects or long-term establishment. Only in the latter case we believe that the issue of ecological host range is addressed.

Acknowledgements

The Royal Veterinary and Agricultural University, The National Environmental Protection Agency, Denmark (Grant no. 7041-0317 and 7041-0081) and EU (BIPESCO, EU FAIR6 CT- 98-4105) supported the studies financially. Christina Wolsted, Rasmus Eliasen, Charlotte Fisher and Karen Marie Kjeldsen performed skilled technical assistance.

References

Bidochka, M.J., Kamp, A.M., Lavenden, T.M., Dekoning, J. & de Croos, J.N.A. 2001: Habitat association in two genetic groups of the insect-pathogenic fungus Metarhizium anisopliae: uncovering cryptic species? – Applied and Environmental Microbiology 67: 1335-1342.

Bidochka, M.J., Menzies, F.V. & Kamp, A.M. 2002: Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences. – Archives of Microbiology 178: 531-537. Hajek, A. & Butler, L. 2000: Predicting the host range of entomopathogenic fungi. – In Follett, P.A. & Duan, J.J. (eds). Nontarget effects of biological control. Kluwer Academic Publishers, Dordrecht: 263-276. Onstad, D.W. & McManus, M.L. 1996: Risks of host range expansion by parasites of insects. – BioScience 46(6): 430-435.

Melolontha

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 21-24

Field experience in the control of Common cockchafer in the Bavarian region Spessart

Ullrich Benker, Bernhard Leuprecht LfL, Institute for Plant Protection, Lange Point 10, D-85354 Freising, Germany

Abstract: The grubs of the Common cockchafer Melolontha melolontha L., 1758, are well known for feeding on the roots of grass and causing seriously damage in grassland. The secondary damage of wild pigs rooting for the grubs compounds the destruction of the turf. Since the summer of 2001, in a sloping grassland site in the region Spessart a greater occurence of chafer grubs was observed for the last decades in Bavaria. First trials with the entomopathogenic fungus Beauveria brongniartii (Sacc.) Petch, 1924, were carried out for the biological control of the grubs (Benker & Leuprecht 2004). Since 2004 the trials were extended to mechanical control using a rotary hoe and chemical control with insecticides like Imidacloprid and Carbofuran. Checks for the success of the treatments were carried out by digging for the grubs and counting the healthy ones and the fungal infected specimens. Up to now the results are: The degree of fungal infestation of the cockchafers and therefore the level of control achieved were quite satisfactory. The level of fungal control was in the end about 80 %. But the best method of controlling common cockchafer grubs showed the mechanical treatment with a decrease of 98 % of the grubs. The chemical treatments seemed not to improve the effect of the rotary hoe. In Bavaria there are still some questions remaining, i.e. how to deal with the Melolontha problem in public and how to realise the long-term strategy of controlling grubs using the Beauveria fungus.

Keywords: Common cockchafer, Melolontha melolontha, grassland, Spessart, biological control, slit seedling machine, Beauveria brongniartii, chemical control, Imidacloprid, Carbofuran, mechanical control, rotary hoe

Introduction

The Bavarian region Spessart is the most north-western part of Bavaria. It is characterised by a sloping landscape including large forests, mainly beech trees, villages at the bottom of the valleys and grassland, meadows, small fields and orchards lying between the villages and the forests. The grassland is used for sheep and horse husbandry and the grass is harvested for cows. The valley of Hessenthal-Mespelbrunn is located about 13 km south-eastern of Aschaffenburg and was in the last years the most infested valley by cockchafer grubs. Cockchafer grubs are well known among the people of the Hessenthal-Mespelbrunn valley because the damage they caused was not hardly to discover. The grubs live in the soil and feed on the roots of grass, orchard trees and other plants. In the worst case the result of the pest’s feeding is a widespread dying of grassland. Without the connexion of the roots between grass and soil the turf is easy to be lifted and in the case of continuous rainfall the slopes are seriously threatened by erosion. The grubs perform a seasonal migration: In the Spessart grassland they remove to a depth of 30-60 cm not only in winter, but also in dry summer periods. But supplied with enough moisture they live from early springtime to late autumn in a depth of 1-2 cm to the turf. A digging at different places in the Hessenthal-Mespelbrunn valley to get an overview, what species of Scarabaeidae are present, gave the result as follows. Melolontha melolontha is the commanding species, followed by the Summer chafer Amphimallon solstitiale (L., 1758),

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the Welsh chafer Hoplia philanthus (Fuessly, 1775) and the Garden chafer Phyllopertha horticola (L., 1758). As a secondary damage of the infestation of the grubs but more severe for the landscape is the damage caused by wild pigs leaving at night the forests for rooting and digging up the grubs to devour them. To avoid all the damages in the grassland it became necessary to fight the real cause, to reduce the grubs to a tolerable level.

Materials and methods

Design of the trial The last cockchafer flight in the Hessenthal-Mespelbrunn valley was in 2003. The treatment started at the end of April 2004 to fight the second larval stage after the first hibernation. In the experiment the grubs should be controlled in three different ways: Firstly in a mechanical way with a rotary hoe/rotary cultivator, secondly with a biological antagonist, the entomopathogenic fungus Beauveria brongniartii, thirdly by chemical treatments with the insecticides Imidacloprid and Carbofuran. In table 1 the six different variants are listed. Every variant besides var. 6 (Imidacloprid, Gasur) was tested in three plots, each plot being of a size of 25 metres in length and 9 metres in width. Gasur was tested in a long strip of nearly 150 m length and 3 m width close-by the other plots. On the day of the treatments the grubs were in a depth of about 2 centimetres.

Table 1. The different variants of the cockchafer control experiment in the Spessart region Hessenthal- Mespelbrunn

Var. Variant Concentration (+) milling way of control 1 Untreated control --- no none (natural) 2 Rotary hoe/cultivator --- yes mechanical 3 Beauveria fungus 50 kg/ha no biological 4 Carbofuran (Carbosip) 10 kg/ha yes chem. + mech. 5 Imidacloprid (Confidor) 0,15 kg/ha yes chem. + mech. 6 Imidacloprid (Gasur) 100 ml/100 kg/ha no chemical

In the untreated control only the natural decrease of the grubs’ population caused by predators, bacterial or fungal diseases could be observed. The Beauveria fungus was applied in the form of Melocont®-Pilzgerste. The kernels were brought into the soil by using a special slit seedling machine. The same seedling machine was used to disperse evenly Imidacloprid in the Gasur form. In these three variants the turf was not milled. To apply both Carbofuran and Imidacloprid in the Confidor form into the soil a rotary cultivator was used. The same rotary hoe was used for milling the mechanically treated plots to fling up the grubs to the daylight, where they died because of ultraviolet radiation or being killed of birds. As in the last three variants the turf was completely destroyed after the treatments the grass has to be reseeded. For measuring the efficacy of a certain treatment the number of the grubs, the healthy ones and the fungal infected ones, in the different plots were counted and the results were compared with the untreated variant. In using a so-called Goettinger frame four times a square metre of the sod was digged up in every plot and the soil was searched for grubs. 23

Results and discussion

The first count of the grubs in the infested site was on the 29th of April 2004. On the same day, in the afternoon, the mechanical, biological and chemical treatments were carried out. The grubs were more or less homogeneously distributed in the plots. With an average of about 200 grubs per m2 the infestation was at a very high level. Among all plots the Beauveria plot was the most infested one. After five weeks, on the 3rd of June, a first monitoring was made to check the success of the treatments. The second and final monitoring of the year 2004 took place on the 31st of August, also in the hope to see long-term effects of the Beauveria fungus. The results of all three counts can be seen in figure 1.

300 Monitoring on 29th of April 1st count on 3rd of June 250 2nd count on 31st of August

200 2

150 Grubs per m 100

0 Var 1Var 2Var 3Var 4Var 5Var 6

Figure 1. Mean values of the cockchafer grubs in the six variants at three different dates (before treatment, after 5 weeks, before hibernation)

On the 3rd of June it was observed that only 50 % of the grubs survived in the untreated plots. The degree of naturally fungal infection with Beauveria brongniartii was in the untreated variant the highest one, even higher than in the Beauveria plots (not to see in figure 1). The rotary hoe showed an overwhelming reduction of the cockchafer’s population. Both Carbofuran and Imidacloprid (Confidor) provided quite good results but it is very uncertain whether the chemical treatment is the assignable cause because both variants were milled, too. The Gasur variant however showed no decreasing effect in comparison to the untreated variant. On the 31st of August it was noticed that the number of the grubs was again reduced to about 50 % in the untreated variant. Furthermore the good effect of the rotary cultivator could be confirmed. The slight increase of the grubs in the variant with the single use of the rotary hoe can be disregarded. But referring to the two insecticides Carbofuran and Imidacloprid (Confidor) the situation was quite different than considered as before. Carbofuran seemed to have no additional effect on the impact of the rotary hoe whereas Confidor yielded the best result. Nearly no grubs could be found at the end of August though the grubs which were 24

detected on the 3rd of June looked at that time very healthy. Maybe the Confidor has a long- term effect on the feeding behaviour and therefore the healthiness of the grubs. It could be assumed that the efficacy of the Beauveria fungus was not satisfactory but in the end there was a decrease of 80 % of the starting number of the grubs in this variant. Such a reduction means for a biological method of pest control an extremely promising result. The big advantage of the use of Beauveria brongniartii is the long-term effect as is known and the conservation of the turf. The result in the Imidacloprid (Gasur) variant of having no effect on the grubs’ population could also be confirmed. All in all it can be summarised that for the moment the single use of a rotary hoe, used at the best possible time, is a practicable method to reduce the grubs of the Common cockchafer in the Hessenthal-Mespelbrunn valley to a tolerable level. The handicap is that the milling destroys the turf. So for a long-time strategy of controlling the grubs the Beauveria fungus will be preferred. Open questions are: How to spread the fungal spores in grassland sites which are too steep for the slit seedling machine? Who pays for all the actions needed in the valley to renew the turf? And last but not least, how to communicate the necessity of controlling Melolontha melolontha to the public because in Bavaria this species is a symbol of intact nature and still regarded as threatened?

Acknowledgements

We thank Kerstin Jung and Gisbert Zimmermann (BBA Darmstadt) for the technical support concerning Beauveria brongniartii. Furthermore we want to thank our collegues Hans-Jürgen Wöppel, Oswald Behl and Konrad Rüdinger (Office for Agriculture in Würzburg) and Niko Versch (Office for Agriculture in Aschaffenburg) for the support in designing the plots and carrying out the different treatments.

References

Benker, U. & Leuprecht, B. 2004: Bekämpfungserfahrungen im Spessart und Vorkommen von Maikäfern und verwandten Scarabaeiden in Bayern. – Nachrichtenbl. Dt. Pflanzenschutzd. 56 (5): 95-98

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 25-29

Isolation of Beauveria brongniartii from soil: Are the available isolation tools neutral?

Jürg Enkerli1, Priska Moosbauer1,2, Franco Widmer1, Silvia Dorn2, Siegfried Keller1 1 Swiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, 8046 Zürich, Switzerland; 2 Institute of Plant Sciences/Applied Entomology, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland

Abstract: For Beauveria brongniartii the fungus used in biological control of Melolontha melolontha, three techniques are routinely applied to monitor or isolate B. brongniartii from soil after BCA application. These are baiting with the host M. melolontha (MB), baiting with Galleria mellonella (GB) or plating soil suspensions on selective medium (SM). It has never been investigated whether any of the three isolation methods may select for certain B. brongniartii strains or whether they are equally suited for the isolation of different strains. We performed two experiments to address this question. First, the virulence of 30 genetically different isolates from Jenaz (eastern Switzerland) were tested in a bioassay with M. melolontha. The collection of 30 isolates consisted of 3 groups of ten isolates, each group consisting of isolates collected with one of the three isolation technique. Although, the virulence of single isolates was significantly different across all isolates the average virulence among the three groups was not significantly different, suggesting that the isolation techniques did not select for isolates with different virulence against M. melolontha. For the second experiment conidia of six B. brongniartii isolates of which always two were obtained with the same isolation techniques were mixed into B. brongniartii free soil. Strains were re-isolated from the soil mixture by using each of the three isolation methods and genotypes were determined by microsatellite analysis. All six genotypes have been re-isolated with the GB method and five genotypes have been re-isolated with either the MB and the SM method. In most cases the observed abundance of the genotypes corresponded with the expected abundance calculated based on equal re-isolation rates for each isolate with each method. A broader spectrum of genotypes will need to be tested in order to confirm the findings and allow statistically valid generalizations. In this study we have described and evaluated a strategy to compare and validate isolation techniques for entomopathogenic soil fungi.

Keywords: isolation techniques, genotype selection, virulence, monitoring

Introduction

The fungus Beauveria brongniartii (Sacc.) Petch is a well established and commercially available biocontrol agent (BCA) to control the larvae of Melolontha melolontha in grasslands and orchards (Keller, 1992; Zelger, 1996). Three techniques are currently available for monitoring and isolation of B. brongniartii from soil samples. The Melolontha bait (MB) or Galleria bait (GB) methods where B. brongniartii is selected from soil samples by baiting either with M. melolontha or Galleria mellonella larvae (Keller et al., 1997; Kessler et al., 2003) and the selective medium (SM) method where soil suspensions are plated on a selective medium (Kessler et al., 2003; Strasser et al., 1996). Although all three techniques are widely applied for pre- and post-treatment monitoring and isolation of new biocontrol strains they have never been compared regarding their selectivity i.e. whether they select for certain B. brongniartii genotypes or whether they allow for equal growth of different strains.

25 26

Particularly, differences in the selective ability of MB and GB methods, which are based on selection for a virulent phenotype, compared to the SM method would be reasonable. Availability of validated selection and isolation techniques is important for reliable monitoring of applied biocontrol agents in the field, selection of new biocontrol strains or investigations of natural population structures and it is crucial to compare selective behavior of different techniques. Sensitive genetic identification tools are an important requirement to perform such comparisons and for B. brongniartii appropriate techniques recently have become available with the development of microsatellite markers (Enkerli et al., 2001). This genetic tool has been applied to investigate genetic diversity in a natural population of B. brongniartii in Switzerland (Enkerli, Keller, and Widmer, unpublished). Preliminary results of this study have suggested that certain genotypes might preferentially be selected by either of the three isolation techniques. The aim of the present study was to investigate whether the three isolation techniques select for certain isolates of B. brongniartii or whether they allow to equally grow different B. brongniartii strains. First, we tested whether genetically different isolates that have been selected with only one of the three isolation techniques differ in their virulence towards M. melolontha. Second, we mixed six genetically different isolates of which always two were isolated with one technique into a soil samples. Subsequently, strains were re-isolated with all three techniques to test whether different techniques select preferentially for isolates with a certain genotype or whether each genotype can be re-isolated at the same rate.

Materials and methods

All fungal isolates originate from a grassland plot of 1ha at Jenaz (eastern Switzerland). The plot was sampled continuously once to twice per year since 1999 to monitor B. brongniartii density and population structure. Isolates were obtained by applying three techniques: (i) Isolation from collected M. melolontha larvae, (ii) baiting with G. mellonella or (iii) plaiting soil suspensions on selective medium and subsequent isolation of single B. brongniartii colonies. For isolation from M. melolontha, larvae were collected from soil, placed individually into plastic cups (4.5 cm ∅, 6 cm high) filled with damped peat and incubated in constant darkness at 22˚C. Subsequently, single isolates were obtained from sporulating mycelium of diseased cadavers. Galleria baiting (GB) as well as isolation from selective medium (SM) were performed as described by Kessler et al. (2003). One hundred and fifty- one B. brongniartii isolates were collected between 1999 and 2001 and genotyped based on 6 microsatellite markers as described by Enkerli et al. (2004). For each isolation technique 10 isolates were selected, which displayed a genotype that was only detected in isolates obtained with a particular technique. This resulted in a collection of 30 isolates with unique genotypes. Virulence of the B. brongniartii strains was determined by dipping 30 M. melolontha larvae per strain in a suspension containing blastospores (107 /ml) for 4 seconds. Excess water was removed with a paper towel and the larvae were placed individually into plastic cups (4.5 cm ∅, 6 cm high) filled with damped peat. The larvae were incubated in constant darkness at 22˚C and fed with sliced carrots. Mortality was recorded daily starting five days after inoculation. Dead insects, with typical signs for fungal infection were moved to a separate moist chamber and incubated until sporulation. Conidia were identified under the microscope. Calculation of average survival time of M. melolontha larvae for each isolate and significance analyses (Kruskal-Wallis H test) were performed with the software SSPS V.10.1. Two isolates per isolation technique, all with comparable growth and virulence characteristics, were selected for the re-isolation experiment (Table 1.) and grown on complete medium (CM) plates (Riba & Ravelojoana, 1984). For each isolate 10 plates were 27

grown in the dark for 3 weeks at 22˚C. Conidia were harvested by washing the plates with 0.1% Tween 80 and resulting suspensions were diluted to a concentration of 107 conidia per ml. Forty ml of each isolate suspension were combined and mixed with 4kg of Beauveria free soil by repeated cycles of spraying of the suspension on the soil (hand spray applicator) and subsequent mixing. This resulted in a soil sample containing a conidia concentration of 105 /g soil for each isolate. The concentration in the soil was verified by applying the SM method and counting colony forming units. Re-isolation of B. brongniartii from the soil was performed by applying the three isolation techniques each in 40 repetitions. For each isolation 30g soil were used. Baiting with M. melolontha (MB) was performed according to the GB method. Eight days post inoculation Melolontha and Galleria larvae were moved to new plastic cups (4.5 cm ∅, 6 cm high) filled with damped peat and further incubated in constant darkness at 22˚C. B. brongniartii strains were re-isolated from diseased larvae up to 70d post- inoculation for MB and 30d for GB. Per diseased larvae one isolate was obtained and subjected to single colony isolation on CM. For re-isolations with the SM method one single colony per SM plate was isolated. Numbers of obtained isolates per isolation technique are listed in Table 2. The genotype of each isolate was determined by applying microsatellite analysis as above.

Table 1. Genotypes of selected B. brongniartii isolates Isolate No. Original isolation Microsatellite loci and allele sizes [bp] Genotype technique Bb1F4 Bb2A3 Bb2F8 Bb4H9 Bb5F4 Bb8D6 1 MB 214 106 196 171 154 172 A 4 MB 214 106 208 165 154 172 B 12 GB 196 103 181 177 157 172 C 19 GB 238 124 217 180 199 172 D 22 SM 238 127 211 177 208 172 E 29 SM 214 106 193 165 151 172 F

Results and discussion

For each isolation technique 10 genetically unique isolates, which were selected with one particular technique only, were tested for their virulence against M. melolontha larvae. Results of the bioassays are shown in Figure 1. The average survival time varied across all isolates from 24d to 62d. The average of the average survival times was 39.6d ± 8.6d for isolates obtained with MB method, 42.9d ± 9.2d for isolates obtained with GB method and 36.2d ± 5.0d for isolates obtained with SM method. Non of the averages differed significantly among the isolation techniques. These results suggest that the three techniques do not select for isolates with different virulence against M. melolontha. Investigations of various growth characteristics such as in vitro biomass, blastospore or oosporein production supported these results (data not shown). No significant differences among averages of the according parameters of the three isolate groups were found (data not shown).

60 50

40 30 20 10 Average survival time [d] 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Isolate no. Figure 1. Virulence against M. melolontha of 30 B. brongniartii isolates obtained either by the MB ( ), GB ( ), or SM ( ) method

Table 2. Abundance of re-isolated genotypes Re-isolation No. of isolates Genotype Original isolation Abundance of genotype technique technique Observed Expected MB 18 A MB 3 (16.7)a 3.0 B MB 7 (38.9) 3.0 C GB 0 ( 0.0) 3.0 D GB 4 (22.2) 3.0 E SM 3 (16.7) 3.0 F SM 1 ( 5.6) 3.0 GB 25 A MB 3 (12.0) 4.2 B MB 3 (12.0) 4.2 C GB 4 (16.0) 4.2 D GB 11 (44.0) 4.2 E SM 1 ( 4.0) 4.2 F SM 3 (12.0) 4.2 SM 40 A MB 9 (22.5) 6.7 B MB 11 (27.5) 6.7 C GB 7 (17.5) 6.7 D GB 11 (27.5) 6.7 E SM 0 ( 0.0) 6.7 F SM 2 ( 5.0) 6.7 a Relative observed abundance of genotypes among isolates re-isolated with the same technique

To test whether the different techniques select certain genotypes or whether they equally selected different genotypes, two isolates of each isolate group were mixed in equal amounts with a soil sample. Subsequently, isolates were re-isolated with the three isolation techniques and the re-isolation rate for each genotype was determined. Eighteen isolates were obtained with the MB method, 25 with the GB method and 40 with the SM method (Table 2.). Four 29

genotypes (A, B, D, and F) were re-isolated with all three techniques, genotype C was not isolated with MB method and genotype E was not isolated with the SM method. In most cases the observed abundance of the genotypes corresponded to the expected abundance calculated based on equal re-isolation rates for each isolate with each method. Genotypes E and F which originally were selected with the SM method displayed the lowest re-isolation rates with the SM method among all six isolates. Due to the low number of repetitions it can not be concluded whether this represents true differences between the methods and/or isolates. To allow for statistically valid conclusions regarding the selectivity of the three selection techniques for certain genotypes of B. brongniartii, a broader spectrum of isolates will need to be tested and the number of re-isolations need to be increased. In this study we have described and evaluated a strategy for validation of isolation methods. As an example we have compared the three B. brongniartii isolation techniques regarding their selectivity for the virulence trait and their selectivity for certain genotypes. Results obtained have demonstrated that this strategy is feasible and well suited to address the question of selectivity for isolation techniques. Availability of sensitive genetic identification tools is a crucial requirement for this type of analyses and the use of microsatellite markers in this study represents another application for this type of genetic marker.

Acknowledgments

We thank Dr. Ph. Kessler for his support with statistical analyses and Ch. Schweizer for assistance in collection of soil samples and isolation of B. brongniartii.

References

Enkerli J., Widmer F., Gessler C. & Keller S. 2001: Strain-specific microsatellite markers in the entomopathogenic fungus Beauveria brongniartii. – Mycol. Res. 105: 1079-1087. Enkerli J., Widmer F. & Keller S. 2004: Long-term field persistence of Beauveria brongniartii strains applied as biocontrol agents against European cockchafer larvae in Switzerland. – Biol. Control 29: 115-123. Keller S. 1992: The Beauveria-Melolontha project: Experiences with regard to locust and grasshopper control. – In: Biological control of locusts and grasshoppers, eds. Lomer & Prior: 279-286. Keller S., Schweizer C., Keller E. & Brenner H. 1997: Control of white grubs (Melolontha melolontha L.) by treating adults with the fungus Beauveria brongniartii. – Biocontrol Sci. Techn. 7: 105-116. Kessler P., Matzke H. & Keller S. 2003: The effect of application time and soil factors on the occurrence of Beauveria brongniartii applied as a biological control agent in soil. – J. Invertebr. Pathol. 84: 15-23. Riba G. & Ravelojoana A.M. 1984: The parasexual cycle in the entomopathogenic fungus Paecilomyces fumoso-roseus (Wize) Brown and Smith. – Can. J. Microbiol. 30: 922-926. Strasser H., Forer A. & Schinner F. 1996: Development of media for the selective isolation and maintenance of virulence of Beauveria brongniartii. – In: Microbial control of soil dwelling pests, eds. Jackson & Glare: 125-130. Zelger R. 1996: The population dynamics of the cockchafer in South Tyrol since 1980 and measures applied for control. – IOBC/wprs Bulletin 19 (2): 109-113. 30 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 31-35

Development of the Melolontha populations in the canton Thurgau, eastern Switzerland, over the last 50 years

Siegfried Keller, Hermann Brenner Agroscope FAL Reckenholz, Reckenholzstrasse 191, CH-8046 Zurich, Switzerland; LBBZ Arenenberg, CH-8268 Salenstein, Switzerland

Abstract: Two Melolontha melolontha L. populations each with a three year life cycle exist in the canton Thurgau. The Bernese cycle (III-1) in the western part and the Uranean cycle (III-2) in the central and eastern part of the canton. Two methods were used to monitor their development: The length of forest borders damaged by swarming adults and number and amount of damages done by white grubs. The data show that the Bernese cycle had its peak period 1972-1975, then collapsed and remained stable at a low level. The Uranean cycle had a peak in the early 50s and 1991-1994. The damages done by white grubs are separated between the two flight regimes but show a good correlation with the damages done by adults in the Uranean flight area. Control measures mainly with the fungus Beauveria brongniartii were only done in the Uranean flight area. A comparison between treated and untreated area demonstrate the long lasting efficacy of this method. Nevertheless, it is concluded that a continuous monitoring of the pest population is a prerequisite for a successful Melolontha management.

Keywords: Melolontha melolontha, population dynamics, Thurgau, damages.

Introduction

Two Melolontha melolontha L. populations each with a three year life cycle exist in the canton Thurgau. However, the cycles of the two populations are shifted by one year: The Bernese cycle (III-1) in the western part and the Uranean cycle (III-2) in the central and eastern part of the canton. The development of the Melolontha populations over the last 50 years is well documented mainly by the length of damaged forest borders and by the extent of damages caused by the white grubs. The aim of this work is a documentation of the development of the cockchafer population in an area which permanently suffered from white grub damages since the mid of the 20th century and to set the population development in relation to control measures with the fungus Beauveria brongniartii. These treatments were mainly carried out in the years 1985 and 1988, when blastospores were sprayed on swarming beetles along forest borders (Keller et al. 1997). In 1985 the Uranean flight area “north” was treated and in 1988 that of the area “south”. The fungus was applied on forest borders in a delimited area where the cockchafers concentrated. In addition to the blastospore treatments, fungus granules (“Beauveria-Schweizer”) were applied in orchards from 1991-1995 followed by the installation of hail nets during subsequent flights (Brenner & Keller, 1996).

Material and methods

At the end of each cockchafer flight the feeding damage of the swarming adults on oaks and beaches along forest borders was assessed using the scores: undamaged, low, medium and heavy damages. The damages were recorded on a 1:25’000 map and the lengths of the damaged forest borders measured on the map.

31 32

Since 1974 the financial losses due to white grub feeding are reimbursed through a specific fund. The amounts allow to draw conclusions on the densities of white grubs. To set the population development in relation to control measures we chose the Uranean flight area “north” (treated 1985) and compared the length of the heavily damaged forest borders inside the treated area with that outside.

Results and discussion

Length of damaged forest borders The length of the forest borders damaged by the swarming adults is recorded since 1955 (Uranean cycle) and 1960 (Bernese cycle) respectively. The two cycles did not develop synchronously. According to the damages of forest borders the Bernese cycle had its peak period 1972-1975. From 1981 onwards the population is low and of minor economic importance. The Uranean cycle had two peaks, one in the early 50s at the beginning of the phase with chemical treatments, and the other between 1991 and 1994. With the exception of the period from 1961-1970, this population always caused significant damages especially in orchards. A chemical treatment was planned for 1973, however public concerns prevented the action. The consequences were 1) the creation of a special fund to compensate the damages done by white grubs and 2) the start for the research on biological control.

Forest borders damaged by cockchafers, Uranean flight

500 heavy 450 400 medium 350 weak 300 250 total km 200 150 100 50 0

955 958 961 964 967 970 973 976 979 982 985 988 991 994 997 000 003 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 Year

Figure 2: Development of the Melolontha population in the Bernese flight area from 1960-2002. No data were recorded 1963-1969 and 1981.

Payments to the farmers to compensate for damages done by white grubs started 1974. The amount of contributions over the years does not reflect both cycles, it rather correlates with the Uranian cycle only. On average the yearly number of damages from 1974-2004 was 75, the maximum was reached 1992 with 340 cases, the minimum was a single case in 1997. The yearly compensations amounted to an average of CHF 237’604.-. On average a single damage was compensated with CHF 3152.-.

Forest borders damaged by cockchafers, Bernese flight

160 haevy 140 medium 120 100 weak 80 total km 60 40 20 0

1960 1963 1966 1969 1972 1975 1978 1981 1984 1987 1990 1993 1996 1999 2002 Year

Figure 2: Development of the Melolontha population in the Bernese flight area from 1960-2002. No data were recorded 1963-1969 and 1981.

Number of damages done by white grubs. Mean of three years

180 160 140 120 100 80 60 damages 40

mean number of 20 0 75-77 78-80 81-83 84-86 87-89 90-92 93-95 96-98 99-01 02-04 Year

Figure 3: Number of damages (3-years average) done by white grubs in the canton Thurgau from 1975-2004.

The development of the number of damages (Figure 3) shows two maxima in the periods 1975-1977 and 1990-1992 respectively. The absolute minimum was reached 1996-1998 with an average of four damages. Since then the number of damages slowly increased. The main reason for the increasing numbers of damages are attributed to the mixed flight regimes which developed over the last ten years. Especially the apple growers are not yet fully aware that

there is no more a strict three year cycle and they neglect the intermediate flights and do not protect the orchards with the existing hail nets. This allows the females of the intermediate flights to deposit their eggs in orchards The drastic decline of the population density and of the damages in the 90s in the Uranean flight area is considered the result of control measures applying the fungus Beauveria brongniartii as a biocontrol agent (spraying blastospores or drilling granules) and /or the installation of hail nets in orchards which prevented cockchafer females from laying eggs in this sensitive crop (Brenner & Keller, 1996).

Influence of the fungus treatment on the population development Within the Uranean flight area “north” the population outside the treated perimeter remained more or less stable until 1994 and collapsed 1997 (Table 1). In 2003 it started to increase. The treated population only remained stable until 1991 and then collapsed. From 1997 onwards there were no more forest borders with heavy damages. The ratio between “outside” and “inside” increased from 2.36 in the year of the treatment to 8.97 in 1994 and can be taken as an indicator for the success of the fungus treatment. The marked increase of the ratio from 1991 to 1994 confirms the previous finding that the fungus needs about two Melolontha generations (6 years) to get established in the host population (Keller, 2004; Keller et al. 1997).

Table 1. Comparison of the population development inside the area treated 1985 (I) and outside (O) the area within the Uranean flight area “north”. The numbers in columns O and I indicate the length of damaged forest borders.

outside (O) inside (I) O/I 1985 42 17.8 2.36 1988 28 9.3 3.01 1991 61 16.6 3.67 1994 35 3.9 8.97 1997 0.8 0 2000 0 0 2003 2 0

Conclusions

Melolontha is a sessile species and relatively easy to monitor by recording the places of damages done by white grubs and the damages done by the swarming adults. These tools show that the Uranean flight area is moving westwards and we can advise the farmers accordingly. Further we have enough experience to recommend the installation of hail nets on apple and berry plantations and the application of B. brongniartii in grassland. The combination of these two control methods reduced the Melolontha population over a long period below the damage threshold but without eradicating the species. Nevertheless, a continuous monitoring of the pest population is necessary.

References

Brenner H. and S. Keller 1996: Protection of orchards from white grubs (Melolontha melolontha L.) by placements of nets. – IOBC/wprs Bull. 19(2): 79-82. Keller, S. 2004. Bekämpfung von Maikäfer-Engerlingen mit dem Pilz Beauveria brongniartii in der Schweiz. – Laimburg Journal 1: 158-164. Keller S., Schweizer C., Keller E. and Brenner H. 1997. Control of white grubs (Melolontha melolontha L.) by treating the adults with the fungus Beauveria brongniartii. – Biocontrol Sci. & Technol. 7: 105-116.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 37-44

1 1 2 1 3 Robert Koller , Kerstin Jung , Stefan Scheu , Gisbert Zimmermann , Joachim Ruther 1 Federal Biological Research Centre for Agriculture and Forestry, Institute for Biological Control, Heinrichstr. 243, D-64287 Darmstadt, Germany 2 Technische Universität Darmstadt, Institute of Zoology, Schnittspanstr. 3, D-64287 Darmstadt, Germany 3 Free University of Berlin, Institute of Biology, Applied Zoology/Animal Ecology, Haderslebener Str. 9, D-12163 Berlin, Germany

Abstract: In the German federal states of Hessen, Rheinland-Pfalz and Baden-Württemberg a massive outbreak of the forest cockchafer, Melolontha hippocastani Fabr. (Coleoptera, Scarabaeidae) endangers approximately 7 500 ha of forests (occurrence on 37 000 ha). As a naturally occurring pathogen, Beauveria brongniartii is a promising candidate for biological control of M. hippocastani. In the present study we investigated, whether spores of B. brongniartii could be spread within the cockchafer population using males as vectors after contamination by passage of an inoculation trap. First, in order to optimise the funnel traps, three different types were compared to a standard trap with respect to their handling in the field and their catch and release characteristics. Each funnel trap was baited with a mixture of the sexual pheromone 1,4-benzoquinone and the sexual kairomone (Z)-3- hexen-1-ol and placed in oak trees (Quercus rubra) infested with cockchafers. Both volatiles are involved in mate finding of forest cockchafers and are attractive to males. During the swarming flight a maximum of 261 males was captured per funnel trap per day. No correlation between the size of the baffle screen and the number of captured males was found. The results suggest that the spatial arrangement of the baffle screen is more important for the capture of males than its dimension. Furthermore, the influence of the position of the funnel trap within the tree on the catching efficacy was studied by placing the traps at different heights. The number of captured males significantly increased with the trap height in the trees. A contamination experiment in flight cages in a forest was performed to evaluate a proposed transfer of spores from (a) trap to male (b) male to female and (c) female to soil/white grubs in the field. Males picked up sufficient spores to become mycosed by B. brongniartii and to transfer the fungus to females successfully. However, spore numbers in the soil did not increase significantly. This confirms that infective spores of B. brongniartii can be disseminated by male cockchafers after the passage through an inoculation trap. Whether the females could transmit the spores in effective numbers into the breeding sites still needs to be proven under field conditions.

Keywords: Scarabaeidae, biological control, 1,4-benzoquinone, (Z)-3-hexen-1-ol, sexual pheromone, sexual kairomone, funnel trap, flight cage

Introduction

Melolontha hippocastani is a polyphageous insect which causes outbreaks every 30-40 years (Altenkirch et al. 2002). The main damage is caused by the white grubs which develop in the soil within 3-4 years, feeding on fine roots of nearly all tree species (Altenkirch et al. 2002, Jung et al., 2005). Adult beetles occur only for about 6 weeks from mid of April until end of May (Ruther et al. 2001). During the swarming flight, a two-step chemically mediated mate

37 38

finding process of M. hippocastani occurs in infested trees (Ruther et al. 2001). Most of the females remain feeding on the host trees (Ruther et al. 2001). Due to the mechanical damage caused by their feeding, green leave volatiles (GLVs) are emitted by the injured tree tissue (Ruther et al. 2004). During the swarming flight, male cockchafers are hovering along the twigs of infested trees orientating towards their mating partner using GLV (Z)-3-hexen-1-ol (Z-3-ol) and the sex pheromone 1,4-benzoquinone (BQ) which enhances the attractiveness of the first one synergistically (Ruther et al. 2001, Ruther et al. 2004). Beauveria brongniartii (Sacc.) Petch (Deuteromycota, Hyphomycetes) is a pathogen causing one of the most important diseases of M. hippocastani and therefore has been used in biological control during the past 100 years (Zimmermann 1998, Altenkirch et al. 2002). Difficulties with soil and aerial application methods of B. brongniartii products within German forests (Jung et al., 2005) have led to a combined use of attractants and insect pathogens as described by Klein & Leacy (1999) and Ruther & Hilker (2003). In this technique, inoculation traps are used to inoculate captured beetles with spores of an entomopathogenic fungus to spread it within the population. In this study we tested catch and release characteristics of different inoculation traps and investigated whether captured males of M. hippocastani can be used as vectors to transfer the fungus to females during the copulation and thus, to establish it in the larval breeding habitats.

Materials and methods

Experiment 1: Comparison of autodissemination traps This experiment evaluated catch and release characteristics of different trap types compared to a standard trap. It was conducted between April 29 and May 6, 2003 in a Quercus rubra stand (about 10 m high) close to Hagenbach (Rheinland-Pfalz, Southern Germany). Differently designed inoculation traps and the standard trap (Table 1, Figure 1) were compared. They were all baited with a membrane dispenser (Wilhelm Biological Plant Protection, Sachsenheim, Germany), filled with 3 ml of a solution of BQ in Z-3-ol (20 mg m-1). In a randomised design, blocks of 3 inoculation traps and the standard trap were placed at least 30 min prior the swarming flight at equivalent positions (5 m above the ground, 1 m minimum distance between traps) in the Q. rubra stand (n = 48). Caught males were counted in the morning after the swarming flight.

Table 1: Characteristics of tested inoculation traps Type developed by Weight Height Baffle screen size Baffle screen [kg] [cm] [cm²] design standard Free University 0.2 66 1224 crossed trap of Berlin BBA Institute for Biological 1.1 88 7500 crossed Control FU Free University 2.0 71 3200 crossed of Berlin HF Hessian Forestry 2.8 47 7200 flat Department

Numbers of captured beetles were analysed by a Friedman ANOVA and consecutive multiple Wilcoxon matched pairs tests with sequential Bonferoni-correction (Sachs, 1992) using Statistica 4,5 scientific software (StatSoft, Hamburg).

Standard trap BBA FU HF

Figure 1: Tested inoculation traps. For details see text and Table 1

Experiment 2: Influence of trap height This experiment evaluated the influence of the height position of the trap within a host tree on the number of captured males. The experiment was conducted between May 8 and 11, 2003 at the same experimental site as experiment 1. The standard trap (Table 1) was hung up in 3, 5 and 6,5 m height in the 10 m high Q. rubra trees (n = 28). Each standard trap was baited as described in experiment 1 and installed at least 30 min prior to the swarming flight in the Q. rubra frontage. Statistical analysis was done as described in experiment 1.

Experiment 3: Dissemination of spores This experiment investigated the proposed transfer of spores from (a) trap to male (b) male to female and (c) female to soil/grub in the field. Between April 25 and May 28 2003, six flight cages (length 3 m, width 3 m, height 2 m) were built up pairwise (treatment and control, distance between the cages 2-4 m) in a Fagus sylvatica understory near Kandel (Rheinland- Pfalz, southern Germany). Each flight cage was equipped with 200 females and 200 males of M. hippocastani. In the treatment cages males were contaminated with an experimental B. brongniartii spore powder formulation (FYTOFITA, Co. Ltd., Czech Republic, 1x109 spores g-1). To simulate natural catching conditions males were thrown individually into the inoculation trap (type BBA; Table 1) during the swarming period. (A) Dead males and females were collected daily and transferred for further examination to the laboratory. The dead beetles were stored on moist soil in plastic dishes (length 16 cm, width 11 cm, height 6.5 cm) under open air conditions but protected from direct sun and rain. Two weeks later the beetles were checked for outgrowth of B. brongniartii. (B) 7 days after the onset of the experiment, 20 living females and males were collected in each flight cage to check for B. brongniartii contamination. The beetles were transferred individually into sterile flasks (Nalgene), washed with 10 ml of sterile Tween 80 (0.1 %) for 20 min by shaking. The suspension was centrifuged for 10 min at 10.000 rpm. 8 ml of the supernatant were decanted and aliquots of the resuspended pellet were spread with a sterile Drigalski spatula on Beauveria-Selective Media (Strasser et al. 1996). After incubation for 2

weeks at 25 °C the plates were checked for the growth of B. brongniartii colonies and the number of spores per individual was calculated from colony forming units (cfu). (C) The first soil samples were taken prior to the treatment, then again 4, 8 and 16 weeks after the treatment in 20-25 cm depth (three samples per cage and date). The samples were stored at 7 °C until analysis. Quantification of the B. brongniartii density in 1 g soil was done as described by Goettel & Inglis (1997). After incubation for 14 days at 25 °C the Petri dishes were checked for B. brongniartii colonies, and the number of cfu of B. brongniartii per 1 g soil was calculated. Infection rate and dose of spores on living males and females after 7 days in the control and treatment cages were analysed by one way ANOVA. Means were compared by using Tukey’s studenized range test for significant differences (Sokal & Rohlf 1995). To analyse possible introduction of spores into the soil log transformed data were analysed by repeated measures ANOVA. SAS (Statistical Analysis System, Version 8.1, SAS Institute Inc. 2000) was used for the statistical analysis.

Results

Experiment 1: Comparison of autodissemination traps During the swarming flight each type of inoculation trap captured males (Figure 2). Only the “HF” trap captured significant less males compared to the others (Figure 2). A maximum number of 261 males was captured in the BBA designed inoculation trap. Catches of the FU, BBA and standard trap did not differ significantly (Figure 2).

Experiment 2: Influence of trap height The number of captured males increased significantly with height of exposure of the traps (Figure 3).

Experiment 3: Dissemination of spores (A) The infection rate of M. hippocastani in the treatment cages was 38.9 %, significantly higher compared to the infection rate in the untreated cages (20.1 %). (B) After 7 days the number of spores (cfu) on males and females were significantly higher in the treatment cages compared to males and females in the control cages (Figure 4). In the treatment cages the number of spores on females was lower compared to males (Figure 4). (C) Spore numbers of B. brongniartii (cfu) in the soil did not differ between control and treatment cages (Table 2).

Table 2: Number of Beauveria brongniartii spores (cfu) per 1 g soil (n = 3 per cage); means ± SD in control and treatment cages. Sample number 1 was taken before the treatment, sample number 2, 3 and 4 were taken 4, 8 and 16 weeks after the treatment, respectively.

Sampling Number Control Cage Treatment Cage 1 65 ± 73 28 ± 39 2 0 ± 0 306 ± 354 3 46 ± 47 46 ± 47 4 676 ± 936 963 ± 1322

70 ay

d a a

& 60

50 a

per trap 40 SE - / 30

10 b ean captues +

M 0 Standard HF FU BBA

Figure 2: Mean captures ± standard error of Melolontha hippocastani males in different types of inoculation traps (Standard, HF = Hessian Forestry Department, FU = Free University of Berlin, BBA = Institute for Biological Control) between April 29 and May 6 2003. Each trap was baited with a dispenser containing 3 ml of 1,4-benzoquinone in (Z)-3-hexen-1-ol (20 mg ml-1). Different letters indicate significant differences (Friedman ANOVA followed multiple Wilcoxon matched pair test with sequential Bonferroni correction, p < 0.05).

160 a 140

120

100

80 b

60 c 40

Mean captures +/- SE per trap & day 20

0 highHoch middleMittel lowNiedrig

Figure 3: Mean captures of Melolontha hippocastani males +/- standard error between May 8 and 11 2003 at different heights (“low” = 3 m, “middle” = 5 m and “high” = 6,5 m height). Each trap was baited with a dispenser containing 3 ml of 1,4-benzoquinone in (Z)-3-hexen-1-ol (20 mg ml-1). Different letters indicate significant differences (Friedman ANOVA followed by multiple Wilcoxon matched pair test with sequential Bonferroni correction, p < 0.05).

Control Treatment * 120

100 * 80

Mean number of spores (cfu) per individual +/- SD Males Females

Figure 4: Mean number of spores per individual +/- standard deviation on living males (n=60) and females (n=60) of Melolontha hippocastani after 7 days in flight cages. * indicates significant differences, ANOVA p < 0.05.

Discussion

In the present study the standard trap and trap types FU and BBA, captured similar numbers of males compared to those described by Ruther & Hilker (2003). Unexpectedly, the number of captured males in the standard trap was similar to the numbers in the trap types BBA and FU (Figure 2) although the baffle screen areas of the latter were much larger (Table 1). Only the HF captured significant lower numbers of males (Figure 2). This suggests that the spatial arrangement of the baffle screen is important for the trap performance. Our results demonstrate that the number of captured males increases with the trap height within a tree. This has consequences for the use of traps for forest cockchafer control since the optimal position of the traps in the top of the trees is difficult to manage in old forest stands with high trees. In the third experiment a transfer of spores from the inoculation trap to the males was shown. By passing through the inoculation traps, males picked up sufficient spores to transfer them successfully to the females. The transmission of infective spores was also shown by a higher infection rate of M. hippocastani in the treatment cages compared to the control cages. The observed high infection rate of M. hippocastani with B. brongniartii in the control cages may be explained by drifting of spores due to wind during dissemination of the male beetles in the treatment cages. Spore numbers in the soil of the treatment cages did not increase significantly and the recommended density of 103-104 cfu/g soil for a successful control of cockchafers (Keller 2002) was not achieved. Favourable abiotic growth conditions (e.g. moist and temperate climate) and high abundance of white grubs are needed for B. brongniartii to become established in soil (Keller et al. 1997, Kessler et al. 2003). Possibly the extreme hot and dry summer in 2003 was responsible for the low abundance of B. brongniartii spores in the soil in our experiment. Strong deviations in cfu/g soil can be explained by the method of

dilution for plating (see above). Furthermore, the artificial situation within the cages affected the behaviour of the beetles as observed by Keller (1978), e.g. maturation feeding of the females was low. Thus, females may not have returned to the soil for egg deposition during the experiment explaining that there were no white grubs in the soil within the cages and that there were no differences between treatment and control cages in B. brongniartii spore numbers.

Acknowledgements

The authors thanks Gabi Dröge, Ivonne Siebecke, Ulrike Gloger and Rony Schmitt for their help during the field work; Dr. Horst Delb and Dr. Jürgen Mattes (Forstliche Versuchs- und Forschungsanstalt Baden-Württemberg) as well as Dr. Joachim Gonschorrek and Dagmar Leisten (Hessen-Forst) for their logistical support. The research was financially supported by the Hessian State Forest Administration.

References

Altenkirch, W., Majunke, C. & Ohnesorge, B. 2002: Waldschutz auf ökologischer Grundlage. – Eugen Ulmer Verlag, Stuttgart. Jung, K., Gonschorrek, J., Ruther, J. & Zimmermann, G. (2005): Field testing of new biocontrol strategies to decrease the population density of Melolontha hippocastani, an important scarab species in Germany. – IOBC/wprs Bulletin 28(3): 85-88. Keller, S. 1978: Infektionsversuche mit dem Pilz Beauveria tenella an adulten Maikäfern (Melolontha melolontha). – Mitt. Schweiz. Entomol. Ges. 51: 13-19. Keller, S., Schweizer, C., Keller, E. & Brenner, H. 1997: Control of white grubs (Melolontha melolontha L.) by treating adults with the fungus Beauveria brongniartii. – Biocontrol Sci. Technol. 7: 105-116. Keller, S., Kessler, P., Jensen, D. & Schweizer, C. 2002: How many spores of Beauveria brongniartii are needed to control Melolontha melolontha? – IOBC/wprs Bull. 25(7): 59-63. Kessler, P., Matzke, H. & Keller, S. 2003: The effect of application time and soil factors on the occurrence of Beauveria brongniartii applied as a biological control agent in soil. – J. Invert. Pathol. 84: 15-23. Klein, M.G. & Lacey, L.A. 1999: An attractant trap for autodissemination of entomopathogenic fungi into populations of the Japan beetle Popillia japonica (Coleoptera: Scarabaeidae). – Biocontrol Sci. Technol. 9: 151-158. Goettel, M. & Douglas, I. 1997: Fungi: Hyphomycetes – In: Lacey, L.A. (ed.): Manual of Techniques in Insect Pathology. Academic Press London: 225-230. Ruther, J., Reinecke, A., Tolasch, T. & Hilker, M. 2001: Make love not war: A common arthropod defence compound as sex pheromone in the forest cockchafer, Melolontha hippocastani. – Oecol. 128: 44-47. Ruther, J. & Hilker, M. 2003: Attraction of forest cockchafer Melolontha hippocastani to (Z)- 3-hexen-1-ol and 1,4-benzoquinone: application aspects. – Entomol. Exp. et Appl. 107: 141-147. Ruther, J., Reinecke, A. & Hilker, M. 2004: Mate finding in the forest cockchafer, Melolontha hippocastaii Fabr., mediated by volatiles. – Laimburg Journal 2: 197-199. Sachs, L. 1992: Angewandte Statistik, 7. Auflage. – Axel Springer Verlag, Berlin. Sokal, R. & Rohlf, F.J. 1995: Biometry. – Freeman & Co, New York.

Strasser, H., Forer, A. & Schinner, F. 1996: Development of media for the selective isolation and maintenance of virulence of Beauveria brongniartii. – Proc. 3rd International Workshop on Microbial Control of Soil Dwelling Pests: 125-130. Zimmermann, G. 1998: Der entomopathogene Pilz Beauveria brongniartii (Sacc.) Petch und Erfahrungen bei seinem Einsatz zur biologischen Bekämpfung von Feld- und Wald- maikäfer. – Nachrichtenbl. Deutsch. Pflanzenschutzd. 50: 249-256.

“New” white grubs

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 47-50

Control of the garden chafer Phyllopertha horticola with GRANMET-P, a new product made of Metarhizium anisopliae

Barbara Pernfuss1, Roland Zelger2, Roberto Kron-Morelli3, Hermann Strasser1 1 Institute of Microbiology, Leopold-Franzens-University Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria; 2 Research Center for Agriculture and Forestry Research Laimburg, 39040 Auer / Pfatten, Italy; 3 Agrifutur s.r.l., Via Campagnole 8, 25020 Alfianello (BS), Italy

Abstract: In summer 2002 four Metarhizium anisopliae isolates, NEMA-GREEN® (Heterorhabditis bacteriophora) and DURSBAN-2E® (chlorpyrifos) were tested in a pilot study in the golf course Igls- Rinn (Austria) which was heavily infested by Phyllopertha horticola. The experimental design of the efficacy study was based on EPPO-standards. Infestation rate was 500 larvae per m2 on the average. Eight weeks post treatment the pest was reduced by 35 % in the plots where the chemical insecticide was used. NEMA-GREEN® caused 19 % mortality of larvae within the same span of time. A slightly lower efficacy between 14 % and 16 % was evaluated for the fungal products based on M. anisopliae. One year afterwards a granular formulation of M. anisopliae as well as NEMA-GREEN® and DURSBAN-2E® were extensively applied on the fairways of the same golf course. The long term effect of these treatments were designated greatly satisfactorily by the staff of the golf course, as in 2004 no harms were observed in the treated areas of the golf course, whereas severe damages caused by P. horticola larvae were observed in the surrounding. Assessment of the M. anisopliae density in soil permited to conclude that the efficacy and the persistence of the fungal product is granted. Up to 1.7 x 105 colony forming units of M. anisopliae per gram soil dry weight were re-isolated from treated fairways. In May 2004 the most aggressive strain of M. anisopliae was brought into registration by the company F. Joh. Kwizda GmbH. The product will biologically fight P. horticola larvae and is named GRANMET-P. We assume a temporary admittance for GRANMET-P still in this year and will force our efforts to subdue the P. horticola calamity in an ecologically and economically friendly way.

Keywords: Metarhizium anisopliae, Phyllopertha horticola, Heterorhabditis bacteriaphora, EPPO- standards, registration, GRANMET-P.

Introduction

For the last years severe harms caused by the garden chafer Phyllopertha horticola (Coleoptera, Scarabaeidae; L.) have considerably increased in amenity areas, pastures and orchards all over Europe. Phyllopertha horticola developes in a one-year life cycle to the adult garden chafer which causes damage by feeding in orchards, deciduous trees, roses and other blooming bushes. In early summer, after the eggs have been laid, the larvae (three stages) feed on roots of grasses, cultivated plants and stock of trees. In mountainous ambiance, damage to pastures entails the risk of serious soil erosion. Damage caused by Phyllopertha horticola is estimated to be hundreds of millions of Euros each year and the pest problem is increasing. Most parts of Europe are strongly concerned.

47 48

The goal of this study was to demonstrate that M. anisopliae is efficacious to fight P. horticola not only under laboratory conditions but also outside - under field conditions. The study was conducted in Austria and was based on the EPPO-Standard PP1/152 (design and analysis of efficacy evaluation trials) and on the German proposal for an EPPO-guideline for testing insecticides on grubs in arable crops (I.22, October 1999).

Material and methods

Experimental design and control agents For the pilot study a full randomised block design was staked off according to EPPO standard [PP 1/152]; two separate fields with two plots for each treatment were used. One single plot ® measured 4 m². Seven different treatments were tested: 1) untreated blank, 2) DURSBAN- 2E , ® 3) NEMA-GREEN , 4) granular formulation (GF) of M. anisopliae Bipesco 6, 5) GF of M. anisopliae - Eric Schweizer Samen AG, 6) GF of M. anisopliae - Andermatt Biocontrol and 7) wettable sporepowder M. anisopliae Bipesco 5. One year after the pilot study the most promising GF of M. anisopliae as well as NEMA- ® ® GREEN and DURSBAN-2E were extensively applied on the fairways of the same golf course.

Figure 1. DURSBAN 2E®: Fairways 12 and 14; GRANMET-P: Fairways 1, 2, 5, 13 and 16; NEMA-GREEN®: Fairways 7, 8, 9 and 11.

The granular formulation of M. anisopliae was applied with a slit seeder to a depth of 2 to 4 cm in a concentration of 50 kg per hectare in August 2003. Fairway No. 16 was treated twice with GRANMET-P (same dosage); in summer 2002 and 2003. NEMA-GREEN® and DURSBAN 2E® were washed into the soil in summer 2003 according to the recommendations of the respective producer.

Evaluation of infestation levels of the garden chafer and effectiveness of insecticides In the pilot study infestation levels of the garden chafer were determined by means of spade sampling as follows: control and test plots (each 4 m2), were assessed by digging three square holes per plot and sampling (20 x 20 cm wide and 20 cm deep) and counting the larvae. The number of larvae was recorded before treatment and 8 weeks post treatment. 49

The effect of extensive treatments were judged by monitoring damage symptoms. This work was carried out by the staffers and labourors of the golf course for the period of more than one year.

Isolation of Metarhizium anisopliae from soil Soil samples were taken to a depth of 10 cm by using a sampling auger, mixed, air-dried and sieved through a 2 mm sieve. Ten gram sub-samples (three replicates) were added to 40 mL 0.1 % (v/v) Tween-80, shaken at 150 rpm for 30 min and then treated in an ultrasonic bath for 30 s. Sabouraud-dextrose agar plates selective for M. anisopliae were inoculated with 50 µL of these soil suspensions and dilutions thereof and were incubated for 14 days at 25 °C and 60 % RH (four replicates per sub-sample). Colonies of M. anisopliae are given as colony forming units (cfu) per gram soil dry weight.

Results and discussion

A quantity of 500 Phyllopertha horticola larvae per m2 on average was assessed in the golf course area in summer 2002. This high infestation rate led to severe damages of the lawn and entailed additional harm by birds, fox and badger, which dug in the soil to feed larvae. In our pilot project four Metarhizium anisopliae isolates, Heterorhabditis bacteriophora ® and DURSBAN-2E (chlorpyrifos) were tested in an efficacy study based on EPPO-standards. Eight weeks post treatment the pest was reduced by 35 % in the plots where the chemical ® insecticide was used. NEMA-GREEN caused 19 % mortality of larvae within the same span of time. A slightly lower efficacy (14 % – 16 %) was evaluated for the fungal products based on M. anisopliae eight weeks post treatment (Strasser et al., 2005). These relatively low efficacy values were attributed to the thick artificial lawn of the golf course that prevented sufficient dispersal of control products and irrigation of soil.

1000000

100000

10000

1000

[cfu per g soil dry weight] 100

10 M. anisopliae 1 untreated control one application two applications GRANMET-P

Figure 2. Abundance of M. anisopliae BCAs in soil samples taken from 0 to 10 cm depth (mean + STD, n = 8; colony forming units - cfu).

In spring 2003 the density of M. anisopliae was determined as an indirect parameter to estimate infectivity of BCA propagules. Ferron (1979) reported a threshold concentration of > 2 x 104 spores g-1 soil dry weight necessary to ensure epidemic levels in pastures. More than this threshold density was brought into soil with two applications of GRANMET-P (Figure 2). Persistence data showed that M. anisopliae is indigenous in Rinn. From untreated soil it was isolated at a concentration of 2.7 x 102 spores g-1 dry weight. In treated plots and fairways the density of spores remained stable or increased. One year post treatment 6 x 103 spores g-1 5 -1 soil dry weight were re-isolated when GRANMET-P was applied once, and 1.7 x 10 spores g soil dry weight when the product was applied twice (Figure 2). These results verify the augmented propagules of GRANMET-P to be persistent. There should be enough infectious propagules in soil to control the soil dwelling stage of P. horticola. Actually, the long term effect and the efficacy of these treatments were designated greatly satisfactorily by the staff of the golf course, as in 2004 no harms were observed in the treated areas, whereas severe damages caused by P. horticola larvae were observed in the surrounding. After the pilot study our expection was that M. anisopliae will reduce the garden chafer population in the same manner as B. brongniartii does for Melolontha spp. (Inglis et al.; 2001). This expection was affirmed by the last two years data, and we will force our efforts to subdue the P. horticola calamity by the registration and application of GRANMET-P.

Acknowledgements

Supported by the European Commission, Quality of Life and Management of Living Resources Programme (QoL) Key Action 1 on Food, Nutrition and Health (Contract n°QLK1-CT-2001-01391)" and by the F. Joh. Kwizda GmbH - Austria.

References

Strasser, H. 2000: Searching for alternative biological pest control agents against Phyllo- pertha horticola (L.). – IOBC/wprs Bulletin 23(2): 23-27. Ferron, P. 1979: Study of the virulence of some mutants of Beauveria brongniartii (Beauveria tenella) fungi infecting Melolontha melolontha. – J. Invertebr. Pathol. 34: 71-77. Strasser, H., Zelger, R., Pernfuss, B., Längle, T., Seger, C. 2005: EPPO based efficacy study to control Phyllopertha horticola in golf courses. – Papierok, B. (ed.). IOBC/wprs Bulletin 28(3): 189-192. Inglis, G.D., Goettel, M.S., Butt, T.M. & Strasser, H. 2001: Use of hyphomycetous fungi for managing insect pests. – In: Fungal Biological Control Agents: Progress, Problems & Potential, eds. Butt, Jackson, and Magan: 23-69.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 51-55

Timing of nematode application to control white grubs (Scarabaeidae)

Arne Peters1, Henk Vlug2 1 E-nema GmbH, Klausdorfer Str. 28-36, D-24223 Raisdorf, Germany 2 Insectconsultancy, Fluitekruidlaan 74; NL-3925 SG, Scherpenzeel, The Netherlands

Abstract: Heterorhabditis bacteriophora is currently applied on more than 200 ha per year in Germany, The Netherlands, Belgium, Denmark and Austria. It is generally advised to apply nematodes from the end of July to the mid of September, when the third instar larvae, which were most susceptible in laboratory studies, are present. A later application in September, although successful in many cases, bears the risk of failure due to dropping soil temperatures and hence reduced infectivity of H. bacteriophora. There is some evidence that applying nematodes earlier can successfully suppress P. horticola populations, even though the grubs are not yet present in the soil. An application in May against grubs of Aphodius contaminatus was sufficient to also control P. horticola. In another experiment, nematodes applied on June 22nd gave better control of P. horticola than those applied at August 9th. This long term effect might be the result of the excellent persistence of H. bacteriophora in the field which was also demonstrated in a strawberry field. Higher soil moisture in spring might be advantageous for nematode reproduction and persistence. For the grub Hoplia philanthus with a two years life cycle, application in spring should be preferred to the August treatment. For principally non- susceptible grub species the timing of application at short periods of higher susceptibility can be the only option o control them: By carefully monitoring the moulting from the first to the second stage larvae and applying H. bacteriophora during that time period, grubs of Amphimallon solstitiale were successfully controlled with H. bacteriophora on two occasions.

Keywords: entomopathogenic nematodes, Heterorhabditis, Steinernema, Hoplia philanthus, Amphi- mallon solstitiale, Phyllopertha horticola

Introduction

The timing for releasing biological control agents is crucial for its success. For applying entomopathogenic nematodes in the field, constraints limiting the time window for application are the soil moisture, which needs to be sufficiently high, a supporting soil temperature, the persistence of the nematodes in the soil and the availability of susceptible stages of the target insect. This paper critically reflects the current recommendations for application timing of Heterorhabditis bacteriophora against different grub species in turf.

Material and methods

Field trials against the garden chafer were performed using the product NEMA-GREEN containing the nematode H. bacteriophora EN01, a mix of 10 New-Jersey isolates and 3 isolates from Germany. Nematodes were applied at a rate of 0.5 million in 0.2 l per m² followed by a post-irrigation of 1 l/m². Two trials were performed against the garden chafer, P. horticola. In the first trial a private lawn near Schwerin (Germany) was treated on June 22nd and on August 9th. The test layout was a complete randomized block design with 4 plots of 2 x 2 m per treatment (June-treatment, August treatment, untreated control). The treatment effect was evaluated on October, 18th by digging out two samples of 25 x 25 cm from each plot. The second trial was performed on a golf-course near Neumünster, Schleswig-Holstein

51 52

(Germany). The first treatment was done on July 18th, the second treatment on September 10th. Evaluation was done on September 25th and 4 weeks after the late treatment by taking out 10 soil cores (10cm diameter and 5 cm depth) per plot and counting the grubs per core. Plot size was 2 x 2m. Since the treatments were done on two different locations on the golf- course, there were plots with untreated controls on each location. There were 8 plots per treatment for the early application and 6 plots per treatment for the late application. In the late application there was also an application done with half the nematode dosage (0.25 million / m²). Larvae of Amphimallon solstitiale were treated at the first larval moult on two occasions. To hit the first moult, about 20 first instar larvae were sampled and checked daily for whether they had moulted or not. When 20% of the larvae had moulted, the field was treated with 0.5 million H. bacteriophora (see above). To enhance nematode penetration through the thatch, a wetting agent (Dispatch from Aquatrols) was added at the recommended rate of 0.7% to the spray-suspension. On the first occasion the treatment was done on 2 ha on a golf-course near Bad Bentheim, Germany on August 1st in 2002. The treatment was done in a rainy week so post-irrigation was not necessary. The effect was checked by evaluating damage by birds, which usually occurs in September, and by occasionally taking samples to look for grubs in September 2002 and in 2003. On the second occasion a soccer field (7000 m²) near Zwolle, The Netherlands, was treated on August 20th, 2003. Post-treatment irrigation was done with 2 l/m². The effect was evaluated by looking for birds damaging the turf and by taking occasional soil samples. There were no untreated controls for these treatments against A. solstitiale.

Results and discussion

Application timing of Heterorhabditis bacteriophora against Phyllopertha horticola In the trial on private lawns near Schwerin the effect of a treatment on June 22. was superior to the treatment on August 9 (Fig. 2). Likewise the earlier application in the trial in 2003 was superior to a treatment in September (Fig. 3). Since the average number of grubs in the untreated control plots did not differ significantly between the two locations the data were pooled in Fig. 3. Interestingly, grub mortality did not differ between the two different nematode doses applied, indicating that 0.25 million / m² is sufficient for controlling P. horticola.

May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Beetle Egg 1st instar 2nd instar 3rd instar Pupa Fig. 1: Life cycle of Phyllopertha horticola (after Sulistyanto et al, 1996).

Currently, the recommended application time for H. bacteriophora against P. horticola is from end of July to the mid of September. It is based on the observation, that third instar larvae, which occur end of July, are most susceptible (Smits, et al., 1994) (Fig. 1). Dropping soil temperatures and the retarded action of the nematodes define the end of the application

window. Field trials with earlier applications have only been done sporadically but with surprisingly good effects. An application before beetle flight in May resulted in 70 and 83% mortality of P. horticola with H. megidis or H. bacteriophora, respectively (Sulistyanto & Ehlers, 1996). Since the golf-course was infected with the dung beetle Aphodius contaminatus in May and 40% of these larvae were controlled, it was concluded that the nematodes had propagated on these hosts and therefore persisted until the third instar of P. horticola appeared. The good effect of the June treatment in trial one was probably also due to the nematode propagating on hosts other than P. horticola.

4 ±1.96*Std. Err. ±1.00*Std. Err. 3 Mean

Number of grubs per sample (n=8) -1 22. June untreated control 09. August

Fig. 2: Number of Phyllopertha horticola in 25 x 25 cm samples after applying Heterorhabditis bacteriophora (0.5 million/m²) to amenity turf on two points of time. Evaluation was done on October 18th (unpublished results, Plant Protection Service Schwerin, Germany).

11 ±1.96*Std. Err. ±1.00*Std. Err. 9 Mean

1 Number of grubs per sample (n=8)

-1 untreated Sep. 0.5 mill./m² Sep. 0.25 mill./m² July 0.5 mill./m²

Fig. 3: Number of Phyllopertha horticola in 10 cm diameter soil cores after applying Heterorhabditis bacteriophora (0,5 million/m²) on a golf course in July and in September. Evaluation was done after 10 weeks for the July-treatment and after 4 weeks for the September treatment (unpublished results, e-nema GmbH).

Application timing of H. bacteriophora against Amphimallon solstitiale The life cycle of the June beetle, A. solstitiale, is similar to that of H. philanthus (Fig. 4, 5). The larvae of this species are, however, not very susceptible to entomopathogenic nematodes (Smits, 1990). Even nematode isolates with high efficacy against grubs failed to control this pest with the exception of S. scarabaei, which affected this species to a similar level than P. horticola in the laboratory (Fischer, et al., 2003). Attempts to cultivate this nematode species outside insects were, however, unsuccessful. The application of H. bacteriophora during the first larval moult, however, gave excellent results. No further grub or bird damage was observed nor on the soccer field in Zwolle neither on the golf-course in Bad Bentheim in the years of treatment and in the following year. Hitting this difficult grub species at the right time can therefore be a control strategy. If a less synchronous population is to be controlled, two treatments should be done in an interval of 1 to 2 weeks.

Life stage J J A S O N D J F M A M J J A S O N D J F M A M J Beetle Egg 1st instar 2nd instar 3rd instar Pupa

Fig. 4: Life cycle of Amphimallon solstitale.

J J A S O N D J F M A M J J A S O N D J F M A M J Beetle Egg 1st instar 2nd instar 3rd instar Pupa

Fig. 5: Life cycle of Hoplia philanthus (after Ansari, et al., 2004).

Application timing of H. bacteriophora against Hoplia philanthus The Welsh chafer, Hoplia philanthus, has become a pest in recent years in Germany, The Netherlands and Belgium. The life cycle was studied in detail by Ansari et al., 2004. The grubs can be found in the upper soil layer in March and are then welcome by birds, which destroy the turf while preying on the grubs. The beetles are flying in mid-June. Due to the two years life cycle the period for nematode application ranges from August in the year of flight to May two years after flight (except for the winter months with too low temperatures for nematodes). In this case, the spring months in the year after flight are the preferred period of treatment since soil moisture is usually high, the soil temperature is sufficient and the nematodes can propagate on the grubs and cause highest pest reduction. The susceptibility of different instars of H. philanthus against H. bacteriophora has not been tested but since turf damage does usually not occur in the first year, mainly 3rd instars were treated in the field (Fig. 5). On a sports field near Bremen a treatment with H. bacteriophora in March 1999 against 2nd instar larvae stopped further grub damage (data not published). In sufficiently humid

summers, like 2004, treatments in July against 3rd instar larvae were successful with 60% control after surface application of H. bacteriophora at 0.25 million/m² (M.A. Ansari, personal communication). With treatments in October, Ansari (2004) achieved 30 to 60% grub reduction 3 weeks after nematode application. Similarly, infected grubs were observed as soon as 10 days after application in the beginning of October on a sports field near The Hague.

Conclusions

The timing of nematode application can be widened if the nematodes propagate on alternative hosts before the susceptible stage of the grub appears. For difficult grub species, the search for susceptible life stages, even if they only occur during a few days of the whole life cycle merit more attention.

References

Ansari, M.A. 2004: Biological control of Hoplia philanthus (Coleoptera: Scarabaeidae) with entomopathogenic nematodes and fungi. – PhD-Thesis, University Ghent, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschapen, Gent: 162 pp. Ansari, M.A., Casteels, H., Tirry, L. & Moens, M. 2004: Life cycle of the white grub Hoplia philanthus F. (Coleoptera: Scarabaeidae), a new and severe pest of turf and ornamentals in Belgium. – Journal of Economic Entomology, submitted. Fischer, R., Strauch, O., Koppenhöfer, A. & Ehlers, R.-U. 2003. Steinernema scarabaei, a highly potent antagonist for Melolontha melolontha, Amphimallon solstitiale and Phyllopertha horticola. – In: 9th European Meeting of the IOBC/WPRS Working Group "Insect Pathogens and Entomoparasitic Nematodes". 23-29 May, 2003, Schloss Salzau, Germany, pp. 28. Smits, P. 1990. Control of white grubs, Phyllopertha horticola and Amphimallon solstitialis (Coleoptera: Scarabaeidae), in grass with Heterorhabditid nematodes. – In: The Use of Pathogenes in Scarab Pest Management. T.A. Jackson and T.R. Glare (eds.). Intercept Publisher Ltd., Andover: 229-235. Smits, P.H., Wiegers, G.L. & Vlug, H.J. 1994. Selection of insect parasitic nematodes for biological control of the garden chafer, Phyllopertha horticola. – Entomol. exp. appl. 70: 77-82. Sulistyanto, D. & Ehlers, R.-U. 1996. Efficacy of the entomopathogenic nematodes Heterorhabditis megidis and Heterorhabditis bacteriophora for the control of grubs (Phyllopertha horticola and Aphodius contaminatus) in golf course turf. – Biocontrol Sci. Technol. 6: 247-250.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 57-61

Metarhizium anisopliae for white grub control in Nepal

Yubak Dhoj GC1, Siegfried Keller2 1 Institute of Agriculture and Animal Sciences, Rampur, Chitwan, Nepal 2 Agroscope FAL Reckenholz, CH.8046 Zürich, Switzerland

Abstract: With an attempt to develop microbial control of white grubs in Nepal, series of experiments were carried out in a complementary way from the mid of 2002 at Tribhuvan University, Institute of Agriculture and Animal Sciences (IAAS), Rampur, Chitwan, Nepal. The first exploratory study proved that, insect pathogenic fungi (Metarhizium anisopliae and Beauveria bassiana) are associated with the white grubs and soils of Nepal. A total of 70 isolates of M. anisopliae and 8 isolates of B. bassiana were recovered between 2003 and 2004. Further works on isolation, re-isolation, culturing into selective medium demonstrated that, these fungi can be detected by Galleria larvae and also may be isolated from infected grub’s cadaver and by plating soil suspension on selective medium. Bioassays with M. anisopliae were undertaken to determine time mortality and dose mortality responses. In the screening study, initially 70 isolates were tested at a concentration of 107 conidia/ml, 8 isolates gave over 80% infected grubs, 65 isolates gave over 50-60% infected grubs and rest of the isolates had a low pathogenicity. The LT50 varied between 2-9 weeks, 12 isolates were highly virulent with an LT50 of 2-4 weeks, 34 isolates had a moderate virulence with an LT 50 of 5-6 weeks and 22 isolates had a low virulence. The most virulent strains were further assessed with 109spores/ml, 105spores/ml, 103 spores/ml and water treatment. Mass production was carried out in autoclavable polycarbonate bags of Swiss and Nepali origin. Marked differences were found in the quality of the fungus producti. That produced in Nepali bags was heavily contaminated irrespective of the test substrate, while 84-94% of the material originating from the Swiss bags were colonized only with M. anisopliae. The study has indicated the opportunity for controlling white grubs with M. anisopliae in Nepal.

Keywords: Entomopathogenic fungi, Metarhizium anisopliae, microbial control, white grubs, Nepal

Introduction

White grubs are economically important pest insects in Nepal, however, a pest management strategy is lacking in the country (GC and Keller, 2002). The soil inhabiting larval stages causes poorly quantifiable losses in the crops. Infestations have been reported across the country and incidences are increasing every year. Control is mainly targeted with the use of chemical pesticides and this trend has been increasing in recent years. Farmers use different types of insecticides including organochlorines, organophosphates and carbamates. Despite all efforts neither traditional methods nor chemical measures are effective in reducing the white grub infestations in Nepal. Some organizations and workers in Nepal have reported pesticide misuses and possible hazards at the farm level. In order to avoid detrimental effects on humans, animals and the environment, the Sustainable Soil Management Programme (SSMP) funded by Swiss development organizations (SDC, Helvetas, Intercooperation) initiated a project with the goal to develop alternative control methods using indigenous antagonists with focus on entomopathogenic fungi.

57 58

Material and methods

Collection of fungal isolates White grubs were collected from farmers’ fields in Gunganagar and Mangalpur of Chitwan district. These sites lie in the south-central region of Nepal at an altitude of 290 m asl. They are predominantly used for the cultivation of maize and millet. The soils are sandy-loamy. After collection the grubs were kept individually in small boxes, fed with slices of potatoes according to their consumption and the substrate was exchanged when needed. Insect pathogenic fungi were isolated from infected grubs as well from soil using the Galleria bait method (GBM) (Zimmermann, 1986) and by plating soil suspension on selective medium (Keller et al, 2003).

Production of fungus material Insect pathogenic fungi were produced in Petri dishes with selective medium adapted from Strasser et al. (1997) with the following composition and preparation: 10 gm peptone, 20 gm glucose, 18 gm agar, all dissolved in 1 liter distilled water and autoclaved at 120°C for 20 minutes. At a temperature of 60°C 0.6 g streptomycin, 0.05 g tetracycline and 0.05 g cyclohexamide previously dissolved in distilled, sterile water and 0.1 ml Dodine were added. The fungus was incubated at 27±2°C and 80±5 %. After 12-15 days, the conidia were harvested either by scrapping off with a loop or by washing off with 0.1% Tween 80. The conidia were suspended in 0.1% Tween 80. The concentration was determined by counting the conidia in a Thoma haemocytometer.

Bioassays with white grubs Time mortality and dose mortality studies were carried out during the period of 2003/04 at the insect pathology laboratory of IAAS, Rampur, Chitwan, Nepal. In the first experiment, 70 different strains of M. anisopliae were assessed with dose of 107 spores/ml. For each strain 30 white grubs of the second instar were dipped into the spore suspension for five seconds (Goettel & Inglis, 1997). Excess liquid was dropped off and the larvae were placed individually in 100 ml plastic boxes half filled with sterile soil and observed in the experimental room at a temperature of 22-24°C. One control was left untreated; the larvae of the other control were dipped in water. The larvae were fed with potatoes and checked every third day for ten weeks. With the most virulent strains dose mortality response studies were conducted with four different doses (109, 105, 102, spores/ml and control) using again the dipping method and 30 larvae per concentration. The white grubs used in the studies were second instars larvae and originated from Chitwan. Since the grub species was unknown, some representative samples of larvae were preserved in ethanol and some other larvae were made adults for later identification.

Mass production of insect pathogenic fungi Mass production was carried out on peeled barley grains in plastic bags. The quality of the fungus production was studied comparing bags of Swiss and of Nepali origin. Swiss bags are polycarbonate bags with a size of 30 x 50 cm, whereas Nepali bags were the same as used to carry grocery items from the markets. The bags were filled with 0.5 kg grains with 250 ml water and autoclaved twice with an interval of 24 hours at 120 psi for 20 minutes. When cooled down. 100 ml fungal suspension was added to each bag and then incubated at a temperature of 22-24°C for 21 days as described by Keller (2004b).

Results and discussion

Collection of fungal isolates Entomopathogenic fungi were isolated from soils or from white grubs in all investigated areas. M. anisopliae is distinctly more frequent than B. bassiana. (Tab. 1). The natural infection of white grubs is low, however, M. anisopliae is wide-spread in soils in Nepal.

Table 1 Origin of the insect pathogenic fungi isolated 2003-2004 from different localities in Nepal. GBM: Galleria bait method; SM: selevtive medium.

Fungus Geographic Number of Isolated from species origin isolates White Soil/GBM Soil/SM grubs M. anisopliae All 70 24 41 5 Chitwan 25 10 15 0 Parbat 35 7 23 5 Tanahun 7 4 3 0 Gaindakot 3 3 0 0 B. bassiana All 8 2 6 0 Chitwan 2 2 5 0 Parbat 1 0 1 0 Tanahun 5 0 0 0

Bioassays with white grubs The time mortality study indicated that 8 isolates gave over 80% infected grubs, 65 isolates gave over 50-60% infected grubs and five isolates had a low pathogenicity. Based on infection rates, fungus strains such as M1, M6, M48, M18, and M50 were found aggressive compared to rest of the strains. The LT50 varied between 2-9 weeks, 12 isolates were highly virulent with an LT50 of 2-4 weeks, 34 isolates had a moderate virulence with an LT 50 of 5- 6 weeks and 22 isolates had a low virulence. The isolates M6 and M1 had the lowest LT50 and were selected as candidates for the biological control of white grubs. The white grub species used in both the experiments were later identified as Maladera spp.

Table 2: Pathogenicity of different strains of M. anisopliae against third instar larvae of Maladera sp. at 22-240C at a concentration of 1*107 spores/ml.

Strain Fungus origin % mortality % mycosis M1 White grub 64.2ab 38.3ab M2 White grub 67.5a 40.8a M6 Soil/GBM 59.2b 26.7b M18 Soil/GBM 58.3b 29.2ab M48 White grub 59.2b 30.8ab LSD (p=0.01) 6.953 10.92 SEM 2.357 2.713 CV% 10.81 23.14 Figures in column followed by same letter are not significantly different at p<0.010 by DMRT

Among the 70 isolates five virulent ones were further assessed with different doses (table 2 and 3). This study showed that M2 was significantly more virulent (P = 0.001) than the other isolates. Table 3 shows a low slope of the dose-mycosis responses.

Table 3: Pathogenicity of different doses of strain M2 against third instar larvae at 22-240C

Strain Concentration % mortality % mycosis M2 109spores/ml 79.3a 52.00a 105spores/ml 72.7ab 45.33a 102spores/ml 70.0b 35.33b untreated 24.7c 0c LSD (p=0.010) 8.483 9.764 SEM 2.108 2.427 CV% 10.81 23.14 Figures in column followed by same letter are not significantly different at p<0.010 by DMRT

Mass production The comparison between Swiss and Nepali bags showed marked a difference which is expressed in the quality (Table 7). The fungus colonized barley grains originating from Nepali bags were heavily contaminated irrespective of the test substrate (wet filter paper or wet peat), while 84-94% of the material originating from the Swiss bags were colonized only with M. anisopliae. However, M. anisopliae rarely covered the whole surface. When the grains were placed on filter paper, the uncolonized area remained uncolonized, but when they were placed on peat, the gaps were often colonized by contaminants which had the tendency to overgrow M. anisopliae. To avoid that this happens in the soil we shall improve the production method in order to get M. anisopliae growing on the whole surface. The contaminants also differed. In the Nepali bags most contaminants were yeasts with the typical smell, while the contaminants in the Swiss bags were Penicillium-like fungi. These marked differences between the two bag types are attributed to material characteristics. It is assumed that the Nepali bags are air-tight while the Swiss bags allow some gas exchange and evaporation of water.

Table 7 Quality of the fungus grains produced in autoclavable plastic bags of Swiss and Nepali origin. M.a. = Metarhizium anisopliae; NP: Nepal; CH: Swiss; FP: filter paper.

Isolate Date set Origin Sub- # grains % grains % grains with % grains Number up of bag strate tested with pure M.a. present with M.a. growth contaminants M1 28.9.04 NP FP 65 0 0 100 M1 ibid CH FP 66 94 100 6 M1 ibid NP peat 52 0 0 100 M1 ibid CH peat 63 92 100 8 M58 22.10.04 NP FP 62 0 0 100 M58 ibid CH FP 67 84 100 16 M58 ibid CH peat 70 90 97 10

Discussion

During this study the entomopathogenic fungi M. anisopliae and B. bassiana were recorded from Nepal for the first time. Earlier GC and Keller, (2003) demonstrated that especially M. anisopliae is widely distributed in Nepal, in cropland as well as in grassland. However, natural infection rates of white grubs proved to be low. The bioassays with the fungal isolates demonstrated that virulence differed among the strains and showed that isolates from white grubs are not a priori more virulent than isolates originating from soils. The Galleria bait method proved to be a suitable method to detect the presence of M. anisopliae and B. bassiana in the soils. The bioassays further demonstrated that more strains of the known insect pathogenic fungi can be found with good potential for white grub control. Although further white grub pathogenic species like B. brongniratii may be found in future investigations in Nepal, the efforts to develop a mycoinsecticide are focused on M. anisopliae. The trials to mass produce M. anisopliae in autoclavable plastic bags demonstrated the feasibility but also the importance of the bag quality. Good fungus quality was only achieved with bags of Swiss origin and used there for many years (Keller, 2004b). Additional efforts must be undertaken to develop a production system which is based only on materials available on the national market.

Acknowledgements

We are greatly indebted to Helvetas for funding the project and to Intercooperation/SSMP for project initiation and local support. We thank Prof. Dr. Peter Nagel, University of Basel, Basel, Switzerland, and Dr. Dirk Ahrens, Deutsches Entomologisches Institute, Germany for identifying white grub species.

References

GC, Y.D. & Keller, S. 2002: Associations of fungal pathogens with white grubs. – In: Integrated pest management in Nepal (Ed.: F.P. Neupane). Himalayan Resources Institute Kathmandu: 37-46. GC, Y.D. & Keller, S. 2003: Towards microbial control of white grubs in Nepal with entomopathogenic fungi. – Bull. Soc. Ent. Suisse 76: 249-258. Goettel, M.S. & Inglis, G.D. 1997: Fungi: Hyphomycetes. – In: Manual of Techniques in Insect Pathology (Ed. L. Lacey), Academic Press: 213-249. Keller, S. 2004a: Bekämpfung von Maikäfer-Engerlingen mit dem Pilz Beauveria brongniartii in der Schweiz. – Laimburg Journal 1: 258-264. Keller, S. 2004b: Versuche zur Bekämpfung von Maikäfer-Engerlingen durch Bodenbehand- lungen mit dem Pilz Beauveria brongniartii (Sacc.) Petch. – Laimburg Journal 1: 265- 269. Keller, S., Kessler, P. & Schweizer, C. 2003: Distribution of insect pathogenic soil fungi in Switzerland with spezial reference to Beauveria brongniartii and Metarhizium anisopliae. – BioControl 48: 307-319. Strasser, H., Forer, A. & Schinner, F. 1997: Development of media for the selective isolation and maintenance of virulence of Beauveria brongniartii. – Proc. 3rd Internat. Workshop Microbial Control of Soil Dwelling Pests: 125-130. Zimmermann, G. 1986: The Galleria bait method for detection of entomopathognic fungi in soil. – J. appl. Ent. 102: 213-215.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 63-69

Screening and selection of virulent isolates of the entomopathogenic fungus Beauveria brongniartii (Sacc.) Petch for the control of scarabs

A. B. Hadapad, A. Reineke and C. P. W. Zebitz University of Hohenheim, Institute of Phytomedicine, Department of Entomology, D-70593 Stuttgart, Germany

Abstract: The melolonthine scarabs, Melolontha melolontha L. and Holotrichia serrata L. are the most important pests of forestry and agricultural system in Europe and the Indian union, respectively. The deuteromycete Beauveria brongniartii (Sacc.) Petch is found to be the most effective natural control agent of M. melolontha. In order to select the most virulent isolates against these scarabs in the Indian union, ten B. brongniartii isolates, obtained from different geographical regions and hosts, were characterised and tested against M. melolontha and H. serrata larvae. The results demonstrated that the size of conidia ranged from 1.94 – 4.39 x 0.92 – 2.29 µm, spore production on Sabouraud Dextrose Agar after 14 days incubation at 25°C ranged from 7.54 x 108 to 2.2 x 109 conidia/cm2 and the highest germination rate of 100 % was recorded at 25°C after 20 h in all isolates except for the isolates ARSEF 1360 and ARSEF 2660. In bioassay studies, all isolates of B. brongniartii with a concentration of 2 x 107 conidia/ml were found to be pathogenic to third instar larvae of M. melolontha and H. serrata with differences in their virulence; three isolates Bbr 50, Bbr 23 and ARSEF 4384 for M. melolontha and two isolates ARSEF 4384 and ARSEF 2660 for H. serrata were shown to be more pathogenic in terms of total mortality, onset of mortality and mycosis. The importance of these characteristics in selecting fungal isolates for further investigations and potential use of these isolates for the management of H. serrata is discussed.

Keywords: Melolontha melolontha, Holotrichia serrata, entomopathogenic fungi, Beauveria brongniartii, virulence, biological control

Introduction

The European cockchafer, Melolontha melolontha L. and the white grub, Holotrichia serrata L. (Coleoptera:Scarabaeidae) are the most important pests of forestry and agricultural systems in Europe and the Indian union, respectively (Keller et al., 1986; Zimmermann, 1998; Vyas et al., 1990). The adult insects are popularly known in Europe as chafer beetles, May or June beetles. White grubs are pests of national importance in India and are a serious constraint to the production of rainy season crops. In endemic areas, the damage to many crops ranges from 20-100% and the presence of one grub/m2 may cause 80-100% plant mortality (Yadava and Sharma, 1995). The entomopathogenic fungi Beauveria brongniartii (Sacc.) Petch (Deuteromycotina:Hyphomycetes) has emerged as an excellent biological control agent for soil-inhibiting scarabs (Reineke and Zebitz, 1996 and Keller et al., 2000). Previous studies have shown that the physiological characteristics and enzyme production of entomopathogenic fungi are related to their virulence (Bidochka and Khachatourians, 1990) or to other characteristics such as conidia viability, speed of germination, infectivity and spore production in response to environmental temperature, relative humidity and UV light and also influence the efficacy of a fungal isolate as a microbial control agent (Milner et al., 1997). In this study, we assessed several characteristics of ten B. brongniartii isolates obtained from wide geographical and host origins with the aim to select highly virulent isolates for efficient biocontrol agent against scarabs.

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Materials and methods

Source of fungal pathogens and insects Ten fungal isolates were obtained from Germany (BBA, Darmstadt) and USDA-ARSEF collection (Ithaca, NY). All isolates were grown and maintained on Sabouraud Dextrose Agar (SDA) and incubated in the dark for 14 days at 25°C. For tests on conidia morphology, conidial suspensions of all isolates were made by scraping conidia with a spatula from 14 day old cultures in 0.01 % Tween 80. Spores were then added to a sterile plastic vial and vortexed for 5 minutes to loosen the conidia. The spore concentration in the resulting suspension was determined using a haemocytometer and the required conidial concentration was accordingly prepared for all the tests. Larvae of M. melolontha and H. serrata were field collected in Baden-Württemberg (Karlsruhe about 150 km from the region of the Kaiserstuhl), Germany and Andhra Pradesh, India, respectively. The grubs were kept at 19°C in the laboratory in natural soil with carrot slices as food before using for the actual bioassays. The grubs were incubated for 1-2 weeks and only healthy third instar grubs were used for the bioassays.

Fungal characteristics For the measurement of the fungal conidia, an ocular micrometer 12.5x (Carl Zeiss) was used. About 20 spores were measured for each isolate; the procedure was repeated thrice with different cultures. Stage micrometer 5+100/100 mm was used to calibrate the ocular micrometer. The speed of germination was determined as the time required for 100 % germination of conidia. For the assay, 24-multiwell plates were used. Each well containing 300µl of a SDA medium amended with 0.08 % Streptomycin. The spore suspension was prepared as described above, 50µl of conidial suspension of 1 x 106 conidia/ml was pipeted and distributed with a miniature spatula in the well of the 24-microwell plate and incubated at 25°C. For each isolate three replications were made. Percent germination was determined using the invert microscope (Carl Zeiss) starting from 4, 6, 8, 10, 12, 18 and 20 h. Only spores with germ tubes longer than their width were considered to have germinated. Three replications were made per treatment in a separate well, with 3 counts of 100 conidia per isolate. The amount of conidia produced by each isolate was estimated using the method described by Varela and Morales (1996). Conidia suspension was prepared as described above; 100µl of conidial suspension (1 x 106 conidia/ml) was plated on SDA overlaid with cellophane film to prevent hyphae penetrating the agar in a 9 cm diameter Petri dish. After incubating at 25°C for 14 days, the conidia were harvested and collected in 1 ml of sterile double distilled water with 0.01 % Tween 80. The conidial suspension was vortexed for 5 minutes to loosen the conidia. The total number of conidia production was estimated by using a haemocytometer. Three counts were made for each plate and quantity of conidia/cm2 was computed. The experiment was repeated twice from a different culture of the isolates. To test the relative pathogenicity, the susceptibility of third instar larvae of M. melolontha and H. serrata were inoculated by placing them on a conidia suspension of 2 x 107 conidia/ml. The technique used in this bioassay as described was by Reineke and Zebitz (1996) with slight modifications. In brief, the aqueous conidial suspension was topically applied on the insect. The appropriate volume of the conidial suspension (1ml/insect) was dispended on the insect with a micropipette and dripped off the excess liquid. Larvae treated with 0.01 % Tween 80 served as a control. The experimental and control individual larvae were kept in vials (~60ml) with appropriate aeration containing native soil and carrot slices as a food and incubated at 20°C with 16:8 (L:D) for 30 days. They were observed every two

days after treatment and were carrot slices replaced by fresh ones. In addition the number of dead insects were noted. The cadavers were surface sterilized with alcohol and were placed in Petri dishes lined with a moist blotting paper to facilitate mycosis. Fifteen insects per replicate and three replicates were used for each isolate. Germination and bioassay data were first subjected to angular transformation before analysis. Analysis of variance (ANOVA) using Systat statistical software (SPSS, 2004) was used to detect differences in conidial size, productivity, mortality and mycosis. Tukey’s HSD multiple comparison followed by significant difference were detected. Probit analysis was used to obtain LT50 and TG50 values.

Results

After 14 days the cultures of all isolates were well established on the SDA plates. Conidia of the B. brongniartii isolates were generally spherical to ellipsoid with the conidia length values ranging from 1.94 to 4.39 µm and width ranging from 0.93 to 2.29 µm (Table 1).

Table 1. Phenotypic characters - conidial size, rate of germination and amount of conidiation among B. brongniartii isolates.

Conidia size (µm) Sporulation Isolate Host TG (95%) x108 conidia Length Width 50 per cm2* Bbr 23 M. melolontha 3.81±0.11ac 1.78±0.08bc 7.82 (7.45-8.19) 19.8±2.92ab Bbr 30 M. hippocastani 4.10±0.05ac 1.98±0.00ab 8.66 (7.18-10.45) 22.7 ±1.76a Bbr 41 H. morose 1.94±0.22b 0.93±0.05d 8.40 (8.03-8.78) 7.54± 1.25c Bbr 50 M. melolontha 4.24±0.10a 2.29±0.05a 6.54 (6.17-6.88) 22 ±2.08a Bbr 59 M. hippocastani 4.10±0.08ac 2.16±0.07a 7.99 (6.85-9.14) 19.8± 4.59ab ARSEF 1072 M. melolontha 4.39±0.72a 2.14±0.17a 6.55 (5.76-7.21) 18.3 ±1.67b ARSEF 1360 --- 1.95±0.09b 0.98±0.01d 13.7(10.98-20.41) 12.7 ±2.50bd ARSEF 2660 Adult coleoptera 4.08±0.05ac 2.22±0.06a 8.39 (6.16-10.38) 8.25 ±1.67c ARSEF 4384 H. parallela 3.19±0.23acd 1.44±0.11c 6.78 (5.48- 7.82) 21.2± 4.17a ARSEF 5358 Melolontha sp. 3.54±0.16acd 2.16±0.06a 7.06 (6.32-7.77) 15.6± 3.34bd Means of conidial size and sporulation with the same letter are not significantly different (Tukey’s HSD multiple comparison (p<0.05). *Standard error of three assays. TG50 – time required for 50% germination.

There was significant difference in spore size among the B. brongniartii isolates (p<0.05), with significantly larger spores found in ARSEF 1072, Bbr 50 and Bbr 30 when compared to the other isolates (Table 1). Compared to other isolates Bbr 41 and ARSEF 1360 isolates were showed smallest conidia size. The highest germination rate of 100 % was obtained at 25°C after 20 h in all the isolates except ARSEF 1360 and ARSEF 2660. For all isolates, spore germination was started at 4 h after inoculation with a TG50 ranging from 6.54 to 13.7 h after inoculation at 25°C (Table 1). The isolates Bbr 50, ARSEF 1072, and ARSEF 4384 have significantly smaller TG50 values than others.

Corrected mortality (%) Fig. 1A Total mycosis (%) 100 a b a a c ab ab a ae ae 80 ac ac

60 6

1 3 7

0 . 4

0 .

5 4 .

0 1 9 40 16.78

8 2 bd cd 15.83

1 bd d d bd bd 20

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

r r r r r 0 3 6 3 3 1 1 2 4 5 b b b b b B B B B B F F F F F E E E E E S S S S S R R R R R A A A A A Isolates

Fig. 1B Corrected mortality(%) Total mycosis (%) 100

a 80 a

60 b b ab b

40 c c c cd 10.14 13.67

20 cd c d d

3 0 1 0 9 2 0 0 4 8 2 3 4 5 5 7 6 6 8 5 r r r r r 0 3 6 3 3 b b b b b 1 1 2 4 5 B B B B B F F F F F E E E E E S S S S S R R R R R A A A A A Isolates

Fig. 1. Pathogenicity of B. brongniartii isolates on 3rd instar larvae of M. melolontha (Fig.1A) and H. serrata (Fig. 1B) at 2 x 107 conidia/ml. (Means ± SE with the same index are not significantly different (Tukey‘s HSD multiple comparison (p<0.05)). LT50 median lethal time in days is given inside bar values; if not given, LT50 could not be calculated because mortality was less than 50%.

The amount of conidia production is directly related to the colony morphology of the isolates (data not shown). Conidia production ranged from 22.7 to 7.54 (x 108 conidia/cm2) for all B. brongniartii isolates and differed significantly among them (p<0.05), with the most virulent isolates Bbr 50, ARSEF 4384 and Bbr 30 (see below) producing significantly more conidia than others (Table 1).

The mean mortality, mycosis and LT50 induced by each isolate at a concentration of 2 x 107 conidia/ml after 30 days for both insects are presented in Fig. 1(A&B). All isolates of B. brongniartii were found to be pathogenic with significant difference in their virulence to M. melolontha (p<0.05) and H. serrata (p<0.05). Three isolates of B. brongniartii (Bbr 50, 23 and ARSEF 4384) were the most virulent towards M. melolontha, causing 95.83, 83.34 and 79.17% mortality in 30 days, respectively (Fig. 1A). It was apparent that isolate Bbr 50 initiated the highest mortality and mycosis levels against M. melolontha represented by LT50 (9.07 days) followed by 14.36 and 15.83 days (at 2 x 107 conidia/ml) obtained from Bbr 23 and ARSEF 4384, respectively (Fig.1A). In case of H. serrata, ARSEF 4384 and ARSEF 2660 showed the highest virulence causing 75.56 and 54.00% mortality in 30 days, 7 respectively. These isolates showed lowest LT50 of 10.14 and 13.67 days at 2 x 10 conidia/ml (Fig. 1B). Moreover, a further comparison of the mycosis rate demonstrated high variation between the isolates towards both tested scarabs. The highest mycosis rate was observed in Bbr 50 showing 100 % in M. melolontha. Almost all isolates showed a good sporulation 2-3 days after the death of larvae except for isolate ARSEF 1360 where no sign of sporulation was evident. In the case of H. serrata, isolates ARSEF 4384 showed highest mycosis (58.89%) compared to isolate ARSEF 2660 (46.36%).

Discussion

There were significant differences among the characterized B. brongniartii isolates in conidial size, spore production, speed of germination and virulence to M. melolontha and H. serrata. In the isolates characterized, those which produced larger conidia and had a higher spore production and faster germination were generally most virulent to third instar larvae of M. melolontha. Although the spores of the four isolates ARSEF 1072, Bbr 30, Bbr 50 and Bbr 59 were almost similar in size, they exhibited different levels of virulence towards scarabs tested. Samuels et al., (1989) reported a negative correlation between morphological parameters (conidial size) and virulence of Metarhizium anisopliae (Metschnikow) Sorokin isolates against brown plant hopper, Nilaparvata lugens Stål. This is in accordance with our studies, where the isolate ARSEF 1072 having larger conidia, more productivity and fast germination was less pathogenic to both scarabs than other isolates. On the other hand, larger conidia of Verticillium lecanii Zimm. Viegas induced a high pathogenicity in aphids (Aphis fabae Scop. and A. gossypii Glover) while isolates with small conidia caused a delayed mortality (Zayed, 1998). Additionally, positive correlation between conidial size and fungal virulence was observed by Alter et al., (1990), where Paecilomyces fumosoroseus (Wize) A. H. S. Brown & G. Smith isolates were tested against diamond back moth, Plutella xylostella L.. Further studies also indicated little or no correlation between conidial size and virulence against the chrysanthmum aphid, Macrosiphoniella sanborni (Gillette) (Drummond et al., 1987). It is therefore difficult to generalize about a potential correlation between conidial size and fungal virulence for a specific host across all fungal isolates. It is believed that fungal isolates with rapid germination and quick infectivity may have an advantage as biological control agents because host infection can potentially occur much more quickly (Varela and Morales, 1999). Rapid spore germination is extremely very important for successful infection to occur because optimal conditions (esp. relative humidity) may not always exist. The ability of conidia to survive short periods of exposure to high temperatures is also highly desirable as such conditions may occur during field application. Ready production of conidia on artificial media facilities mass production and therefore enhances an isolates commercial potential.

During this study, selectivity among the isolates to one group rather than the other was found. All B. brongniartii isolates tested were apparently more pathogenic to third instar larvae of M. melolontha compared to H. serrata. This indicated that B. brongniartii isolates obtained from different geographical and host origins showed higher host specificity to M. melolontha compared to H. serrata. However, those isolates obtained from Holotrichia sp (Bbr 41 and ARSEF 4384) or from an undetermined coleopteran insect (ARSEF 2660) showed greater pathogenic to H. serrata. This may indicate that B. brongniartii has a narrow ecological host range. A difference in pathogenicity of different isolates of B. brongniartii was related to their abilities to penetrate the cuticle of the host. Reineke and Zebitz (1996) classified 31 isolates of B. brongniartii according to differences in enzymatic patterns of the isolates. Similarly, there was a difference in pathogencity of different B. brongniartii isolates tested against M. melolontha (Darwish et al., 2000) and H. serrata (Sharma et al.,1999). The last observation could partially be in agreement with our results concerning the selectivity between the isolates. In summary, all B. brongniartii isolates varied in virulence towards both tested scarabs. However, little or no relationship could be demonstrated among the examined characteristics nor between any one trait and the virulence. It is essential to base final isolate selection for field-testing on a variety of criteria, some of which we have evaluated in this study. Of the ten isolates B. brongniartii tested, three isolates for M. melolontha and one isolate for H. serrata showed a greater potential for field use on the basis of these criteria and warrant further assessment under field conditions.

Acknowledgements

ABH is thankful to the DAAD Bonn, Germany for research fellowship. We thank to M. Froeschle (State institute for plant protection, Stuttgart), Dr. K. Uma Devi and her staff (Andhra University, India) for kind help during the grubs collection. We also thank Dr. G. Zimmermann (BBA, Darmstadt) and Dr. R. Humber (USDA-ARS, Ithaca, NY) for providing strains of B. brongniartii.

References

Alter, J.A., Vandenberg, J.D. & Cantone, F.A.1999: Pathogencity of Paecilomyces fumosoroseus isolates to diamondback moth, Plutella xylostella: correlation with spore size, germination speed, and attachment to cuticle. – J. Invertebr. Pathol. 73: 332-338. Bidochka, M.J. & Khachatourians, G. 1990: Identification of Beauveria bassiana extra- cellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanoplus sanguinipes. – J. Invertebr. Pathol. 56: 362-370. Darwish, E., Zebitz, C.P.W. & Zayed, A. 2000: The combined action of a neem exctract and Beauveria brongniartii (Sacc.) on the larvae of Melolontha melolontha L. (Coleoptera: Scarabaeidae). – In: Abou el Ela, R.G. & M.E. Naeem (eds.): Proc. 1st Int. Conf. of Applied Entomology, Cairo University, Fac. of Science, Dept. of Entomology, held at Giza - Egypt, 11-12 March 2000: 29-37. Drummond, J., Heale, J.B. & Gillespie, A.T. 1987: Germination and effect of reduced humidity on expression of pathogencity in Verticillium lecanii against the glasshouse whitefly Trialeurodes vaporariorum. – Ann. Appl. Biol. 111: 193-201. Keller, S., Forrer, H.R., Fried, P. M., Alfoldi, T., Lockeretz, W. & Niggli, U. 2000: Experiences in white grub [Melolontha melolontha] control with the fungus Beauveria

brongniartii. IFOAM 2000: the world grows organic. – Proceedings 13th International IFOAM Scientific Conference, Basel, Switzerland, 28 to 31 August, 2000: 133. Keller, S., Keller, E. & Auden, J.A.L. 1986: Ein Grossversuch zur Bekämpfung des Maikäfers (Melolontha melolontha L.) mit dem Pilz Beauveria brongniartii (Sacc.) Petch. – Mitteilungen der Schweizerischen Entomologischen Gesellschaft 59(1-2): 47-56. Milner, R.J., Staples, J.A. & Lutton, G.G., 1997: The effect of humidity on germination and infection of termites by the Hyphomycete, Metarhizium anisopliae. – J. Invertebr. Pathol. 69, 64-69. Reineke, A. & Zebitz, C.P.W. 1996: Protein and isoenzyme patterns among isolates of Beauveria brongniartii with different virulence to European cockchafer larvae (Melolontha melolontha L.). – J. appl. Ent. 120(5): 307-315. Samules, K.D.Z., Pinnock, D.E. & Bull, R.M. 1990: Scarabeid larvae control in sugarcane using Metarhizium anisopliae. – J. Invertebr. Pathol. 55: 135-137. Sharma, S., Gupta, R.B.L. & Yadava, C.P.S. 1999: Effect of certain soil fungi on Metarhizium and Beauveria spp. and their pathogenicity against Holotrichia consanguinea. – Indian Phytopathology. 52 (2): 196-197. SPSS (Systat statistical software) 2004: Statistical product and service solution, system user’s guide Version 12. Varela, A. & Morales, E. 1996: Characterization of some Beauveria bassiana isolates and their virulence towards the coffee berry borer, Hypothenemus hampii. – J. Invertbr. Pathol. 67: 147-152. Vyas, R.V., Yadav, D.N. & Patel, R.J. 1990: Studies on the efficacy of Beauveria brongniartii against white grub. – Annals of Biology, Ludhiana 6(2): 123-128. Yadava, C.P.S. & Sharma, G.K. 1995: Indian white grubs and their management. – Technical bulletin No.2. Project Coordinating Center, AICRP on white grubs, Durgapuara, Jaipur India. 26 pp. Zayed, A. 1998: Characterization of four entomopathogenic isolates of the fungus Verticillium lecanii with different pathogenicity levels towards two aphid species. – Ph.D Thesis, Fac. of Agric., University of Hohenheim, Stuttgart, Germany. 107 pp. Zimmermann, G. 1998: The entomopathogenic fungus Beauveria brongniartii (Sacc.) Petch and experiences in its use for biological control of the European field and forest cockchafer. – Nachrichtenblatt des Deutschen Pflanzenschutzdienstes. 50(10): 249-256.

Wireworms

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 73-79

European wireworms (Agriotes spp.) in North America: Distribution, damage, monitoring, and alternative integrated pest management strategies

Robert S. Vernon1, Wim Van Herk1 and Jeff Tolman2 1 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, P.O. Box 1000, Agassiz, British Columbia, CANADA V0M 1A0 2 Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St., London, Ontario CANADA N5V 4T3

Abstract: Three species of wireworms, notably Agriotes obscurus, A. lineatus, and A. sputator, were introduced to North America from Europe about a century ago. Recent surveys using pheromone traps have shown that all three species are present in the Maritime provinces of Eastern Canada, and A. obscurus and A. lineatus are present in the westernmost province of British Columbia, and in the state of Washington in the USA. In recent years, these species have become major pests of a variety of crops. Of major concern in Canada is that all of the most commonly used insecticides for wireworm control are no longer available, and attempts to register new wireworm insecticides are proving difficult. In addition, some of the leading candidate wireworm insecticides, including the thianicotinyls thiamethoxam and clothianidin, and the cloronicotinyl imidacloprid, may not be as effective at reducing wireworm populations as suggested by some studies. Our data, involving laboratory toxicity (LD50), and field efficacy studies on cereals and potatoes, suggests that wireworms (A. obscurus) may actually enter a long-term state of intoxication upon contact with the thianicotinyl clothianidin (and thiamethoxam in the laboratory), and the cloronicotinyl imidacloprid, and recover later on in the growing season. The various stages of insecticide intoxication leading to revival or death in wireworms are described in this paper for fipronil and clothianidin. Alternative control strategies also under investigation at our research centre include: cultural controls (field flooding and lethal trap crops); physical controls (barriers to adult movement); semiochemical controls (mass trapping); natural products (plant extract toxicity and repellency); and biological control (lead by Todd Kabaluk) using Metarhizium anisopliae. With assistance from Dr. Miklos Tóth (Hungary), pheromone traps have been developed for the three exotic European species, which have been used in North America for surveys as well as in the development of IPM programs in British Columbia. A wireworm risk index has been developed for strawberry fields that combines click beetle and wireworm sampling techniques, and pheromone blends have been developed to capture both A. lineatus and A. obscurus for mass trapping. Data on all of the above research topics is presented.

Keywords: Wireworms, Agriotes obscurus, A. lineatus, distribution, control, monitoring, pheromone traps

Introduction

The dusky wireworm, A. obscurus (L.), and the lined click beetle, A. lineatus (L.) were introduced to North America from Europe, probably in the 1800s via soil in ship ballast, and are now established in the westernmost province of British Columbia (BC), and the easternmost (Maritime) provinces of Nova Scotia and Newfoundland in Canada (Eidt 1953; Vernon et al. 2001). They have also recently been found in Washington state in the USA (Vernon et al. 2001). The potato wireworm, A. sputator (L.), has also been introduced from Europe to Nova Scotia and possibly to other Maritime provinces in Canada. Reports of damage to various cultivated crops by these and other indigenous species are growing, with their greatest impact traditionally being on corn, Zea mays L. (Gramineae) and potatoes,

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Solanum tuberosum L. (Solanaceae) (Wilkinson et al. 1976). In potatoes, European wireworms are causing increasing damage to crops destined for fresh market or processing plants in BC and Nova Scotia. Efforts to control wireworms in southwestern BC using the granular insecticide Thimet 15G (=phorate) in potatoes have also been implicated in the direct poisoning of waterfowl and the secondary poisoning of 30-40 bald eagles in the early 1990s. As a result, Thimet (the only effective registered insecticide remaining for wireworm control in Canada) has been removed from BC, and the registration for Thimet in the rest of Canada will expire in 2006. In small fruit crops, wireworm damage was sporadic before 1990, but has been increasing in recent years. Damage has been most pronounced in strawberries, Fragaria x ananassa Duchesne (Rosaceae), where new plantings are sustaining heavy seedling mortality or reduced vigour due to wireworms tunnelling into transplanted crowns or feeding on the young roots (Vernon et al. 2000). Of even greater concern is that European wireworms, (Agriotes spp.) will feed upon and completely enter strawberry fruit destined for fresh market or for the processing industry which have a zero tolerance for insect contaminants. Currently the options for control of wireworms in most susceptible crops in Canada are limited or non-existent, and will be even more limited with the removal of all effective wireworm insecticides in the near future. This paper describes some of the work underway at the Agriculture and Agri-Food Canada (AAFC) research centres in Agassiz, BC, and London Ontario, and is divided into: a) basic biological/ecological studies; b) development of control methods; and c) development of monitoring procedures.

Basic biological and ecological studies Surveys: General presence or absence surveys for A. obscurus, A. lineatus and A. sputator in North America have been greatly simplified in recent years by the commercial development of species specific Agriotes pheromones in Europe (Tóth et al. 2003). From this work, a new pheromone trap was designed (Vernon and Tóth, submitted; Vernon 2004), and is currently being used in delimitation surveys for these species in the Maritime provinces (Prince Edward Island, Newfoundland, New Brunswick and Nova Scotia) and British Columbia, and will be expanded to all other provinces in Canada in 2005. Surveys for A. obscurus and A. lineatus are currently underway in Washington and Oregon as well as in the northeastern United States, and will likely be expanded for all three species in 2005. In British Columbia and Nova Scotia, it has been found that where the exotic species are established, they have largely replaced the indigenous species in numbers and in pest status. Mobility: Until recently, researchers studying European wireworms in Canada have claimed that A. lineatus and A. obscurus click beetles do not fly (Eidt 1953; Wilkinson et al. 1973). This is contrary to observations in Europe, and has implications on how effectively these species can disperse and on how certain control strategies will work (i.e. adult exclusion or mass trapping). In 2002, however, both sexes of these species were observed in flight in southwestern BC on several occasions (Crozier et al. 2002), and it was found in the laboratory that they can be stimulated to fly by increasing the ambient temperature to 24oC. Spatial and temporal field distribution and abundance: Pheromone traps for adults and bait traps for wireworms were placed in systematic arrays in over 50 strawberry fields between 2000-2002 to study the within-field distribution of A. lineatus and A. obscurus adults and larvae, and to determine if pheromone trap catches can be correlated with bait trap catches. As expected, the distribution of wireworm populations was generally random and quite unpredictable. The relative spatial distribution of A. lineatus and A. obscurus adults, however, could vary considerably both within and between fields over time depending on farming 75

activities (i.e. cultivation and spraying) and proximity to alternative adjacent field and headland habitats (Agriotes reservoirs). Populations of A. lineatus and A. obscurus are also temporally asynchronous in BC, with initial and peak catches of male A. obscurus in 2001 occurring 15.6 and 18.7 days, respectively, before A. lineatus (Vernon et al. 2001). A. obscurus populations have consistently emerged and peaked before A. lineatus in all surveys conducted between 2000-2004. Host Preferences: In field studies conducted in the spring of 1998, A. obscurus wireworms aggregated in similar numbers at wheat, oat, barley, and fall rye planted in circular (127-cm2) bait stations containing 100 seeds planted 3 cm deep. Similar levels of aggregation also occurred at 11 varieties of wheat, with spring wheat cultivars (high germination rates) appearing to be slightly more effective than winter wheat varieties (lower germination rates). From this study, it was concluded that any of the cereal crops tested would be suitable for monitoring A. obscurus wireworm populations in the spring if planted in circular bait stations at 100 seeds/127-cm2, and would also be effective for aggregating wireworms by means of a trap crop (Vernon et al. 2003).

Development of control strategies Chemical controls: Field studies to determine the efficacy of lower risk alternative insecticides to replace the higher risk (i.e. lindane, fonofos and phorate) insecticides for wireworm control in cereal crops and potatoes have been underway in Agassiz, BC since 1996. In recent years, considerable attention has been paid to the thianicotinoids, thiamethoxam (e.g. Cruiser) and clothianidin (e.g. Poncho), and to the cloronicotinoid imidacloprid (e.g. Genesis) for the control of several insect pests of potato, including wireworms. In field trials, we have found that imidacloprid (seed piece treatment) provides poor control of damage caused by A. obscurus, whereas thiamethoxam and clothianidin (seed piece treatments) provide reductions in damage similar to phorate. Further investigations, however, have indicated that reductions in tuber damage by the thianicotinoids may not directly relate to reductions in wireworm populations. In laboratory studies (contact LD50 trials using a Potter spray tower at AAFC London, Ontario), A. obscurus consistently entered a rapid and long- term state of intoxication upon contact with imidacloprid, thiamethoxam and clothianidin, which in some cases could last throughout the typical growing period for potatoes (90-100 days). Long-term intoxication was not observed in other insecticides tested, including diazinon, chlorpyrifos, tefluthrin, lindane, spinosad and fipronil. The long-term contact effects of clothianidin relative to fipronil are shown in Fig. 1. These laboratory data are interesting, because it was also found that wireworm populations in potato and wheat plots treated with imidacloprid and clothianidin in 2003 were similar in number to wireworm populations in untreated check plots when sampled the following spring in 2004. This suggests that the cloro- and thianicotinoids, although providing levels of crop protection from wireworm damage for a growing season (due possibly to wireworms entering a long term state of intoxication), may not ultimately result in population decreases due to their eventual recovery. This hypothesis, however, is based on only one year of studies, and additional work is underway to confirm or refute these observations. Studies are also underway at PARC, Agassiz to study the toxic and repellent effects of essential oils and various insecticides against A. obscurus, A. lineatus and other indigenous pest wireworms.

CATEGORIZATION OF MORBIDITY CATEGORIZATION OF MORBIDITY FIPRONIL (0.001%) CLOTHIANIDIN (0.1%) ALI VE WRITHING LEG & MOUTH MOUTH DEAD ALIVE WRITHING LEG & MOUTH MOUTH DEAD 100 100

80 80

60 60 % % 40 40

20 20

0 0 1 DAT 7 DAT 35 DAT 91 DAT 1 DAT 7 DAT 35 DAT 91 DAT

Figure 1. The long term effects of applying fipronil and clothianidin (contact LD50 studies using a Potter spray tower) to late instar A. obscurus wireworms. Data are shown for 1, 7, 35 and 91 days after treatment. Wireworm health is divided into five categories: alive and healthy; writhing; immobile except for leg and mouthpart movement upon prodding; immobile except for mouthpart movement upon prodding; and dead.

Cultural controls: Trap crops. Rows of wheat planted as trap crops 1 week in advance of planting strawberries (between the rows of wheat), effectively and inexpensively reduced strawberry seedling mortality from 43% to 5% (Vernon et al. 2000). Treating wheat seed with insecticide (i.e. Agrox DL Plus, containing lindane and diazinon) and planting the wheat as a trap crop at increasing seeding densities resulted in increasing wireworm aggregation and mortality (Vernon, unpublished data). It was determined that a seeding rate of 2.4 treated seeds/cm in rows spaced 0.5 m apart would provide optimum attraction and mortality of A. obscurus wireworms when used in fallowed fields in the spring. This practice was used by strawberry growers in BC for wireworm population reduction until 2003, at which time all cereal seed treatments containing lindane were de-registered in Canada. Work is currently underway at AAFC, Agassiz, to identify and register new insecticide seed treatments for cereal trap crops that will control wireworms with similar efficacy to lindane. Lethal trap crops are also being investigated for use in potatoes, where treated wheat seed is applied in-furrow at the time of seeding. This ’attract-and-kill’ strategy has been quite effective with insecticides such as fipronil, where wireworm control similar to that of phorate is possible at low doses of insecticide/ha. Field Flooding. Laboratory studies were completed in 2004 at AAFC Agassiz, to determine the optimum parameters by which wireworm populations can be controlled by field flooding. The LT50 (T = time) values for control of A. obscurus were determined in various soil types and at different temperatures. As has been observed for other wireworm species, the LT50 was reduced as the water temperature (in flooded soil) was increased from 5-20°C. Differences in LT50 were also observed between soil types, where soils with higher salt content had lower LT50 values. It was concluded that the majority of wireworms would be killed within 7-10 days if fields could be flooded when soil temperatures are between 15-20°C (late Summer in BC). This strategy will be tested by a number of organic potato growers in southwestern BC in 2005.

Physical/mechanical controls: With respect to physical controls, a moulded plastic exclusion trench device has been developed and patented at PARC, Agassiz for mass trapping Colorado potato beetles, and has also been found to intercept and mass trap European click beetles. A study conducted in 2001 at AAFC Agassiz, demonstrated that about 65% fewer A. obscurus click beetles entered fields of grass enclosed by these exclusion trenches than in non-enclosed fields. It was decided, however, that the rate of exclusion efficacy was too low and the cost too high to warrant further work. In terms of mechanical controls, attempts to kill wireworms assembled at wheat trap crops by heating the soil with propane flamers were totally ineffective.

Semiochemical controls: The use of semiochemicals in various ways to control elaterids has been investigated only in Russia, primarily against Agriotes spp. In an abstract by Ivashchenko and Chernova (1995), pheromones applied in an undisclosed manner at the rate of 120 g pheromone per hectare caused the “disorientation” (confusion) of male Agriotes (species not disclosed), resulting in over 70% of females remaining unmated. This abstract also alluded to mass trapping, but the technique was not discussed except to state that it was less effective than disorientation. Another abstract by Balkov and Ismailov (1991) stated that effective direct control of A. sputator and A. gurgistanus can be achieved by intensive use of pheromone traps over 3-4 years. In other work, Balkov (1991) found that 30 A. sputator pheromone traps/ha were sufficient at medium or low levels of infestation (up to 5 individuals/m2), but 120 traps/ha were required at higher levels of infestation (over 10 individuals/m2). In that study, larvae had been reduced by 86% after 4 years of trapping in a field with a medium infestation. These reports suggest that mass trapping and/or mating disruption might warrant investigation as alternative control strategies against A. obscurus and A. lineatus in environmentally sensitive areas in BC. In 2001, a number of mark-release recapture studies were conducted at AAFC, Agassiz, to study how efficient male A. obscurus and A. lineatus are in locating pheromone traps. It was found that marked A. obscurus beetles released 10-12 m away from a pheromone trap could locate the trap within 16 hours, and in another study, 9% of marked A. lineatus males had located a pheromone trap 50 m away after 24 hours. It was also found that pheromone traps set at a density of 1 trap per 25 m2 could capture 32% of released males within 24 hours, and 38% within 48 hours. The high percentage recapture of released males in such a relatively short period of time suggested that these pheromone traps might be used to mass trap males and reduce the fecundity of the remaining females. This was tested in a larger scale replicated study in 2004, where pheromone traps were set out at a density of 1 trap per 10 m2 along a grassy dyke in southwestern BC. Of populations of marked beetles uniformly released into these areas, 83% of A. obscurus, and 80% of A. lineatus were recaptured within the first 3 weeks after release. This study will be repeated on a larger scale in 2005, and the mating success of females will be determined. If effective, mass trapping could be used to gradually reduce wireworm populations in reservoir areas surrounding arable fields, and diminish the threat of continuous reinfestation of fields by these pests.

Development of monitoring procedures Click beetle monitoring traps: The commercial development of species specific Agriotes pheromones in Europe (Tóth et al. 2003) was instrumental in the development of a pheromone trap (Vernon, 2004) for the survey and monitoring of A. obscurus, A. lineatus and A. sputator in North America. In addition to their utility as survey tools, the A. obscurus and A. lineatus traps have also been evaluated as IPM monitoring tools to determine if the number 78

of adults caught can reliably predict the risk of wireworm damage in individual fields. Pheromone traps and bait traps were placed in over 50 strawberry fields in BC between 2000- 2002, and the number of click beetles relative to wireworms trapped were analyzed. The data indicate that high or low numbers of adults in pheromone traps in individual fields are often correlated with high or low numbers of wireworms in bait traps. However, click beetle catch did not always correlate with wireworm catch, and it is tentatively concluded that pheromone trap catch alone is not sufficient to consistently predict the risk of wireworm damage in individual fields. As a more conservative approach, however, we have found that the deployment of A. obscurus and A. lineatus pheromone traps alongside bait traps at a minimum of 5 sites per field, and using the formula: Wireworm Risk = # wireworms (# A. obscurus + # A. lineatus), will provide a more consistent index of wireworm risk. This system is currently being used by the strawberry processing industry in BC to identify fields at greatest and lowest risk of strawberry contamination. Click beetle mass traps: The traps developed for monitoring and surveying click beetles are too expensive for use in mass trapping strategies. To address this problem, lower cost pheromone trap designs have been developed that could eventually be used for mass trapping. In addition, a blended pheromone lure has been developed that will capture both A. obscurus and A. lineatus in a single trap, which would further reduce the cost of mass trapping programs.

Acknowledgements

We thank: the Matching Investment Initiative of AAFC; the Fraser Valley Strawberry Growers Assn.; the Potato Industry Development Committee; Canadian Wildlife Service; Environment Canada; Ducks Unlimited Canada; B.C. Waterfowl Society, E.S. Cropconsult Ltd.; Phero Tech Inc.; Canadian Food Inspection Agency; BCARC; University College of the Fraser Valley; Lower Mainland Horticultural Improvement Assn., Bayer AgroScience; Syngenta; and Zeneca Agro.

References

Balkov, V.I. 1991: Attractant traps for control of wireworms. – Zashchita Rastenii Moskova 10: 30-31. Balkov, V.I. & Ismailov, V.Y. 1991: Attractant traps for elaterids. – Zashchita Rastenii 10: 21. Crozier, S.A., Tanaka, A. & Vernon, R.S. 2003: Flight activity of A. obscurus L. and A. lineatus L. (Coleoptera: Elateridae) in the field. – Journal of the Entomological Society of British Columbia 100: 91-92. Eidt, D.C. 1953: European wireworms in Canada with particular reference to Nova Scotian infestations. – The Canadian Entomologist 85: 408-414. Ivashchenko, I.I. & Chernova, S.V. 1995: Biologically active substances against click beetles. – Zashchita Rastenii Moskova 9:16-17. Tóth, M., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Imrei, Z., Tolasch, T., Franke, W. & Jossi, W. 2003: Identification of pheromones and optimization of bait composition for click beetles pests (Coleoptera: Elateridae) in Central and Western Europe. – Pest Manag. Sci. 59: 417-425. Vernon, R.S., Kabaluk, J.T. & Behringer, A.M. 2000: Movement of Agriotes obscurus (Coleoptera: Elateridae) in strawberry (Rosaceae) plantings with wheat (Gramineae) as a trap crop. – The Canadian Entomologist 132: 231-241. 79

Vernon, R.S., LaGasa, E. & Philip, H. 2001: Geographic and temporal distribution of Agriotes obscurus and A. lineatus (Coleoptera: Elateridae) in British Columbia and Washington as determined by pheromone trap surveys. – Journal of the Entomological Society of British Columbia 98: 257-265. Vernon, R.S., Kabaluk, J.T. & Behringer, A.M. 2003: Aggregation of Agriotes obscurus (Coleoptera: Elateridae) at cereal bait stations in the field. – The Canadian Entomologist 135: 379-389. Vernon, R.S. 2004: A ground-based pheromone trap for monitoring Agriotes lineatus and A. obscurus (Coleoptera: Elateridae). – Journal of the Entomological Society of British Columbia. In Press. Wilkinson, A.T.S., Finlayson, D.G. & Campbell, C.J. 1976: Controlling the European wireworm, Agriotes obscurus L., in corn in British Columbia. – Journal of the Entomological Society of British Columbia 73: 3-5.

80 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 81-85

Monitoring and control of Agriotes lineatus and A. obscurus in arable crops in the Netherlands

Albert Ester, Klaas van Rozen Applied Plant Research (PPO-AGV) Wageningen University and Research, P.O. Box 430, 8200 AK Lelystad, The Netherlands. [email protected]

Abstract: Wireworms can cause severe damage in potatoes and several other crops. To reduce wireworm attack Applied Plant Research (PPO-AGV) in Lelystad and Plant Research Institute (PRI) in Wageningen have conducted an integrated crop protection system to reduce wireworm populations and potato damage. Not the wireworm, but the adult beetle is the target to attack. Field research in 2002 resulted in reliable decrease of click beetles after spraying with insecticides.

Keywords: integrated crop protection, wireworms, Agriotes lineatus, A. obscurus

Introduction

Wireworms (larvae of Agriotes spp.) are known to be destructive after long-term grassland in following crops like potatoes up to four years. But recently more damage occur in all-arable rotations (Parker & Howard, 2001). Usually crops are protected against wireworms with soil treated insecticides. This is the most logical method as larvae will live several years in the soil before pupating into the adult stage. Soil treatment just before planting potatoes takes a firm line with wireworms. Nevertheless, sharping new environmental demands, Mocap (a.i. ethoprofos) is the only soil insecticide available in the Netherlands. The current Dutch way of detection wireworms before planting is not waterproof for advising soil treatment, putting split potatoes into the soil before planting at several places in the field. No or some wireworms indicates certain damage during the season, but does not predict damage at the end of the season (Parker, 1996). Pesticide policy and uncertain wireworm detection advanced the search for an alternative. The aim of this study includes detection Agriotes lineatus and A. obscurus using sex pheromones and fight the beetles after monitoring on a catch peak, before egg deposit starts. This may reduce the population density of wireworms in following crops and decrease crop damage.

Material and methods

Species specific attractants A natural sex pheromone consists a variety of chemical components. Plant Research International (PRI Wageningen) is capable of producing an artificial sex pheromone consisting some important chemicals of the natural sex pheromone (Ester et al, 2002). Nearly 100 percent of the caught click beetles are species-specific. For the sake of convenience we use the word sex pheromone in stead of components. With artificial sex pheromones it is possible to catch easily male click beetles.

81 82

Pitfall traps Agriotes spp. are soil dwelling insects and may fly occasionally. Pitfall traps are used to catch the beetles. These insect traps, already used in horticulture, can be dug in the soil. The top of the pitfall is placed equally to the soil surface. To attract the click beetles, sex pheromones are attached in the centre beneath of the lid. In the Netherlands A. lineatus and A. obscurus wireworms are the most harmful species. Up to more than several hundreds of beetles were caught in one week. Testing the sex pheromones of these two species resulted in good specific catches.

Host plants Towards specific detection and control of click beetles potential host plants were monitored on the presence of five click beetle species. Large amount of A. lineatus and A. obscurus were captured in grass for seed production and summer barley (Ester et al, 2002).

Field trial 2002 Six fields of 4 to 7 ha were selected divided in two sides, treated and untreated. Two fields grass seed was growing, the other four fields was sown with summer barley. Both sides consisted of six pheromone traps, three for each specimen. The traps were placed randomly in one line, 20 meters from each other. Twice a week the traps were emptied. Numbers of specimen were collected and counted. After an increase of the caught population, the decision should be made to spray the treated side with a pyrethroïd, all fields at the same time. Pyrethroïds as Decis EC, a.i. deltamethrin, and Karate, a.i. lambda-cyhalothrin, may be applied in host plants, subject to permission for protection against insects in a certain crop in the Netherlands. Decis EC was applied in the recommended dose of 0.3 l ha-1 in grass seed and 0.25 l ha -1 in two fields of summer barley. The other two summer barley fields were treated with 0.2 l ha-1 Karate. The insecticides were applied with 400 l water ha-1. Moment of spraying was the15th or 16th of May in the evening with dry and relatively warm weather with maximum and minimum temperatures of 21 C respectively 9 C on a dry crop. Monitoring continued until harvesting time. To monitor the flight of the beetles makes sure the treatment will be effective.

Farmers introduction 2004 Sixty farmers introduced the integrated system in different arable rotations. Instead of 2002, commercial available funnel traps were used. These insect traps are placed in the soil, the undercarriage equal to the soil surface and contains 10 % water of the container. To attract the click beetles, sex pheromones were attached in the centre of the lid of the funnel traps. In opposite to the pitfall traps, Agriotes spp. need to crawl five centimetres up to fall or flying into the funnel traps. The funnel trap caught significant more A. lineatus than pitfall traps, but A. obscurus captures were equal to pitfall traps (unpublished data, 2004). Per five ha four numbers of traps were used, two for each Agriotes spp. Each five more ha two more traps were recommended. Supplementary, farmers were advised to situate the traps in length, twenty metres apart from each other, in the centre of the fields. Approximately twice a week the traps were emptied by farmers and recorded. Beetles were counted per specimen and removed.

Statistical analysis Beetle catches were statistical analysed using analysis of variance (ANOVA) in Genstat 6. From the ANOVA means, least significant differences (LSD) and F-probabilities were obtained. 83

Results and discussion

Before spraying no striking difference in catches of A. lineatus and A. obscurus captures between treated and untreated were recorded, accept for two times when significant more A. obscurus were caught in the untreated side (Table). Overall catches from 19th of April until the 17th of May no difference were found between treated and untreated. On the 17th of May no differences were found between the treated and untreated side, one or two days after treatment. This may be due to the last catch on the 14th of may and the timing of spraying afterwards. But from on the five or six days after spraying, both species showed significant lower numbers of beetles in the treated than the untreated side until the 21st of June, with the exception of 28 May for A. obscurus and 11 June for A. lineatus. On 28th of June and 2nd of July, approximately 43 and 47 days after treatment, significant more A. obscurus were found in the untreated side. After spraying the overall numbers A. lineatus and A. obscurus per trap were for both significant lower in the treated in comparison to untreated sides. Overall reduction per specie was respectively 88 % and 81 %.

Table. Average numbers of Agriotes spp. per trap in the treated and untreated sides of six fields, 2002.

DATA A. LINEATUS A. OBSCURUS Treated Untreated F-prob.Lsd Treated Untreated F-prob. Lsd 19 April 0,2 0,0 0,163 0,24 0,3 0,1 0,217 0,41 23 2,8 1,3 0,231 2,41 3,8 3,9 0,880 2,30 26 2,8 3,4 0,596 2,60 6,4 7,1 0,730 4,00 29 0,6 0,8 0,699 0,89 0,7 2,3 0,005 1,05 3 May 1,8 0,9 0,160 1,20 2,4 2,2 0,706 1,38 7 S 1,3 1,8 0,424 1,29 1,3 2,8 0,007 1,10 10 6,8 6,8 0,978 6,36 6,0 6,8 0,734 4,57 14 7,6 7,7 0,974 5,34 6,8 6,9 0,925 3,66 17* 6,9 6,2 0,816 5,96 5,5 7,2 0,500 5,11 21 1,2 9,2 0,008 5,63 1,7 12,9 0,013 8,61 24 0,9 6,1 < 0,001 2,49 2,1 8,1 0,002 3,54 28 0,6 4,3 0,025 3,22 1,0 4,3 0,053 3,39 31 1,0 17,9 < 0,001 7,29 1,9 17,5 < 0,001 6,51 3 June 1,4 8,1 < 0,001 3,43 2,1 11,5 < 0,001 3,09 7 2,2 12,8 0,016 8,34 1,3 8,3 < 0,001 3,36 11 1,2 8,2 0,119 9,00 0,9 5,9 0,003 3,11 14 0,00 5,25 0,014 4,02 0,6 2,1 0,040 1,43 18 1,4 17,9 0,023 13,99 1,0 5,3 0,012 3,17 21 0,4 4,7 < 0,001 1,88 0,4 2,7 0,004 1,42 25 0,3 2,3 0,181 3,15 1,2 2,8 0,092 1,87 28 0,1 2,0 0,140 2,62 0,8 1,8 0,025 0,86 2 July 0,0 1,3 0,142 1,72 0,3 2,1 0,026 1,58 9 1,1 4,0 0,087 3,39 1,5 2,7 0,163 1,69 ≤17/5 31 29 0,838 17,8 33 39 0,331 12,8 21/5-9/7 12 104 < 0,001 37,1 17 88 < 0,001 18,3 Treatment 15th and 16th of May The bold numbers differ significant between the treated and untreated sides.

In general the numbers of Agriotes spp. counted by farmers were comparable to the field trials in 2002 (figure 1 and 2). Differences may be related to variation in timing of treatments, 84

amount of water used for application, weather conditions, inadequate use of the pheromones and traps and the situation of placing of the funnel traps in the field. Overall complain was the intensively labour put in. This resulted in placing of the traps near the edges of the fields, which may have attracted Agriotes spp. from outside the fields. Another complain was the high numbers of mice caught, which decaying very rapidly. Monitoring the flight after insecticide application was considered an advantage, data showed positive and negative effects of the treatment. Nevertheless, this completely new method of protecting crops against larvae of Agriotes spp. needed and needs extensively information and accompaniment at introduction to the farmers.

Winter wheat 50 40 A. lineatus spp. 30 A. obscurus M 20 10 Numbers of Agriotes 0 6 6 7- 10-5 17-5 24-5 31-5 14- 21-6 28-6 M = timing treatment Data of monitoring

Figure 1. Practical treatment with 0.3 l ha-1 Decis EC in winter wheat, 2004.

Grass seed 50 M 40 A. lineatus

spp. 30 A. obscurus 20

Numbers of 10 Agriotes 0

5 6 6 3- 7- 10-5 17-5 24-5 31-5 14-6 21- 28-6 M = timing treatment Data of monitoring

Figure 2. Practical treatment of 0.2 l ha-1 Karate in grass seed, 2004.

One can conclude that pyrethroïd control can reduce Agriotes spp. significantly, based on monitoring and is often needed to control aphids or Mayetiola schoberi B. in cereals or grass for seed production. Timing of application may be quite sufficient through monitoring, but more research is needed to obtain optimal threshold information to decide whether or not an application is necessary. For the moment insecticides may be used to much based on low captures, which will not improve environmental demands. Also, no significant results about decreasing damage by wireworms after three or four years are available. This system aims to protect the crop against wireworm damage and may replace soil treatment in time by one or two treatments against the adult beetle. This may prevent the build up of the wireworm population in host crops. So far more research is inevitable. 85

References

Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the U.K. – Agricultural and Forest Entomo- logy 3: 85-98. Parker, W.E. 1996: The development of baiting techniques to detect wireworms (Agriotes spp., Coleoptera: Elateridae) in the field, and the relationship between bait-trap catches and wireworm damage to potato. – Crop Protection 15(6): 521-527. Ester, A., Rozen, K. van & Griepink, F.C. 2002: Monitoring of Agriotes spp. with sex pheromones in arable crops. – Sixth International Conference on Pests in Agriculture, 4-6 December 2002. 86 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 87-90

Practical implementation of a wireworm management strategy – lessons from the UK potato industry

William E. Parker ADAS, Woodthorne, Wergs Road, Wolverhampton, WV6 8TQ, UK

Abstract: wireworms, principally Agriotes obscurus, A. sputator and A. lineatus, have become increasingly important pests of potato in the United Kingdom (UK) in recent years. The reasons for this are not entirely clear, but are probably are combination of biological factors, such as long-term changes in agronomic practice which favour wireworm survival, and market factors such as higher tuber quality demands from processors and major retailers. This has required the development of new risk assessment tools, principally bait trapping for wireworms and pheromone trapping for adults, which have required extensive testing with potato growers and advisers to ensure their uptake by the industry. The priority now is to develop alternatives to soil pesticides for in-crop control of wireworms.

Key words: wireworm, potato, risk assessment, control, IPM

Introduction

Wireworms, the soil-dwelling larvae of click beetles, are widely distributed throughout the UK. They have the potential to attack a wide range of crops including cereals, sugar beet, carrot and other vegetables (Miles, 1942) and soft fruit, but the most serious damage usually occurs on potato. Potato crops are particularly susceptible to attack as wireworm damage to tubers reduces crop quality rather than gross yield, and even low populations (<100,000 ha-1) can cause an economic level of damage (see reviews by Jansson & Seal, 1994; Parker & Howard, 2001). Click beetles in the UK have long life cycles, and wireworms in the soil take up to four years to complete their development, making them a very persistent problem. Traditionally, most potato crops are grown in arable farming areas in eastern England where rotations that include long-term grass (a crop usually carrying high wireworm populations) are uncommon. Consequently, wireworms were until recently regarded as a minor but locally important pest in mixed arable and livestock farming areas (e.g. western England and Wales) where grassland is still common. However in the last 15 years, wireworm damage has become an increasing problem for all potato growers in England (including those in the east) and Wales and to some extent in Scotland. More regular damage has also occurred in fields with no history of long-term grass. The reasons for this apparent shift in pest status of wireworms are not entirely clear. Contributory factors may include increasingly stringent quality demands from retailers and the renting of wireworm-infested grass fields as ‘clean’ potato land free of soil-borne disease and potato cyst nematode. There is also some evidence that long-term set-aside (one to five years fallow) provides a suitable habitat for wireworms (Hancock et al., 1992). Another common perception is that the long-term decline in soil residues of persistent organochlorine insecticides such as aldrin, lindane and DDT may also be allowing more wireworms to survive. Although residues of such insecticides are known to persist for at least six years after the last application (Strickland et al., 1962), there is no evidence of a causal link between long-term wireworm population changes and soil

87 88

insecticide residues. Nonetheless, the use of organochlorine insecticides (principally lindane and aldrin) remained the mainstay of wireworm control in the UK until well into the 1990s, and their apparent effectiveness effectively stifled further research into alternative control strategies for wireworms for nearly 30 years (1960s, 70s and 80s). However on potato, the problem of wireworm control on potato in the UK was brought into sharp focus in 1988/89 by the withdrawal of registrations for the use of aldrin on potato for wireworm control. This precipitated the development of new risk assessment and control strategies, and this paper summarises the developments that have occurred between 1990 and the present (2004).

Risk assessment techniques

Post-1990, it became clear that the potato industry required more effective risk-assessment techniques to allow wireworm-infested fields to be identified reliably, as the only truly effective method of preventing wireworm damage was to grow potatoes in uninfested fields. Since the 1940s, assessing wireworm populations in soil had been achieved by soil sampling and extraction of wireworms from soil by flotation (Cockbill et al., 1945). This technique was labour-intensive, prone to high levels of sampling error at low population densities (Yates & Finney, 1942), and was therefore not sufficiently reliable for modern-day needs, particularly with regard to predicting the level of wireworm damage to a potato crop from a given level of wireworm infestation.

Bait trapping Given the limitations of soil sampling, a more reliable wireworm sampling technique was required, and a new bait trapping system (based on earlier work in North America) was developed during the early 1990s (Parker, 1994; Parker, 1996). This work showed that a trap system using a cereal bait could be more effective in detecting wireworms than soil sampling provided soil temperatures were high enough (>5°C) and no alternative food source was available (i.e. the soil was bare). A joint effort between ADAS and (then) Rhône-Poulenc Agriculture (now Bayer CropScience) in 1993 put the system out to a wide-scale test with potato growers and advisers (Parker et al., 1994), and a modified version of the bait-trap system is still widely used in the UK today (now promoted by Bayer CropScience). However, despite the uptake of bait trapping by the industry, there was no useable relationship between bait trap catches and subsequent damage to potato (Parker, 1996), and so bait trapping remained in essence a presence/absence test. As with soil sampling, there was therefore no possibility of rationalising insecticide use through the development of a threshold-based risk assessment system.

Site characteristics Accurate assessment of the risk of wireworm infestation could in principle be improved by identifying a suite of site factors that could be easily measured by growers and advisers. These could be used as a back-up to soil sampling or bait trapping where the sampling systems had failed to identify the presence of a wireworm population. The best indicator of wireworm presence or absence is the duration of grassland in the cropping history of individual fields. The proportion of grass fields infested with wireworms is relatively constant at c. 60 to 70% once grass age had exceeded 10 to 15 years. Although a range of other site factors can be used to enhance the risk assessment process, none (singly or in combination) reliably predict wireworm infestation status (Parker & Seeney, 1997).

Pheromone trapping In 1999/2000, work was started in the UK to evaluate the click beetle pheromone trapping system that had become available in Europe. This had already proved to be both effective and reliable against a range of European Agriotes species (Furlan et al., 1997; Furlan & Toth, 1999) and clearly offered the potential for a simple method of assessing click beetle (and hence wireworm) populations without the difficulties associated with sampling soil (either directly or via bait trapping) for wireworms. Work in 2000 and 2001 showed that the three main Agriotes species found in the UK were still Agriotes obscurus, A. lineatus and A. sputator, and that the pheromone traps were both specific and highly sensitive (Furlan et al., 2002). A large-scale test of the system with potato growers in the UK in 2002/03 showed that there was a potentially useful relationship between click beetle trap catches and residual wireworm populations in the soil, and that low wireworm populations could be easily detected in most fields. The work also showed that wireworms were commonly present at low levels in arable fields with no grass history. The pheromone trap system does have some practical limitations. In particular, because the main peak of adult beetle activity is after the optimum time for potato planting, pheromone traps have to be used as an ‘early warning system’ up to one year in advance of planting potatoes. This is a drawback for those potato growers who regularly rent ‘clean’ land for potato production, as they do not make field selection decisions one year in advance. However, the pheromone traps were well-received by potato growers and advisers in 2003, and the traps will be available commercially in the UK for 2005. The success of this risk assessment work has highlighted 1) that such trap systems have to be both useful and economic for growers to take them up. This requires practically-orientated research and the direct involvement of growers and agronomists; 2) the involvement of trap manufacturers and agrochemical interests is required to ensure the trap system is commercially available. The introduction of the pheromone trap system has also raised many new research questions, including the need to understand the factors influencing wireworm survival and build-up in arable rotations.

Wireworm control Effective, environmentally friendly means of controlling wireworms in the potato crop are still lacking. Although in the UK alternative pesticides following the withdrawal of aldrin are available (ethoprophos and fosthiazate) and often as effective as aldrin (e.g. Parker et al., 1990), these organophosphorous (OP) insecticides only reduce wireworm damage. They are also seen by some as environmentally undesirable. Work on the future is likely to concentrate on biocontrols and novel biofumigants rather than alternative conventional chemistry. However, novel solutions require considerably more research and will almost certainly have to be used in conjunction with other control measures in an integrated wireworm management programme.

Acknowledgements

Work on wireworms in the UK summarised here was variously funded by the British Potato Council, Rhône-Poulenc Agriculture Ltd and the UK Department for the Environment, Food and Rural Affairs (Defra, formerly MAFF).

References

Cockbill, G.F., Henderson, V.E., Ross, D.M. & Stapely, J.H. 1945: Wireworm populations in relation to crop production. I. A large-scale flotation method for extracting wireworms from soil samples and results from a survey of 600 fields. – Annals of Applied Biology 32: 136-148. Furlan, L. & Toth, M. 1999: Evaluation of the effectiveness of the new Agriotes sex pheromone traps in different European countries. – Proceedings of the XX IWGO Conference: 171-175. Furlan, L., Toth, M., Parker, W.E., Ivezic, M., Pancic, S., Brmez, M., Dobrincic, R., Barcic, J.I., Muresan, F., Subchev, M., Toshova, T., Molnar, Z., Ditsch, B. & Voigt, D. 2002: The efficacy of the new Agriotes sex pheromone traps in detecting wireworm population levels in different European countries. – Proceedings of the XX1st IWGO Conference: 293-303. Furlan, L., Toth, M. & Ujvary, I. 1997: The suitability of sex pheromone traps for implementing IPM strategies against Agriotes populations (Coleoptera: Elateridae). – Proceedings of the XIX IWGO Conference (Abstract): 6-7. Hancock, M., Ellis, S., Green, D.B. & Oakley, J.N. 1992: The effects of short- and long-term set-aside on cereal pests. – BCPC Monograph No. 50 ‘Set Aside’: 195-200. Jansson, R.K. & Seal, D.R. 1994: Biology and management of wireworm on potato. – Proceedings of the International Conference on 'Advances in Potato Pest Biology and Management': 31-53. Miles, H.W. 1942: Wireworms and Agriculture. – Journal of the Royal Agricultural Society of England 102: 1-13. Parker, W.E. 1994: Evaluation of the use of food baits for detecting wireworms (Agriotes spp, Coleoptera: Elateridae) in fields intended for arable crop production. – Crop Protection 13: 271-276. Parker, W.E. 1996: The development of baiting techniques to detect wireworms (Agriotes spp, Coleoptera: Elateridae) in the field, and the relationship between bait-trap catches and wireworm damage to potatoes. – Crop Protection 15: 521-527. Parker, W.E., Clarke, A., Ellis, S.A. & Oakley, J.N. 1990: Evaluation of insecticides for the control of wireworms (Agriotes spp) on potato. – Tests of Agrochemicals and Cultivars 11: 28-29. Parker, W.E., Cox, T. & James, D. 1994: Evaluation of the use of baited traps to assess the risk of wireworm damage to potato. – Proceedings of the Brighton Crop Protection Conference - Pests & Diseases: 199-204. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the United Kingdom. – Agricultural & Forest Entomology 3: 85-98. Parker, W.E. & Seeney, F.M. 1997: An investigation into the use of multiple site characteristics to predict the presence and infestation levels of wireworms (Agriotes spp, Coleoptera: Elateridae) in individual grass fields. – Annals of Applied Biology 130: 409- 425. Strickland, A.H., Bardner, H.M. & Waines, R.A. 1962: Wireworm damage and insecticide treatment of the ware potato crop in England and Wales. – Plant Pathology 11: 93-107. Yates, F. & Finney, D.J. 1942: Statistical problems in field sampling for wireworms. – Annals of Applied Biology 29: 156-167. Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 91-100

An IPM approach targeted against wireworms: What has been done and what has to be done

Lorenzo Furlan Department of Agronomy, Entomology, University of Padova, Agripolis, via Romea 16, Legnaro PD; Italy; e-mail: [email protected]

Abstract: The implementation of IPM strategies against wireworms has been extremely difficult until a few years ago because of the shortage of reliable information on the key aspects of the various species. Knowledge needed can be described as follows: a) taxonomic criteria for species determination; b) species distribution; c) biology/ecology of each species; d) methods to predict population density; e) economic thresholds for the different crops and f) effectiveness of different control strategies. A review of the research done in Europe, particularly in Italy, over the last twenty years, will be given together with a description of what still needs to be done to complete the information needed to implement IPM strategies in different European countries.

Keywords: wireworms, Elateridae, Agriotes brevis, A. litigiosus, A. lineatus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. proximus, Coleoptera, IPM strategies

Introduction

Generally speaking, wireworms can be described as strong, thin, yellow-brown larvae living in the soil and damaging seeds, seedlings and young plants. Unfortunately, most of the knowledge generated over the past years concerning this pest is generic in nature, without indication of species actually involved. Varying by location and agronomic factors they are larvae mainly belonging to the family Elateridae. From a practical point of view most of the larvae belong to genus Agriotes. Sometimes, however, pest populations can be a mix of different genera including Synaptus, Athous, Melanotus, Hemicrepidius and others. Wireworms are polyphagous and harmful to many important crops in all of Europe. Nevertheless the main damage resulting from the presence of wireworms is not the reduction of yield or of product quality but, indirectly, the effects of pollution caused by the large quantities of insecticides applied to protect crops with limited assessment of the actual presence of economic populations. Extensive studies conducted in Northeastern Italy have demonstrated that less than 5% of the fields planted with maize and sugar beet need to be treated with soil insecticides to control wireworms (Furlan, 1989; Furlan, 1990; Furlan et. al., 1992 b). Despite this, most of the farmers use a soil insecticide or insecticide-treated seed when planting their crops. They do this mainly to control wireworm populations since the information needed to implement an IPM strategy are missing or unknown to them. The knowledge needed to implement an IPM strategy can be described as follows: a) taxonomic criteria for species determination; b) species distribution; c) biology/ecology of each species; d) methods to predict population density; e) economic thresholds for the different crops; and f) effectiveness of different control strategies.

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Results and discussion a. Taxonomy Adults Information available: all species that are important from an agricultural point of view have been well described and several excellent taxonomists have worked and/or are actively working on them (Cate and Platia, 1997; Jeuniaux, 1996; Jagemann, 1955; Laibner, 2000; Leisegneur, 1972; Lohse, 1979; Platia, 1994; Zeising, 1984). Despite the fact that good descriptions are available, the differences between some species may be considered negligible and sometimes may lead to mistakes in identification. For example distinguishing Agriotes sputator from Agriotes brevis or Agriotes lineatus from Agriotes proximus can sometimes be difficult. Several keys to identify the adults are available but they all refer to specific regions and do not include one or more important species present at other sites.

What has to be done: an identification key valid for all of Europe, which is suitable for use by a large group of interested people should be prepared. Currently the closest key to accomplishing this is one developed by Laibner, 2000. It includes the highest number of species presently found in an identification key in Europe.

Larvae Information available: in order to study the biology and the ecology of the different species it is essential to determine the species of larvae collected within fields. Unfortunately there are few taxonomists who have worked or currently work on larval determinations. Those who do usually operate within specific regions, which do not include some important species (Dolin, 1964; Dolin, 1978; Emden, 1945; Kausnitzer, 1994; Rudolph, 1974). In most cases the determination of larvae to species is more difficult than that of wireworm beetles. Sometimes very small morphological differences between some species suggest doubts as to their actual separation. This problem may be solved by rearing the “supposed” different species and studying their progeny. However, this is obviously difficult and time consuming. Indirectly information can be obtained by studying sex pheromone communications within the populations. For example two closely related species whose larvae, and sometimes beetles, can hardly be separated by using morphological characters like with A. brevis Candèze and A. sputator L., present substantial differences in their sex pheromone composition. The males of the species are attracted by different blends of pheromone compounds (Tóth et al., 2002a; 2003; Yatsinin et al., 1986). But, the same pheromones actively attract Agriotes proximus Schwarz and Agriotes lineatus L. males (unpublished data) which are considered two different species despite the fact that there are only slight differences between the adults and the larvae look the same. This happens also with Agriotes litigiosus Rossi; rearing the var. laichartingi and the f. typ. it is possible ascertain that the larvae of the two varieties can be clearly distinguished (different shape of the 9th abdominal segment) but adult females present the same pheromone composition and the males of both, presenting different colours, are attracted by the same compound - geranyl isovalerate (Furlan et al., 2001b; Tóth et al., 2003). Similarly Agriotes ustulatus Schäller shows two forms, dark and light coloured (Honek and Furlan, 1995). But the other morphological characteristics of both beetles and larvae are the same and the sex pheromone composition does not differ as well. On the other hand, the same sex pheromone (geranyl hexanoate) actively attracts both Agriotes sordidus Illiger and Agriotes rufipalpis Brullé (Tóth et al., 2002 b) despite the fact that adults of the two species can be clearly separated (yet, the larvae look the same).

What has to be done: a taxonomic key for all of Europe. The variability of the main discriminating characters need to be taken into consideration. The main problems are the separation between the species of the sordidus group (Agriotes sordidus Illiger; Agriotes rufipalpis Brullè, Agriotes medvedevi Dolin…..) and the between Agriotes proximus and Agriotes lineatus and other very close species. In order for more reliable species determinations to be made, the classical morphological approach should be integrated with the genetic one. PCR analyses of males might help to clarify differences between uncertain beetle species and at the same time allow for a reliable determination of larvae, which are very difficult to separate based on morphological characters. b. Species distribution Information available: in some regions, general historical information about harmful wireworm species is available (Dolin, 1964; Platia, 1994; Rusek, 1972a; Tóth, 1984); this information may be corroborated by the observations of the specimens in museums and private collections. Yet in most cases, no precise data about the actual distribution of species in different rural areas are available. Since species behavioural differences can occur and damage to crops can happen at different times during the growing season precise information on the species present within each area would be useful. Currently, pheromone traps suitable for monitoring all the most important Agriotes species in Europe are available. They have proven to be effective in detecting the presence of species, even at very low population levels, and have made it possible to draw the first comprehensive maps of species distribution (Furlan et al., 1997; Furlan et al., 2001; Furlan et al., 2001 a; Furlan et al., 2001 b; Karabatsas et al., 2001; Kudryavtsev et al., 1993; Subchev et al., 2004; Tóth et al., 2001).

What has to be done: in most of the European countries maps accurately describing the distribution of the Agriotes species are not available. Distribution maps should be defined by using the sex pheromone traps within each region previously divided into main zones according to prevalent crop rotations, organic matter content, soil type and precipitation. c) Biology Information available: life cycles differ dramatically between species. Detailed information on the biology of each species forms the bases for implementing an effective IPM strategy. Wireworm species can be divided into two main groups. A) species with adults which do not overwinter, live a few days and lay eggs a few days after swarming: Agriotes ustulatus Schäller, A. litigiosus Rossi, Synaptus filiformis F. B) species with adults which overwinter and live for months. These lay eggs for a long period after adult hardening: Agriotes sordidus Illiger, A. brevis Candeze, A. lineatus L., A. sputator L., A. obscurus L., A. rufipalpis Brullè, A. proximus Schwarz. Reliable information concerning the biology of the different species can be obtained by studying concurrently the phases of their life cycle under laboratory conditions (rearing chambers at constant temperatures), in rearing cages close to natural conditions and in open fields (Furlan 1996; Furlan, 1998, Furlan, 2004). When biological information obtained with the different methods are in agreement, then the overall understanding of the behaviour of the species can be achieved. This may also allow for the extension of this information to different regions, but concurrent biological studies provide for a better overall understanding of the biology under different environments (Furlan et al., 2004). Currently, good biological information is available for the following species: Agriotes ustulatus (Furlan, 1994; 1996; Furlan 1998; Hinkin, 1983), A. sordidus (Furlan, 2004; Furlan et al. 2004), A. brevis (the study conducted in Italy has been completed and is ready to be 94

published; some information has already been made available by Masler, 1982; Rusek, 1972b) A. litigiosus (the study has been completed and ready to be published, some information for the variety tauricus has already been made available by Kosmacevsky, 1955). Crop rotation and availability of food resources through the season, climatic-agronomic conditions (mainly organic matter content) and soil characteristics are the main factors influencing the composition of species communities and larval population density. For the species studied in Italy, the most important factor appears to be crop rotation (Furlan and Talon, 1997; Furlan et al., 2000; Furlan et al., 2002); this is the situation in other regions as well (e.g. Szarukàn, 1977). The presence of meadows and double cropping within the rotation cycle results in a population increase of species belonging to the group B (overwintering as adults).

What has to be done: insufficient information is available for Agriotes obscurus (Langen- buch, 1932; Regnier, 1928; Roberts, 1919; 1921; 1922; 1928; Subklew, 1934), A. sputator (Roberts, 1919; 1922; Kosmacevsky, 1955), A. lineatus (Langenbuch, 1932; Roberts, 1919; Subklew, 1934), while very little data regarding A. proximus, A. rufipalpis, Synaptus filiformis and the other wireworm genera have been collected. Therefore, studies following the methods implemented for the species whose biology has already been completed, should be carried out for all species. Comparative biological studies under different climatic conditions should be conducted for species whose biology has already been described. d) Methods to predict population levels Information available: On the base of the agronomic and climatic characteristics of a field, it is possible to predict if high population densities of the predominant Agriotes species may be present; in order to ascertain the actual population densities the following methods are currently available: soil sampling, bait traps for larvae and adult sex pheromone traps. The oldest method is soil sampling. A shovel or specific soil-sampler is used. Usually, a soil core sample that is 12 cm in diameter and 30 to 60 cm deep is taken depending on the time of the season. After collection, soil cores are placed into Tullgren funnels fitted with a 0.5 cm mesh screen at the bottom. The soil is allowed to dry for at least 30 days in a sheltered place and the larvae that fall into the collecting vials are counted and identified. This collection method is very time consuming and expensive. Also, because of the aggregated distribution of the larvae (Furlan and Burgio, 1999), it is necessary to collect a high number of soil cores to obtain a reliable population estimate. This method, which is useful for research purposes, is not usually applied under farm conditions. In comparison with soil sampling, bait traps are more sensitive, less time consuming, but can be used only in defined conditions. Usually, they are placed in a field using a grid-like soil sampling scheme, provided the soil is bare (traps only work properly if there is no/low presence of CO2-producing roots). More often the traps are made and used according to the description given by Chabert and Blot (1992) – a modified version of traps described by Kirfman et al. (1986). These are comprised of a plastic pot 11cm in diameter with holes in the bottom; the pots are filled with vermiculite, 30 ml of wheat seeds and 30 ml of maize seeds. The pots are moistened before being placed into the soil at just below the soil surface and are covered with an 18 cm diameter plastic lid placed a few cm above the rim of the pot. Traps are checked by hand-sorting the contents after 10 - 15 days. In order to recover most of the larvae and increase capture-precision, the trap contents can be put into Tullgren funnels and processed as described for soil cores. Other similar bait traps models have been suggested by Parker, 1994; Parker, 1996; Parker et al., 1994. The mean processing times of bait traps are faster than soil core samples (Parker, 1994; Furlan, unpublished data). The main limitation of bait traps is that the tangled mass of rootlets of the germinated seeds makes it necessary to search carefully for the wireworms. Moreover, the 95

bait traps will only work at proper temperatures. By far the most sensitive tool is the pheromone trap, as first described by Russian researchers (Oleschenko et al., 1987), but the information supplied is related to the next years’ data. After seven years of work, a “high capacity” trap and different lures suitable for monitoring the most important European Agriotes species (A. brevis, A. lineatus, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus) have been made available for all the European countries (Furlan et al., 2001a).

What has to be done: the bait traps currently available for larval monitoring may not give accurate results depending on the accuracy displayed by those making the counts. A bait trap suitable for catching larvae, but keeping them separated from the tangled mass of rootlets of the germinated seeds, would provide for a quicker and more accurate estimation of wireworm population densities. e) Economic thresholds for the different crops Information available: the sensitivity of crops to wireworm attack is highly variable (Furlan and Toffanin, 1996). Providing the same population density in controlled conditions, it is possible to describe a precise scale of plant sensitivity; for example 10-20 larvae of A. ustulatus will completely destroy two lettuce plants, but will cause no plant lose for cabbage or capsicum (Furlan, unpublished data). The sensitivity of single seedlings or plants and the sensitivity of the crop (agronomic sensitivity) should be also distinguished. Thresholds have to be established for each of the different associations, that being crop-wireworm species and methods used to assess population levels. In available literature it has been possible to find thresholds expressed as number of larvae per sq m with a generic reference to wireworms without any specification as to the species actually involved (e.g. Hinkin, 1976). The first indicative thresholds were also expressed as number of larvae caught by bait traps (Chabert and Blot, 1992). Agriotes species showed a different response to bait traps in one study so it is necessary to assess thresholds for each of the wireworm species (Furlan, unpublished data). In Italy for maize, data collected over 15 years allowed for the defining of significant correlations between the number of larvae per sq m or the average number of larvae per bait trap and the number of damaged maize plants by Agriotes brevis, A. sordidus, A. ustulatus (the threshold for this species is 3-4 times higher than A. brevis - Furlan, unpublished data). Thresholds must also to be established for the crop sowing period: in late spring, very high A. ustulatus populations cannot damage maize stands because most of the larvae are in a non- feeding phase (Furlan, 1998). Virtually non-sensitive or low sensitive crops can be planted in infested fields, while the remaining cultivated soils may be planted with any other crop. Rotation and correct allocation of the crops within a farm might be sufficient to avoid economic damage to the crops without using any specific control tool. The most effective biological strategy is planting where no economic populations are present. Generally speaking, a rational IPM strategy should be based on: A) locating high risk areas for wireworm attack by considering agronomic factors and sex pheromone trap captures; B) planting of sensitive crops in low risk areas; C) among areas at high risk for wireworm attacks, locating areas with actual Agriotes populations over threshold levels by using the new bait traps for larvae (easy, quick, objective): C1: zones where no economic larval population is present: it is possibile to sow sensitive crops without any treatment C2: zones where economic larval populations have been found: 96

C2a: no sensitive crops in the same season or application of an insecticide in the most suitable period to control larvae at the stage(s) suitable for damaging crops; C2b: sensitive crops late in the season or in next year interference with Agriotes life cycle: – tillage in the most suitable period to cause high mortality (maximum presence of eggs and newly hatched larvae) – application of biological treatments including biocidal plants or meals (Furlan et al, 2005) in the period of population development (rotation may be planned taking into consideration these aspects too).

What has to be done: the tool which potentially can facilitate the implementation of IPM strategies is the use of sex pheromone traps since they are easy to use and not time and/or money consuming. Their practical use requires: 1) establishing the biological significance of the pheromone trap catches; the determination of the actual range of attractiveness (studies under way) and of the relationship between males captured and level of the female population. The pheromone traps catch females as well and it is possible, at least for some species, to develop traps which catch mainly females by using floral volatiles (e.g. the same volatiles suitable for attracting Diabrotica females, Tóth and Furlan, unpublished data); it is therefore possible to estimate the swarming of female populations and thus obtain data with a higher biological significance. 2) establishing a reliable correlation between adult trap catches and subsequent larval populations for all the species in different climatic and agronomic (mainly rotation) conditions. The correlation between beetle captures and subsequent larval populations is influenced by many biotic and abiotic factors. Since many variables are involved, it is not possible to pool and analyse all the data, but it is necessary to identify categories. For each category the correlation between beetle captures and larval population density or crop damage has to be studied. In order to define the categories, reliable data in regard to rotation, rain, type of soil, etc., have to be collected for each field where the pheromone traps are deployed. More species can be studied in the same field so that information for one may be valid for several species. For both these aspects, studies are already underway and encouraging data have been obtained (Furlan et al., 1996; Furlan et al., 1997; Furlan et. al., 2001 a; Furlan et al., 2001 b; Furlan et. al., 2001 c). Test replications in many different conditions are needed to meet the practical requirements of the effective implementation of an IPM strategy. In order to identify more precisely the areas with wireworm populations over the threshold where the sex pheromone traps have detected high beetle population densities, thresholds for larvae populations levels (expressed as number of larvae/bait trap) should be found with reference to each combination crop- wireworm species. f) Effectiveness of different control strategies Information available: where economic wireworm populations have been found and where there is no possibility to move the sensitive crop to non-infested fields, different protection options may be considered. The chemical approach to wireworm control has been implemented all over the world and has caused severe side effects (Furlan and Girolami, 1991) and in some cases failed to adequately protect crops (Furlan, 1989; Furlan, 1990; Furlan et al., 1992; Furlan and Toffanin, 1994). The mechanism and the actual effectiveness of the different control methods can be precisely evaluated under controlled conditions (Furlan and Toffanin, 1998; Furlan and Campagna, 2002). Unfortunately, biological, effective, practical and low cost strategies suitable for protecting sensitive crops from wireworm attack in these fields have been lacking. Biocidal plants and meals (Furlan et al., 97

2005) and Metharizium spores are particularly promising. Their potential may be considered comparable to that of chemical insecticides, especially if they are used as a means to interfere in population development, not simply like a substance reducing wireworm populations just before or concurrently to crop planting. Intercropping with wheat or other plants may also be included in the IPM strategy (Furlan and Toffanin, 1996; Vernon et al., 2000).

What has to be done: we need to define a reliable life table: abiotic factors are the main cause of wireworm mortality, but more information on how to increase their effectiveness, and also which parasites might be used from a practical point view to keep wireworm populations under the threshold, are needed. The actual effect of biocidal plants and meals in open field conditions also has to be thoroughly investigated.

Acknowledgements

I would like to thank Dr. Giuseppe Platia for the great help in the taxonomic evaluations and Prof. Richard Edwards for the revision of the manuscript.

References

Cate, P.C. & Platia, G. 1997: New species of Agriotes Eschscholtz (Coleoptera: Elateridae) from Greece, Turkey and Syria. – Zeitschrift der Arbeitsgemeinschaft Österreichischer Entomologen, 49(3-4): 109-113. Chabert, A. & Blot, Y. 1992: Estimation des populations larvaires de taupins par un piège attractif. – Phytoma 436: 26-30. Dolin, V.G. 1964: Litschinki zhuchov-stschelkunov (provolotschniki) evropeiski tschasti SSSR. Kijev, “Urozhaj”: 206 pp.. Dolin, V.G. 1978: Opredelitel licinok zukov – scelkunov fauny SSSR – Kiew (Russian) Emden, F.I. 1945: Larve of British beetles. – 5. Elateridae. – Entomol. Monthly Mag. 81: 13- 37. Furlan, L. 1989: Analisi delle possibilità di riduzione dell’impiego di geosidisinfestanti nella coltura del mais nel Veneto. – L’Informatore Agrario 17:107-115. Furlan, L. 1990: Analisi della possibilità di riduzione dell'impiego dei geodisinfestanti nella bietola da zucchero. – L'Informatore Agrario 5: 73-80. Furlan, L. & Girolami, V. 1991: Limits and side effects of the chemical control of soil insects and necessity of biological control. –IOBC/wprs Bulletin 14(1): 51-52. Furlan, L., Talon, G., Toffanin, F. 1992: Valutazione, in condizioni controllate, dell'azione insetticida di diversi geodisinfestanti sulle larve di elateridi (Agriotes spp.). – Atti Giornate Fitopatologiche 1992(1): 247-256. Furlan, L. 1994: Il ciclo biologico di Agriotes ustulatus Schäller (Coleoptera:Elateridae) nell'Italia Nord-orientale. – XVII Congresso Nazionale di Entomologia, Udine 13-18 giugno 1994: 601-604. Furlan, L. & Toffanin, F. 1994: Valutazione dell'efficacia di differenti strategie di lotta contro le larve di elateridi di due specie diverse (Agriotes ustulatus Schäller, Agriotes brevis Candeze). – Atti giornate fitopatologiche 1994(2): 187-194. Furlan, L. 1996: The biology of Agriotes ustulatus Schäller (Col., Elateridae). I. Adults and oviposition. – Journal of Applied Entomology 120: 269-274. Furlan, L. & Toffanin, F. 1996: Suscettibilità di alcune colture erbacee agli attacchi di diverse specie del genere Agriotes e valutazione dell' efficacia di alcune strategie di protezione biologica. – Atti Giornate Fitopatologiche 1996(1): 215-222. 98

Furlan, L., Tóth, M., Ujvary, I. & Toffanin, F., 1996: L' utilizzo di feromoni sessuali per la razionalizzazione della lotta agli elateridi del genere Agriotes: prime sperimentazioni in Italia. – Atti Giornate Fitopatologiche 1996(1): 133-140. Furlan, L., Tóth, M. & Ujváry, I. 1997: The suitability of sex pheromone traps for implementing IPM strategies against Agriotes populations (Coleoptera: Elateridae). – Proceedings of XIX IWGO Conference, Guimaraes, August 30 - September 5: 173-182. Furlan, L. & Talon, G. 1997: Aspetti entomologici: influenza dei sistemi colturali sulla evolu- zione delle popolazioni dei fitofagi ipogei ed in particolare di Agriotes sordidus Illiger in Modelli Agricoli e Impatto Ambientale, valutazioni aziendali e territoriali. – Raisa, UNIPRESS, Padova: 11-16. Furlan, L. 1998: The biology of Agriotes ustulatus Schäller (Col., Elateridae). II. Larval development, pupation, whole cycle description and practical implications. – J. Appl. Ent. 122: Furlan, L. & Toffanin, F. 1998: Effectiveness of new insecticides used as seed dressing (Imidacloprid and Fipronil) against wireworms in controlled environment. – Atti Giornate Fitopatologiche 1998: 195-200. Furlan, L. & Burgio, G. 1999: Distribuzione spaziale e campionamento di Agriotes ustulatus Schäller, A. brevis Candeze, A. sordidus Illiger (Coleoptera, Elateridae) in Nord Italia. – Boll. Ist. Ent. "G.Grandi" Univ. Bologna 53: 29-38. Furlan, L., Curto, G., Ferrari, R., Boriani, L., Bourlot, G. & Turchi, A. 2000: Wireworm species damaging crops in Po Valley. – Informatore Fitopatologico 5: 53-59. Furlan, L., Tóth, M., Yatsinin, V. & Ujvary, I. 2001 a: The project to implement IPM strategies against Agriotes species in Europe: what has been done and what is still to be done. – Proceedings of XXI IWGO Conference, Legnaro Italia, 27 ottobre – 3 Novembre 2001: 253-262. Furlan, L., Tóth, M., Parker, W.E., Ivezic, M., Pancia, S., Brmez, M., Dobrincic, R., Barcic, J.I., Muresan, F., Subchev, M., Toshova, T., Molnar, Z., Ditsch B. & Voigt, D. 2001 b: The efficacy of the new Agriotes sex pheromone traps in detecting wireworm population levels in different european countries. – Proceedings of XXI IWGO Conference, Legnaro, Italia, 27 ottobre – 3 Novembre 2001: 293-304. Furlan, L., Di Bernardo, A., Maini, S., Ferrari R., Boriani, L., Boriani, M., Nobili, P., Bourlot, G., Turchi, A., Vacante, V., Monsignore, C., Figlioli, G. & Tóth, M. 2001 c: First practical results of click beetle trapping with pheromone traps in Italy. Proceedings of XXI IWGO Conference, Legnaro, Italia, 27 ottobre – 3 Novembre 2001: 277-282. Furlan, L., Di Bernardo, A. & Boriani, M. 2002: Proteggere il seme di mais solo quando serve. – L’Informatore Agrario 8:131-140. Furlan, L. & Campagna, G. 2002: Study on the efficacy of imidacloprid and fipronil as seed dressing in controlling wireworms. – Atti Giornate Fitopatologiche 2002(1): 499-504. Furlan, L. 2004: The biology of Agriotes sordidus Illiger (Col., Elateridae). – J. Appl. Ent. 128(9/10): 696-706. Furlan, L., Garofalo, N. & Tóth, M. 2004: Biologia comparata di Agriotes sordidus Illiger nel Nord e Centro-sud d'Italia. – L'Informatore Fitopatologico 2004(11): 32-37. Furlan, L., Bonetto, C., Patalano, G. & Lazzeri, L. 2005: Potential of biocidal meals to control wireworm populations. – Agroindustria, ISCI, Bologna: in print. Hinkin, S. 1976: Determining the density of wireworms. Rastitelma Zashchita 24(10): 22-25. Hinkin, S. 1983: Biology and ecology of western click beetle Agriotes ustulatus Schäller (Elateridae, Coleoptera). – Rasteniev dni nauki 20(1): 155-122. Honek, A. & Furlan, L. 1995: Colour polymorphism in Agriotes ustulatus (Coleoptera: Elater- idae): Absence of geographic and temporal variation. – Eur. J. Entomol. 92: 437-442. 99

Jagemann, E. 1955: Kovaríkovití - Elateridae. – Fauna CSR, zv.4, Praha, CSAV: 262-263. Jeuniaux, C. 1996: Faune de Belgique. Élatérides (Elateridae). – Institut Royal des Sciences Naturelles de Belgique. Bruxelles: 172 pp. Karabatsas, K., Tsakiris, V., Zarpas, K., Tsitsipis, J.A., Furlan, L. & Tóth, M. 2001: Seasonal fluctuation of adult and larvae Agriotes spp. – Proceedings of XXI WGO Conference, Legnaro Italia, 27 ottobre – 3 Novembre 2001: 269-276. Kamm, J.A.., Davis, H.G. & McDonough, L.M. 1983: Attractants for several genera and species of wireworms (Coleoptera:Elateridae). – Coleopt Bull 37:16-18. Kirfman, G.W., Keaster, A.J. & Story, R.N. 1986. An improved wireworm (Coleoptera: Elateridae) sampling technique for midwest cornfields. – J. Kans. Entomol. Soc. 59(1): 37-41. Klausnitzer, B. 1994: Die Larven der Käfer Mitteleuropas. Vol. 2. Myxophaga, Polyphaga Teil1. – Goecke & Evers Verlag, Krefeld: 118-189. Kosmacevskij, A.S. 1955: Nekotoryje voprosy biologii i ekologii scelkunov. – Uc. zap. Krasnodar. gos. ped. inst. 14: 3-22. Kudryavtsev, I., Siirde, K., Lääts, K., Ismailov, V. & Pristavko, V. 1993: Determination of distribution of harmful click beetle species (Coleoptera, Elateridae) by synthetic sex pheromones. – J. Chem. Ecol. 19:1607-1611. Laibner, S. 2000: Elateridae of the Cszech and Slovak Republics Ceské a Slovenské repu- bliky. – Kabourek Ed.: 292 pp. Langenbuch, R. 1932: Beiträge zur Kenntnis der Biologie von Agriotes obscurus L. und Agriotes lineatus L. – Zeitschrift für angewandte Entomologie 19: 278-300. Leseigneur, L. 1972: Coléoptères Elateridae de la faune de France continentale e de Corse. – Bulletin Mensuel de la Société Linnéenne de Lyon 41: 327. Lohse, G.A. 1979: 34. Familie Elateridae. –In: Freude, H., Harde, K.W. & Lohse, G.A. (eds.): Die Käfer Mitteleuropas. Vol. 6. Diversicornia: 103-186. Masler, V. 1982: Skodlivé druhy kovácikovitych (Coleoptera,Elateridae) na Slovensku a ochrana proti nim. – Polnohospodárska veda 3/82, Bratislava: 126 pp. Oleschenko, I.N., Ismailov, V.Y., Soone, J.H., Laats, K.V. & Kudryavtsev, I.B. 1987. A trap for pests. – Byll. Izobretenii 11: 299(in Russian). Parker, W.E. 1994: Evaluation of the use of food baits for detecting wireworms (Agriotes spp., Coleoptera: Elateridae) in fields intended for arable crop production. – Crop Protection 13: 271-276. Parker, W.E. 1996: The development of baiting techniques to detect wireworms (Agriotes spp., Coleoptera: Elateridae) in the field, and the relationship between bait-trap catches and wireworm damage to potato. – Crop Protection 15: 521-527. Parker, W.E., Cox, T. & James, D. 1994: Evaluation of the use of baited traps to assess the risk of wireworm damage to potato. – Proceeding of the Brighton Crop Protection Conference, Pest and Diseases 1994: 199-204. Platia, G. 1994: Coleoptera, Elateridae. –Fauna d' Italia, Vol. 33, Edizioni Calderini, Bologna. Regnier, R. 1928: Les taupins nuisibles en grande culture. Contribution à l’étude de l’Agriotes obscurus L. – Revue de Pathologie végétale et d’Entomologie agricole 15: 40-47. Roberts, A.W.R. 1919: On the life history of wireworms of the genus Agriotes Esch., with some notes on that of Athous haemorroidalis F. Part I. – Ann. Appl. Biol. 6: 116-135. Roberts, A.W.R. 1921: On the life history of wireworms of the genus Agriotes Esch., with some notes on that of Athous haemorroidalis F. Part II. – Ann. Appl. Biol. 8: 193-215. Roberts, A.W.R. 1922: On the life history of wireworms of the genus Agriotes Esch., with some notes on that of Athous haemorroidalis F. Part III. – Ann. Appl. Biol. 9: 306-324. 100

Roberts, A.W.R. 1928: On the life history of wireworms of the genus Agriotes Esch., Part IV. – Ann. Appl. Biol. 15: 90-94. Rudolph, K. 1974: Beitrag zur Kenntnis der Elateridenlarven der Fauna der DDR und der BRD. – Zool. Jb. Syst. 101: 1-151. Rusek, J. 1972 a: Agriotes brevis und Agriotes sordidus (Coleoptera Elateridae) Schalinge in N-Italien. – Redia 53: 321-329. Rusek, J. 1972 b: Die mitteleuropäischen Agriotes- und Ectinus-Arten (Coleoptera, Elater- idae) mit besonderer Berücksichtigung von A. brevis und den in Feldkulturen lebenden Arten. – Academia Praha: 90 pp. Subchev, M., Toshova, T., Tóth, M. & Furlan, L. 2004: Click Beetles (Coleoptera: Elateridae) and their swarming period as established by pheromone traps in different plant habitats in Bulgaria: 1. Meadow. – Acta zool. Bulg. 56 (2): 187-198. Subklew, W. 1934: Agriotes lineatus L. und Agriotes obscurus L. (Ein Beitrag zu ihrer Mor- phologie und Biologie). – Z. ang. Ent. 21: 55-70. Szarukàn, I. 1977: Pajorok (Melolonthidae) és drótférgek (Elateridae) a kite taggazdasàgok talajaiban 195-ben. – Novenyvedelem 13(2): 49-54. Tóth, Z. 1984: Click beetles (Elateridae) in the soils of Central Europe. Their distribution and description. Part I (Gen. Agriotes). – Acta Phytop. Acad. Scient. Hung. 19: 13-29. Tóth, M., Imrei, Z., Szarukan, I., Korosi, R. & Furlan, L. 2001: First results of click beetle trapping with pheromone traps in Hungary 1998-2000. – Proceedings of XXI IWGO Conference, Legnaro Italia, 27 ottobre – 3 Novembre 2001: 263-268. Tóth, M., Imrei, Z., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Subchev, M., Tolasch, T. & Francke, W. 2002a: Identification of the sex pheromone composition of the click beetle Agriotes brevis Candeze (Coleoptera: Elateridae). – J. Chem. Ecol. 28: 1641-1652. Tóth, M., Furlan, L., Szarukán, I. & Ujváry, I. 2002b: Geranyl hexanoate attracting males of click beetles Agriotes rufipalpis Brullé and A. sordidus Illiger (Coleoptera: Elateridae). – J. Appl. Ent. 126: 312-314. Tóth, M., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Imrei, Z., Tolasch, T., Francke, W. & Jossi, W. 2003: Identification of pheromones and optimization of bait composition for click beetle pests in Central and Western Europe (Coleoptera: Elateridae). – Pest Manag. Sci. 59: 1-9. Vernon, R.S., Kabaluk, T. & Behringer, A. 2000: Movement of Agriotes obscurus (Coleo- ptera:Elateridae) in strawberry (Rosaceae) plantings with wheat (graminae) as a trap crop. – The Canadian Entomologist 132: 1-11. Yatsynin, V.G., Karpenko, N.N. & Orlov, V.N. 1986: Sex pheromone of the click beetle Agriotes sputator L. (Coleoptera: Elateridae). – Khim Komm Zhivot, Edition Moskva, Nauka: 53-57 (in Russian). Zeising, M. 1984: Bemerkenswerte Elateridenfunde aus Österreich, der CSSR, Frankreich und Deutschland (Elat.). – Entomologische Blätter für Biologie und Systematik der Käfer 80(1): 61-62. Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 101-104

Strategies to regulate the infestation of wireworms (Agriotes spp.) in organic potato farming: Results

Ute Schepl, Andreas Paffrath Chamber of Agriculture of North Rhine-Westphalia, Department for Organic Farming and Horticulture, Endenicher Allee 60, D-53115 Bonn, Germany

Abstract: Wireworms are a common problem in organic potato farming. Some observations and experiments were made in order to test some approaches which can regulate the wireworm infestation. Four possibilities were tested: Crop rotation, tillage, the use of Neemcake and early harvest. Two different crop rotations were designed. The first one was characterised by a high part of summer crops some like field beans, white cabbage, potatoes and carrots and catch crops. In the second one there were cultivated clover grass, celery, winter wheat, potatoes, winter rye and clover undersowing. In this one the feeding damage by wireworms were obvious higher than in the first crop rotation. Two different times of tillage were chosen in a further experiment. It could be noticed that an immediate tillage before planting had the better regulation effects on wireworms than a tillage five weeks before: potatoes were lower damaged. An additional use of Neemcake had the best regulation effects. In the last experiment the green potato tops were cut off about four weeks before they would have died naturally. Therefore an early harvest could follow. These potatoes were lower damaged by wireworms than the other one which were harvested later.

Keywords: wireworm, potato, crop rotation, tillage, Neemcake, early harvest

Introduction

The potato is the most important root crop in organic farming. It is very good for direct marketing and obtains high market values, but only if the harvested potatoes are not damaged. Wireworms the larvae of click beetles cause feeding damages. The infestation can be seen by the 2 mm holes in the tubers. The most unusual characteristic of the click beetle’s larva is the fact that the larva remains for several years (up to 5 years) instead of undergoing a complete metamorphosis at least once a year. During this time they only feed on organic material. There are different species of plant damaging click beetles in Germany. Agriotes spp. L. is the genus which can be found most often. More and more organic farmers harvest potatoes which are damaged by wireworms. Therefore observations and experiments have been designed in the Centre of Horticulture in Cologne-Auweiler for some years.

Material and methods

The experiments were conducted in the experimental Centre of Horticulture in Cologne- Auweiler in randomized block design with 4 replications each. After clearing an organic orchard with grass underseed the areas were prepared with clover grass and cereals during several years for experiments with stockless organic farming. Most of the experiments were not designed for wireworm control. They had other aims like the comparision of two crop rotations with two different fertilization or the long-term effect of ploughing and not ploughing. In order to detect the feeding damage in potatoes by wireworms composite samples of 100 tubers were scored. Sample means were calculated and ploted.

101 102

Results and discussion

Crop rotation: Since 1998 there is a trial in which two crop rotations will be compared with each other. Many crops in crop rotation one are cultivated in spring: Crop rotation one: field beans+catch crop, white cabbage, potatoes, winter wheat+catch crop and carrots. In crop rotation two cereals occur twice in addition to grass-clover mixture. Crop rotation two: clover grass, celery, winter wheat+undersowing, potatoes, winter rye+undersowing The infestation with wireworms was in crop rotation two significantly higher than in crop rotation one over a period of four years. For both crop rotations obtained the same preconditions. Apparently wireworms had better development conditions in crop rotation two under grass-clover and two winter cereals with undersowing than in crop rotation one without grass-clover and four summer crops. The feeding damage by wireworms was in both crop rotations in 2002 and 2003 at a same level. Probably the very dry field conditions mainly in 2003 stimulated wireworms feeding behaviour in August and September (fig. 1).

% damage by wireworms 100

80 76

60 50 44 42 40 32 24 20 13 13 14 10 6 2 0 1999 2000 2001 2002 2003 2004 CR 1 CR2

Figure 1. Crop rotation and potatoes damaged by wireworms

Usually it is recommended that potatoes should be planted directly in the first year after clover grass. The larvae could feed on living organic matter from the previous year. However in another trial the potato infestation with wireworms also increased in the first year after perennial grass-clover so that no marketable potatoes could be lifted. Presumably there lived quite a few generations of wireworms in the soil due to the preceding perennial cultivation of clover grass.

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Soil tillage and Neemcake: The effect on the potato infestation with wireworms was tested in a tillage experiment by ploughing up clover grass at two different times. The first variant was ploughed five weeks before potato planting in the end of march (early ploughing). In the second variant the soil was ploughed directly before the potatoes were planted in the beginning of May (late ploughing). The least feeding damage of 28% by wireworms was noticed in the second variant. An early tillage entailed significantly an infestation of 36%. Probably more elaterid larvae were active in the upper 30 cm of soil in the beginning of May and could be killed mechanical by ploughing. Through a fertilization with Neemcake (80 kg N/ha) with a content of 6% nitrogen, 3% phosphate and 1% potassium the potato yields could not only be increased significantly, but also the health of tubers could be improved. With a wireworm infestation of 4 % it was remarkably better than in the control (28%) (fig. 2). It still has to be tested if this impact is due to a phytosanitary effect or due to the additional disposition of organic material.

% damage by wireworms 100 + Neemcake 80

40 36 20 28 28 4 0 early ploughing late ploughing early ploughing late ploughing

Figure 2. Tillage, Neemcake and potatoes damaged by wireworms

Early harvest: On an experimental field with an infestation by wireworms, which was known to be high, the potato tops were partially scythed off eight weeks before the estimated harvest date. These potatoes were lifted four weeks later when the skin was firm enough. The potato tops of the remaining potato plants died naturally. These potatoes lifted at the estimated harvest date had always higher damages by wireworms than the tubers lifted earlier (fig. 3). No significant difference in yields could be proved between the two different diggings.

104

% damage by wireworms 100

early harvest late harvest 80 77 77 60 72

40 50

20 28

8 0 30.07.2002 07.08.2003 12.08.2004 04.09.2002 10.09.2003 16.09.2004

Figure 3. Early/late harvest and potatoes damaged by wireworms

Acknowledgements

We thank Theo Pütz and Wilfried Mehl for technical assistance.

References

Anonym 1948: Wireworms and Food Production. A wireworm survey of England and Wales (1939 – 1942). Ministry of Agriculture, Fisheries and Food, HMSO, London, Bulletin No. 128. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to U.K.. – Agricultural and Forest Entomology 3: 85-98. Paffrath, A. 2002: Drahtwurmbefall an Kartoffeln. – Bioland Verbandszeitung 01/2002: 23. Radtke, W., Rieckmann, W. & Brendler, F. 2000: Kartoffeln. Krankheiten – Schädlinge – Unkräuter. – Verlag Th. Mann, Gelsenkirchen: 272 pp. Schepl, U. & Paffrath, A. 2003: Entwicklung von Strategien zur Regulierung des Drahtwurm- befalls (Agriotes spp. L.) im Ökologischen Kartoffelanbau. – In: Beiträge zur 7. Wissen- schaftstagung zum Ökologischen Landbau, Ökologischer Landbau der Zukunft. B. Freyer (ed.), Universität für Bodenkultur, Wien: 133-136. Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 105-108

Status-Quo-Analysis and development of strategies to regulate the infestation of wireworms (Agriotes spp. L.) in organic potato farming

Ute Schepl, Andreas Paffrath Chamber of Agriculture of North Rhine-Westphalia, Department for Ecological Farming and Horticulture, Endenicher Allee 60, D-53115 Bonn, Germany

Abstract: More and more organic farmers lift potatoes which are damaged by wireworms. Therefore a nationwide status quo-analysis was carried out by the Chamber of Agriculture of North Rhine- Westphalia in 2002 and 2003. The survey included an intensive literature research, a questionnaire with 26 relevant questions on site assessment and on farm trials. The results of the evaluation of the questionnaire will be presented here. It was noticed that a long period of grass-clover cultivation in the crop rotation had a negative effect on the quality of potato tubers. Peas and lupines seemed to be the better previous crops for a good quality of tubers in contrast to field beans and red clover. An autumn fertilization with manure resulted in a low feeding damage on potatoes by wireworms. Many perennial weeds some like twich-grass, dock and field thistle had a negative effect on the quality of potatoes fed by wireworms.

Keywords: potato, wireworm, grass-clover mixture, legumes, crop rotation, manure, weed control

Introduction

More and more organic farmers lift potatoes which are damaged by wireworms. Wireworms are the larvae of click beetles. They feed on organic material during their long time of development - up to 5 years. Tubers will be not marketable as food potatoes if feeding damage by wireworms is over 5 %. Therefore a nationwide status quo-analysis was carried out by the Chamber of Agriculture of North Rhine-Westphalia in 2002 and 2003. There were compiled potential reasons and extent of the infestation of wireworms in organic potato farming. It arose new information and development of new strategies against wireworms for advice centres and practice.

Material and methods

A nationwide survey among organic potato growers started in October 2002. Different agricultural advice centres in Germany were involved, too. In addition, the causes for wireworm infestation were analysed in detail on 25 farms. The wireworm damage was delimited from other pests and diseases with similar damage symptoms. New strategies were developed for wireworm reduction. In order to estimate the feeding damage of wireworms potatoes were scored after harvest. 10 kg potatoes on average were scored on holes fed by wireworms.

Results and discussion

Grass-clover in crop rortation A nationwide survey was conducted on reasons, complexity and steps taken for a successful combat against wireworms. Four possibilties were chosen out of the survey in order to find a way to control wireworms. Farmers were asked for the crop rotation they have. First it was

105 106

noticed that grass-clover in crop rotation caused always in a high damage by wireworms. The longer grass clover was part of the crop rotation the higher became damage on tubers (fig. 1). Perhaps there live several generations of elaterid larvae in perennial grass-clover feeding on organic matter.

% damage by wireworms

39 40

30 23 20 13 10 10

0 without grass-clover annual grass-clover two years old grass- triennial grass- clover clover N=44 N=44 N=23 N=7

Figure 1: Grass-clover in crop rotation and potatoes damaged by wireworms

% damage by wireworms

without grass-clover in crop rotation 50 with grass-clover in crop rotation

30 25

19 20 18 11 9 10

2 4 3 0 Vicia faba Pisum sativum Lupinus luteus Trifolium pratense

N=8 N=7 N=4 N=6 N=3 N=9 N=5 N=1

Figure 2: Different legumes as previous crops and potatoes damaged by wireworms 107

Different legumes as previous crops Secondly it was founded out that different previous crops had varied effects on infestation by wireworms. Peas and lupines seemed to be the best previous crops for a good quality of tubers – 2 respectively 3%. Potatoes had a high feeding damage – 18 respectively 19% when field beans and red clover were previous crops. When legumes some like field beans, bush beans, lupines or red clover were cultivated in addition to grass-clover in the crop rotation the feeding damage on potatoes was mostly higher than with legumes only (fig. 2). Some crops have ingredients which have an attractive or deterrent effect on soil pests. Which kind of causes in legumes are given must be investigated yet.

Manure application at different times Farmers were asked for their manuring habits. It seems to depend on the time of manure fertilization whether feeding damages by wireworms occur. An autumn fertilization with manure resulted in a low feeding damage on potatoes by wireworms in the next year. Manure application in spring, summer and winter induced higher defects on potato tubers (fig. 3). Perhaps the condition of short dung is important for the development of the click beetle’s larvae. It has to be investigated if different rotting states of manure can influence the growing of soil pests.

Altitude of weed infestation Farmers were asked for the altitude of weed infestation on potato land they cultivated. It was obvious that many perennial weeds some like twich-grass, dock and field thistle had a negative effect on the quality of potatoes fed by wireworms (fig. 4). Click beetle females will be attracted by the densely leaved weeds and the soil beneath them which don’t dry out quickly. They like it to lay their eggs into such stands. It can go by that high weed densities in agricultural land offer favourable conditions for the development from the egg to the first instars.

% damage by wireworms

21 20 18 15

10 5

0 spring summer autumn winter N=17 N=16 N=11 N=22

Figure 3: Manure at different times and potatoes damaged by wireworms 108

% damage by wireworms 60

30 23

20 15 14

0 low middle strong N=67 N=42 N=8

Figure 4: Perannual weed density and potatoes damaged by wireworms

Acknowledgements

The project was financed by the Federal Ministry of Consumer Protection, Food and Agri- culture in the “Federal Program Organic Agriculture“.

References

Anonym 1948: Wireworms and Food Production. A wireworm survey of England and Wales (1939 – 1942). – Ministry of Agriculture, Fisheries and Food, HMSO, London. Bulletin No. 128. Kolbe, W. 1999: Kulturgeschichte der Kartoffel und ihrer Schaderreger. – Verlag Dr. W.A. Kolbe, Burscheid: 120 pp. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to U.K.. – Agricultural and Forest Entomology 3: 85-98. Paffrath, A. 2002: Drahtwurmbefall an Kartoffeln. – Bioland Verbandszeitung 01/2002: 23. Radtke, W., Rieckmann, W. & Brendler, F. 2000: Kartoffeln. Krankheiten – Schädlinge – Unkräuter. – Verlag Th. Mann, Gelsenkirchen: 272 pp. Schepl, U. & Paffrath, A. 2003: Entwicklung von Strategien zur Regulierung des Drahtwurm- befalls (Agriotes spp. L.) im Ökologischen Kartoffelanbau. – In: Beiträge zur 7. Wissen- schaftstagung zum Ökologischen Landbau, Ökologischer Landbau der Zukunft. B. Freyer (ed.). Universität für Bodenkultur, Wien: 133-136. Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 109-115

Metarhizium anisopliae as a biological control for wireworms and a report of some other naturally-occurring parasites

Todd Kabaluk1, Mark Goettel2, Martin Erlandson3, Jerry Ericsson1, Grant Duke2, Bob Vernon1 Agriculture and Agri-Food Canada: 1 Pacific Agri-Food Research Centre, Box 1000, Agassiz, British Columbia, Canada V0M 1A0; 2 Lethbridge Research Centre, Box 3000, Lethbridge, Alberta, Canada T1J 4B1; 3 Saskatoon Research Centre, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2

Abstract: The discovery of unique isolates of Metarhizium anisopliae from wireworm cadavers gave rise to four years of research to explore the potential of this fungus as a biological control. Field trials alluded to 30% reduction in wireworm damage to potato tubers and resulted in significant in-field infection and mortality of wireworms. The testing of fourteen new isolates against three wireworm species in laboratory bioassays has shown isolate x species interactions far superior than observed in the past, with LT50 (T=time) values as short as 8 days using 106 conidia/g soil. Adults were also found to be highly susceptible to M. anisopliae infection. High concentrations of M. anisopliae conidia in soil caused wireworms to emigrate, but less so when a food source was present. Thermogradient profiling characterized isolates for estimating their activity for use at different soil temperatures, as well as for their potential under commercial production. Investigations into the emergence of M. anisopliae infections in non-inoculated wireworms are being carried out by studying their immunological responses through the development of a method for measuring phenoloxidase activity in the haemolymph. During the course of all of these investigations, several parasitic enemies have been observed and identified.

Keywords: biological control, mycoinsecticide, wireworm, entomopathology, Metarhizium anisopliae

Introduction

In Canada, a few notable efforts have been made to explore Metarhizium anisopliae as a biocontrol for wireworms. Fox and Jaques (1958) reported the occurrence of the fungus in populations of Agriotes sputator and A. lineatus in Nova Scotia in 1951, and carried out bioassays using this enemy-host combination. Zacharuk and Tinline (1960 and 1968, for example) carried out extensive bioassays and Zacharuk (1973) detailed aspects of pathogenesis of M. anisopliae in relating to wireworms. In 1999, an epizootic occurring near Agassiz, British Columbia was exploited and several M. anisopliae isolates were collected from cadavers of A. obscurus and produced en masse for testing and development as a biocontrol agent through laboratory and field experiments that have extended to the present day. During the course of this work, several other isolates and strains were collected from farm fields, acquired from other researchers, and ordered from microbial gene banks. The M. anisopliae / wireworm project encompasses dose-response bioassays and associated wireworm behavioural studies, wireworm immunology, basic studies involving the influence of biotic and abiotic factors and host susceptibility / fungal pathogenicity, and various field trials. Research activities take place among Agriculture and Agri-Food Canada research centres in Agassiz, British Columbia; Lethbridge, Alberta; and Saskatoon, Saskat- chewan. This article reports findings from a sample of studies carried out from 2000-2004.

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Materials and methods

Soil Soil used in all laboratory experiments except for the M. anisopliae isolate x wireworm species bioassays was field-collected silt-loam. Prior to use, it was autoclaved to ensure sterility, followed by screening through fine mesh. Moisture content for all experiments was adjusted to 13% + 2% using tap water. Soil type in the M. anisopliae isolate x wireworm species bioassays was sand.

Wireworms Wireworms were collected from farm fields with no record of pesticide applications. The stock collections were housed in plastic tubs filled with field soil and maintained at 5°C. For A. obscurus and A. lineatus, worms in the 0.028g – 0.039g weight range were used. Ctenicera pruinina were selected at random. All experiments used A. obscurus unless specified otherwise.

Temperature and exposure experiment M. anisopliae conidia were mixed with soil to achieve 106/g air-dry soil. The soil was divided into three batches, and placed in growth chambers set at 6°C, 12°C, and 18°C. Wireworms were placed in the soil. At increasing exposure times, wireworms were removed from the soil, rinsed vigorously with water and incubated individually in sterile soil at 18°C.

Emigration – deterrence of wireworms To create two chambers, two film canisters were glued end to end, with a 0.75cm hole drilled between the chambers. One chamber was filled with sterile soil only (untreated chamber), the other end (treated chamber) filled with: sterile soil with 0, 106, 107, or 108 conidia/g (wet wt) soil, partially germinated wheat seed / no wheat seed. To determine if wireworms were repelled from a M. anisopliae environment, wireworms were placed in the treated chamber and emigration to the untreated chamber measured after 24h. To determine if wireworms were deterred from entering a M. anisopliae environment, wireworms were placed in the untreated chamber and their location measured after 24h.

M. anisopliae isolate x wireworm species bioassays Fourteen isolates of M. anisopliae acquired from wireworm cadavers and various collections of fungi were bioassayed against three species of wireworms: Agriotes obscurus, A. lineatus, and C. pruinina. Wireworms were bioassayed in sterile sandy soil maintained at 9% moisture with conidial concentrations of 106/g wet soil. Conidia were acquired from fresh cultures.

Thermogradient profiling of M. anisopliae isolates Experiments were carried out by pipetting 5.0x103 conidia of each of 14 M. anisopliae isolates onto plates of potato dextrose agar. After an initial 48h incubation period, the isolates were grown in a thermal gradient plate apparatus at 5, 10, 15, 20, 25, 30, 35, and 40°C and colony radial growth was measured every 48h, for up to 10 days.

Click beetle bioassay 0.005 g of M. anisopliae conidia were evenly spread on 100mm diameter filter paper that was placed inside a Petri dish of equal size. To transfer conidia onto adult click beetles, all ten A. obscurus or A. lineatus beetles were left to crawl on the conidia-treated filter paper surface for 20 minutes. For another treatment, two of ten beetles were left to crawl on the filter paper. Following this exposure, beetles were placed in a clean Petri dish with untreated moistened filter paper and food and mortality measured over time.

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Mortality of wireworms from field treatments The experimental unit was a core of soil measuring 15cm diameter and 12cm deep. The first treatment consisted of 3.68g of M. anisopliae granules (formulated conidia) spread 1cm deep within the 15cm diameter circular area. The resulting conidia concentration was 63,775,510 conidia/cm2. One hundred wheat seeds were added to this area as a wireworm attractant. The second treatment consisted of free conidia mixed with soil from the entire core resulting in 4,193,550 conidia/cm3. Wheat seeds were added to the area 1cm below the circular surface as above. The third treatment consisted of 100 wheat seeds coated with conidia (4.16 x 107 conidia/seed) and distributed as for seeds above. Wireworm field mortality and mortality of living wireworms from the cores and incubated in the laboratory were measured over time.

Field applications of broadcast preplant incorporated (BCPPI) M. anisopliae granules In six field experiments over 4 years, BCPPI granules were applied to achieve 2.5 x 1014 conidia/hectare and immediately incorporated into the soil using either a rototiller or s-tine implement. Potatoes were planted into the treated area and the number of wireworm feeding holes in the seed tuber or new tubers measured, as was yield and size of new tubers.

Non-target organisms Carabus granulatus, Agonum sp., Pterostichus melanarius, Hippodamia convergens, and predateous-, plant-, fungus-, and bacteria-feeding nematodes were exposed to conidia of M. anisopliae in a variety of bioassays. Methods of treatment included dusting, bathing, submersing, or exposure to conidia in soil.

Phenoloxidase activity Larvae of A. obscurus were injected between the 3rd and 4th anterior segments with 1.43 x 107 conidia/mL suspended in 0.005% Triton X-100 PBS (carrier and non-injected controls were also included). Phenoloxidase was measured spectrophotometrically at increasing time intervals following injection.

Results and discussion

Temperature and exposure experiment Figure 1 shows that at 18°C, wireworms must be exposed to conidia-treated soil for a minimum of 24h before infection will take place (or in the range 12-24h, which remains untested). At 12°C, this minimum was 48h, and the progress of infection was significant delayed. No morality resulted from exposure to conidia-treated soil at 6°C (data not shown).

100 35 90 18C 12C 30 80 Duration of exposure 25 70 0 hours 60 12 hours 20 240 hours (total mortality) 50 24 hours 40 48 hours 15

% MAM 96 hours 30 10 20 240 hours 5 10 0 0 0204060800 20406080 Number of days incubated (from beginning of exposure period)

Figure 1. Percent Metarhizium-associated mortality (MAM) of Agriotes obscurus larvae in response to an increasing duration of exposure to conidia-treated soil at 18oC and 12oC. 112

Emigration – deterrence of wireworms The rate of wireworm emigration from a soil-conidia environment was proportional to the concentration of conidia, and according to the presence of food (Figure 2). Because emigration was measured after 24h, a high proportion of wireworms (25-40% (no food); 15- 20% (food)) may not achieve the minimum 24h exposure time for infection to occur as shown in Figure 1. Conversely, wireworms appeared slightly deterred from entering a soil-conidia environment, but not significantly.

M. anisopliae isolate x wireworm species bioassays C. pruinina was susceptible to almost all of the isolates, with LT50s ranging from 8 to 20 days. Only one isolate failed to induce more than 50% mortality. A. obscurus was highly susceptible to four isolates, with 100% mortality occurring in 16-25 days. Overall LT50s for this species ranged from 11-28 days. A. lineatus was the species most resistant to the isolates in general. The three most pathogenic isolates showed 80-95% mortality in 28-30 days. LT50s ranged from 11-32 days.

45 80 Emigration from M. anisopliae environment Deterence from entering a M. anisopliae environment 40 70

35 60

30 50

ration 25 g 40 20 No Travel % deterred 30 % emi 15 Returnees TTl Deterred 20 10 No food in Met environment 10 5 Food in Met environment

0 0 6 7 8 7 8 01010100 10 10 10 010100 10 10 Conidia / g soil

Figure 2. The effect of a soil-conidia environment on Agriotes obscurus emigration when wireworms were placed in this environment (left graph) and their deterrence from entering this environment (right graph). Total deterred (TTl Deterred) is the sum of worms that did not travel to the Metarhizium anisopliae environment (No Travel) and those that travelled there, but returned (Returnees).

Thermogradient profiling of M. anisopliae isolates The mean maximum radial growth rate of the colonies was 8.81mm/48h with a mean deviation of 2.14mm/48h. The maximum growth rate for the majority of isolates occurred at 30°C. The two remaining isolates showed a maximum growth rate at 25°C. Among all maximum growth rates, the fastest growth was 12.25mm/48h and the slowest was 3.58mm/48h.

Click beetle bioassay Both A. obscurus and A. lineatus adults were susceptible to infection by conidia of M. anisopliae (Figure 3) with 100% mortality occurring in 17 days when all beetles were treated. Conidia were transferred from beetle to beetle, as high mortality occurred when only two beetles were treated. 113

100 100 Total 80 mortality MAM 80 all beetles treated two beetles treated 60 60 not treated Agriotes lineatus 40 40 % mortality 20 Agriotes obscurus 20

0 0 30-May 2-Jun 4-Jun 10-Jun 16-Jun 30-May 2-Jun 4-Jun 10-Jun 16-Jun

Figure 3. Mortality of adult Agriotes obscurus and A. lineatus following exposure to conidia of Metarhizium anisopliae. MAM is Metarhizium-associated mortality.

Mortality of wireworms from field treatments

Wireworms in the field Field wireworms incubated in the lab 70 70 60 After 27 days in the field 60

y 50 After 48 days in the field 50 40 40 30 30 % mortalit % 20 20 10 10 0 0 Granules Conidia- Coated Control Granules Conidia- Coated Control soil mix seed soil mix seed

Figure 4. Mortality of wireworms exposed to inundative applications of Metarhizium anisopliae conidia in the field. Left graph shows infection in the field; right graph shows surviving wireworms from field treatments that were incubated in the laboratory.

Applications of M. anisopliae under field conditions resulted in mortality of wireworms that were attracted to the treatment area by wheat seed (Figure 4). Even though some wireworms did not die, they were determined to be infected, as a high proportion died following incubation in the laboratory. Granular conidia likely provided the highest localized concentration of conidia, and therefore the highest mortality.

Field applications of BCPPI M. anisopliae granules Applications of BCPPI M. anisopliae granules showed a consistent effect of reducing the number of holes per potato tuber, although in individual experiments, the effect was not significant. However, applications did significantly increase the yield of potato (Table 1).

Non-target organisms There were no deaths attributed to M. anisopliae after Carabid granulatus, Agonum sp., Pterostichus melanarius, and Hippodamia convergens were exposed to conidia, nor did M. anisopliae affect fecundity of H. convergens. Soil applied M. anisopliae showed no effect on diversity indices or soil levels of predateous-, plant-, fungus-, and bacteria-feeding nematodes. 114

Table 1. The effect of broadcast preplant incorporated (BCPPI) applications of Metarhizium anisopliae granules on the number of holes per potato tuber and tuber size. Statistically significant difference detected for tuber size only (alpha=0.05).

Tuber Number of holes per cm2 on potato tubers size (g) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 2 Treatment (seed (new (new (new (seed (new (new tubers) tubers) tubers) tubers) tubers) tubers) tubers) BCPPI 8.1a 6.9a 7.6a 4.1a 7.2a 6.3a 76.6a Control 10.3a 12.2a 10.1a 6.0a 12.0a 10.3a 56.0b

Phenoloxidase activity We are looking at phenoloxidase activity and haemocyte load to assess the immunological response elicited by exposure to injected fungal propagules. Phenoloxidase activity was detected within the haemolymph of A. obscurus larvae 15 and 30 minutes after conidia- injections, but was not detected 1, 2, 4, 8, 16, 24, 48, and 72h after injection. These preliminary results suggest that phenoloxidase activity is short-lived within the haemolymph and it is suspected that its activity might be limited the injection site. Injection of conidia caused 100% mortality within 6 days.

Other naturally occurring parasites During the course of this research, several other parasites were encountered. Both Beauveria bassiana and Tolypocladium cylindrosporum were isolated and confirmed pathogenic to A. obscurus. During the course of examining abdominal contents of A. obscurus adults, a dipteran pupa measuring approximately 0.7mm x 1.7mm with one pair of 0.2mm long breathing tubes was discovered inside the abdominal cavity, suggesting that the beetle was the egg laying site of a parasitic fly. The occurrence of Mermithidae nematodes were numerous and have been observed routinely in stock collections of A. obscurus for the past four years. When emerged from the wireworm, these Mermithids are approximately 10cm long and white in colour. In a few field collections of A. obscurus, deutonymphs of mites in the family Acaridae were documented. These mites are phoretic, and non-parasitic to the larvae (Behan- Pelletier, personal communication).

Acknowledgements

We thank Taryn Carlton and Kelly Holowka for their technical contributions to this work. Markus Clodius and Wim van Herk discovered the Dipteran pupa inside the abdominal cavity and the Acaridae mite deutonymphs.

References

Fox, J.S. & Jaques, R.P. 1958: Note on the Green-Muscardine fungus, Metarrhizium anisopliae (Metch.) Sor., as a control for wireworms. – The Canadian Entomologist 89: 314- 315. Zacharuk, R.Y. 1973. Penetration of the cuticular layers of Elaterid larvae (Coleoptera) by the fungus Metarrhizium anisopliae, and notes on a bacterial invasion. – Journal of Inverte- brate Pathology 21: 101-106. 115

Zacharuk, R.Y. & Tinline, R.D. 1968. Pathogenicity of Metarrhizium anisopliae, and other fungi, for five Elaterids (Coleoptera) in Saskatchewan. – Journal of Invertebrate Patho- logy 12: 294-309. Zacharuk, R.Y. & Tinline, R.D. 1960. Pathogenicity of Metarrhizium anisopliae (Metch.) Sor. and Beauveria bassiana (Bals.) Vuill. to two species of Elateridae. – Nature 187: 794-795.

116 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 117-122

Evaluation of different sampling techniques for wireworms (Coleoptera, Elateridae) in arable land

Nina Brunner1, Bernhard Kromp1, Peter Meindl1, Christian Pázmándi2, Michael Traugott2 1 Ludwig Boltzmann Institute of Organic Agriculture and Applied Ecology, Rinnböckstraße 15, 1110 Wien, Austria, 2 Centre for Mountain Agriculture and Institute of Zoology and Limnology, University of Innsbruck, Technikerstraße 13, A-6020 Innsbruck, Austria

Abstract: Wireworms, the larvae of click beetles (Coleoptera: Elateridae), are abundant dwellers of arable soils, feeding on potato tubers, maize roots and other arable crops, thereby often causing economically severe damage. Since no pesticides are allowed for direct control of wireworms in organic farming, assessment systems for detecting wireworm infestation levels and forecasting damage thresholds are urgently needed. However, the basis of any wireworm risk assessment is a proper sampling technique for recording wireworm occurrence and abundance in the soil. The present study addresses this issue as it aims to evaluate the effectiveness of three baiting methods: (1) potato halves, (2) mesh-bags baited with a cereal mixture, and (3) a newly developed, baited subterranean pitfall trap. Moreover, wireworm densities were determined within soil samples which were extracted by a modified Kempson apparatus. The study was carried out in a potato field in Vienna, and a potato and a clover-grass field in middle Burgenland, Eastern Austria. Heavy wireworm infestations were reported for both fields in the previous years. Bi-weekly samples were taken from a grid of 20 sampling points per field in autumn 2003 and from end of April until end of September in 2004. In total, 271 wireworms from five genera were captured in the three study sites. The extraction of the soil samples revealed that wireworm densities were low at all three sites. By comparing the three baiting methods for wireworm catches from 2004, baited mesh-bags and subterranean pitfall traps were more effective in detecting wireworms than potato halves. Our results suggest that the most feasible way to assess the wireworm infestation of a field are cereal baited traps, while the use of potato halves is not effective.

Keywords: Agriotes sp., Adrastus sp., Hemicrepidius sp., sampling techniques, bait traps, subterranean pitfall traps

Introduction

Wireworms, the larvae of click beetles (Coleoptera, Elateridae) are a quite severe pest on potato and maize, especially in organic farming where there are no insecticides allowed to be applied for direct control. The abundance and feeding activity of the larvae is thought to depend on several factors such as crop rotation, ploughing, occurrence of weeds, elaterid species composition, the age structure of larvae as well as soil environmental factors such as humus content, moisture, and temperature (Parker & Howard 2001, Pázmándi & Traugott, 2005). There are two main periods of wireworm activity in Central-Europe, one from April to May and a second from September to October (Parker & Howard 2001). Risk assessment for wireworm infestation levels is needed to estimate the danger of attack for susceptible crops (Parker 1996, Jossi & Bigler 1997, Parker & Howard 2001, Samson & Calder 2003). Therefore, accurate sampling techniques are needed to assess the

117 118

presence and abundance of wireworm populations in arable soils. In general, wireworms can be sampled by two methods, soil samples and bait traps (Parker & Howard 2001). Soil samples extracted by a heat-light extractor may provide reliable estimates of population density since also larvae of the early instars are collected (Jones 1937). The main drawback of this methodology is its labour-intensiveness (high effort of taking and transporting the samples) and limited capacity of the extraction facilities. Our study is based on several investigations evaluating different sampling methods for wireworms mainly from UK and USA (e.g. Ward & Kaester 1977, Doane 1981, Jansson & Lecrone 1989, Parker 1994, Parker 1996, Simmons et al. 1998, Horton & Landolt 2002). Laboratory and field experiments have shown that especially germinating wheat seeds are superior attractants for wireworms (Horton & Landolt 2002). The present study aimed at finding out the most efficient among four different sampling methods by testing them in field trials, including buried potato halves, a method still recommended to farmers by cultivation manuals.

Material and methods

Investigation sites The three investigation sites, two potato fields and one clover-grass field, were located in Eastern Austria. Potato field B in Breitenlee/Vienna had a soil of sandy-loamy silt, potato field D1 and clover-grass field D2 in Draßmarkt/Burgenland were situated on sandy loamy- clayey brown earth. The sites were chosen because of high wireworm occurrence in previous years. In field B, potato was following wheat, in D1 the previous crop was clover-grass. Sampling was performed in September and October 2003 and from April to September in 2004.

Sampling methods Four sampling methods were used: two types of baited traps, mesh-bag and subterranean pitfall-trap, as well as potato halves and soil cores. The bait mixture consisted of wheat and barley (10 ml each, soaked in water for 24 hours) and about 100 ml of soil from the respective fields. The mesh-bags consisted of plastic net (net width 3 x 3 mm), glued together on two sides to produce a pouch of 200 x 200 mm that was baited and tied with a string (Horton & Landolt 2002). The mesh-bags were buried in a depth of 140 mm and taken out with a spade after two weeks of exposure in the soil. The soil surrounding the mesh-bags (“mesh-bag outside”) and the bait-mixture from inside the mesh-bags (“mesh-bag inside”) were taken as separate samples. The subterranean pitfall-trap (developed by M. Traugott and manufactured by the Bavarian State Research Centre for Agriculture) is a plastic tube of 300 mm length and 130 mm in diameter perforated by holes (15 mm in diameter) evenly spread in the upper 200 mm of the tube, with a removable tin can on the bottom and covered by a plastic lid on the top. It was installed at ground level using a drilling machine for fence posts to make a hole of approximately the diameter and length of the tube and then fitted in thoroughly by hand. The bait mixture was filled into the tin can and removed for examination after two weeks. The potato halves were buried in a depth of 140 mm with the cut surface downwards. They were inspected for wireworms and feeding holes after two weeks. The soil cores (140 mm deep, 150 mm in diameter) were taken in monthly intervalls.

Sample examination The soil samples were extracted for wireworms by heat-light extraction in a modified Kempson apparatus (Meyer 1980, 1995). The “mesh-bag outside” soil samples were extracted in a Berlese apparatus. The bait mixtures from inside the mesh-bags and from the 119

subterranean pitfall-traps were hand-sorted for wireworms or extracted by Berlese. Hand- sorting was done after rinsing the material with water over a sieve of 0.5-1 mm net-width to get rid of fine particles and make the investigation of entangled sprouts easier. All wireworms sampled were determined to species level as well as to larval stage by measuring the head- capsule widths (Klausnitzer 1994, Kaupp & Wurst 1997).

Sampling design In the potato fields B and D1, all sampling was performed in the potato planting rows in bi- weekly intervalls. In the clover-grass field D2, samples were taken monthly, except subterranean pitfall-traps, which were inspected bi-weekly. Samples were taken from a grid of 20 sampling positions, situated in four transects, running from border to field centre of the potato fields. There were five sampling positions within each transect. The distance between transects was 30 m and between sampling positions 7 m. Due to the narrow shape of the clover-grass field two transects of 10 positions were chosen and only 10 subterranean pitfall-traps installed. The distance between transects was 20 m and between positions 7 m. At each sampling date, meshbags and potato halves were placed and soil samples were taken from within a 7 × 7 m area around the subterranean pitfall traps so that the same spot was only sampled once. There was at least a distance of 1 m among potato halves, meshbags and soil samples at each sampling date. The subterranean pitfall-traps stayed in their places during the whole sampling season.

Results and discussion

A total of 271 wireworms were captured by the four methods in all three fields (Tab. 1). In the two potato fields, similar total numbers of wireworms were found. In field B Adrastus sp., Agriotes obscurus and Hemicrepidius sp. were dominating, followed by the less abundant Agrypnus murinus and Athous sp. and Agriotes lineatus. In field D1, however, A. obscurus was the only abundant species. In the clover-grass field D2, less wireworms were captured which is partly due to a smaller number of samples, again with A. obscurus being most abundant. The mean catches per meshbag were similar in the three fields, subterranean pitfall trap and potato half did not work due to unfavourable moisture conditions in the clover grass field. As to the efficacy of the used sampling methods, in all three fields the baited mesh-bags turned out to work best. Both in the potato field B and in the clover-grass field D2 a higher number of wireworms was extracted from the surrounding soil than from the bait-mixture inside the mesh-bags, possibly due to a more accurate extraction by Berlese than by hand- sorting. In the potato fields the subterranean pitfall-traps were working second best, while the potato halves were not attractive for wireworms. In the clover-grass field, however, only a single wireworm was caught in the subterranean pitfall-traps. This can be explained by unfavourable soil as well as weather conditions in 2004 when heavy rainfalls flooded the tin cans, and the bait mixture rotted. The same was true for the potato halves. Likewise, in the soil cores only a few larvae could be detected by extraction indicating that overall wireworm densities were low in all three study sites. Fig. 1 shows the seasonal occurrence of the different larval stages of A. obscurus for field D1. During the whole sampling season, the A. obscurus population included larvae of several stages, from three up to five different stages per date. The presence of different cohorts indicates that the field was used for reproduction by this species at least during the last four years. This in turn confirms that wireworm species like A. obscurus are capable of reproducing and infesting field sites which have been under arable cultivation for many years. 120

Furthermore, A. obscurus larvae can also be baited during summer which indicates that the activity and damage of elaterid larvae is not restricted to spring and autumn.

Table 1. Wireworm species composition and individual numbers sampled by meshbags (MB; meshbags outside: MBo, meshbags inside: MBi), subterranean pitfall traps (SPT), potato halves (PH), and soil cores (SC) in two potato fields (B, D1) and one clover-grass field (D2) in Eastern Austria from April to September 2004. n = number of samples taken. X = mean number of wireworms per sample. SE = standard error of mean.

Mbi SPT SC Field Species Mbo n=200 PH n=200 Total n=200 n=200 n=120

B Adrastus sp. 19 9 13 0 0 41 Agriotes obscurus 12 9 2 2 1 27 Hemicrepidius sp. 4 6 15 0 0 25 Agrypnus murinus 2 6 0 0 0 8 Athous sp. 2 3 1 0 0 6 Agriotes lineatus 1 2 0 0 1 4 total 40 35 31 2 2 111 ± SE 0.2 ± 0.04 0.18 ± 0.04 0.16 ± 0.03 0.01 ± 0.01 0.02 ± 0.01 n=180 n=180 n=180 n=180 n=120 D1 Agriotes obscurus 32 35 11 3 1 86 Agriotes ustulatus 5 2 3 0 4 14 Agriotes lineatus 1 7 1 0 1 10 Hemicrepidius sp. 0 0 1 0 0 1 Athous sp. 1 0 0 0 0 1 Agriotes sputator 0 0 0 0 1 1 total 39 44 16 3 7 113 ± SE 0.22 ± 0.04 0.24 ± 0.04 0.08 ± 0.03 0.02 ± 0.01 0.07 ± 0.03 n=100 n=100 n=110 n=100 n=100 D2 Agriotes obscurus 13 12 1 0 1 27 Agriotes ustulatus 4 1 0 0 2 7 Adrastus sp. 6 1 0 0 0 7 Agriotes lineatus 2 1 0 0 3 6 total 25 15 1 0 6 47 ± E 0.25 ± 0.07 0.15 ± 0.05 0.01 ± 0.01 0 0.06 ± 0.03

In concluding our sampling results, wireworms seem to be detected properly in arable soils only by baiting. The low numbers of wireworms extracted from soil cores in comparison to those derived by baited traps was reported earlier by several authors. In arable fields for instance Parker (1994) found 70% of total wireworms in cereal baited traps, followed by 20% in vegetable baited ones, whereas only 8% were extracted from soil cores and 2% from unbaited traps . 121

0 11.05. 27.05. 09.06. 24.06. 08.07. 23.07. 05.08. 20.08. 02.09.

L2 L3 L4 L5 L6 L7 L8

Figure 1. Seasonal occurrence of different instars (L) of Agriotes obscurus larvae in potato field D1/Draßmarkt (Burgenland) in 2004 based on the total catch of all four sampling methods.

For assessing wireworm populations, the main disadvantage of baited traps compared to soil cores of known surface is that bait catches cannot be used to give an estimation of population density per unit area. In Parker’s study (1994), they did not correlate with wireworm numbers derived from soil cores. On the other hand, according to Parker (1994) a standard set of 20 soil cores (10 cm in diameter, 15 cm in depth) has a limit of detection of 62 500 wireworms per hectare, a level of infestation still high enough to cause economic damage, especially in potatoes. Additionally, since the evaluation of bait trap catches is much less labour-intensive than the processing of soil cores, Parker (1994), Jossi & Bigler (1997), Simmons et al. (1998), Horton & Landolt (2002) and other authors recommend cereal-baited traps as most effective method for assessing wireworm populations in fields intended for cultivation of susceptible arable crops.

Acknowledgements

For help with the field work, thanks are due to M. Diethart, E.M. Grünbacher, P. Hann, S. Hofbauer, M. Hofer, M. Kienegger, J. Laibl, J. Prasch, A. Rothmann and C. Trska. For providing the Kempson and the Berlese facilities, we are grateful to E. Meyer, University of Innsbruck/Institute of Zoology, and to W. Waitzbauer, University of Vienna/Institute of Ecology and Conservation Biology, respectively. The study sites were provided and cultivated considerately by the farm managers K. Mayer (Vienna) and F. Gruber (Draßmarkt), which we appreciated greatly. This work was supported by a grant of the University of Innsbruck.

References

Doane, J.F. 1981: Evaluation of a larval trap and baits for monitoring the seasonal activity of wireworms in Saskatchewan. – Environmental Entomology 10: 335-342. 122

Horton, D.R. & Landolt, P.J. 2002: Orientation response of Pacific Coast wireworm (Coleoptera: Elateridae) to food baits in laboratory and effectiveness of baits in field. – The Canadian Entomologist 134: 357-364. Jansson, R.K. & Lecrone, S.H. 1989: Evaluation of food baits for pre-plant sampling of wireworms in potato fields in southern Florida. – Florida Entomologist 72: 503-510. Jones, E.W. 1937: Practical field methods of sampling soil for wireworms. – Journal of Agricultural Research 54: 123-134. Jossi, W. & Bigler, F. 1997: Auftreten und Schadenprognose von Drahtwürmern in Feldkul- turen. – Agrarforschung 4(4): 157-160. Kaupp, A. & Wurst, C. 1997: Nachträge und Ergänzungen. 42. Familie Elateridae. – In: Klausnitzer, B. (ed.): Die Larven der Käfer Mitteleuropas. 4. Band, Gustav Fischer Verlag, Jena, Germany: 330-344. Klausnitzer, B. 1994: 42. Familie Elateridae. – In: Klausnitzer, B. (ed.): Die Larven der Käfer Mitteleuropas. 2. Band, Myxophaga/Polyphaga. Gustav Fischer Verlag, Jena, Germany: 118-189. Meyer, E. 1980: Aktivitätsdichte, Abundanz und Biomasse der Makrofauna. – In: Ökologi- sche Untersuchungen an Wirbellosen des zentralalpinen Hochgebirges (Obergurgl, Tirol), Vol. IV. Janetschek (ed.). Veröffentlichungen der Universität Innsbruck, Austria: 1-54. Meyer, E. 1995: Endogeic Macrofauna. – In: Methods in soil biology. Schinner, Öhlinger, Kandeler & Margesin (eds.). Springer Verlag, Berlin & Heidelberg, Germany: 346-354. Parker, W.E. 1994: Evaluation of the use of food baits for detecting wireworms (Agriotes spp., Coleoptera: Elateridae) in fields intended for arable crop production. – Crop Protection 13(4): 271-276. Parker, W.E. 1996: The development of baiting techniques to detect wireworms (Agriotes spp.) in the field, and the relationship between bait-trap catches and wireworm damage to potato. – Crop Protection 15(6): 521-527. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the U.K. – Agricultural and Forest Entomo- logy 3: 85-98. Pázmándi, C. & Traugott, M. 2005: A stable isotope analysis of wireworms puts new light on their dietary choices in arable land. – IOBC/wprs Bulletin 28(2): 127-132. Samson, P.R. & Calder, A.A. 2003: Wireworm (Coleoptera: Elateridae) identity, monitoring and damage in sugarcane. – Australian Journal of Entomology 42(3): 298-303. Simmons, C.L. & Pedigo, L.P. & Rice, M.E. 1998: Evaluation of 7 sampling techniques for wireworms. – Environmental Entomology 27(5): 1062-1068 Ward, R.H. & Kaester, A.J. 1977: Wireworm baiting: Use of solar energy to enhance early detection of Melanotus depressus, M. verberans and Aeolus mellillus in Midwest cornfields. – Journal of Economic Entomology 70: 403-406

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 123-126

Bait and pheromone trapping of Agriotes sp. in Lower Austria (first results)

Marion Landl1, Lorenzo Furlan2, Johann Glauninger1 1 Institute of Plant Protection (IPS), Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna 2 Department of Agronomy, Entomology, University of Padova; Via Romea 16, I-35020 Legnaro (PD).

Abstract: In some parts of Austria wireworms are an important insect pest, especially in potato crops. Most of the harmful species belong to the genus Agriotes (Coleoptera, Elateridae).The appearance and the distribution of this genus in Austria are unknown in most cases. In the beginning of further extensive studies on species composition in different habitats we monitored a field in one of the main potato production areas of Austria by using the new sex pheromone traps and bait traps for larvae. The following species were caught: A. ustulatus, A. lineatus, A. brevis, A. sputator and A. obscurus. A. ustulatus, A. lineatus and A. brevis were the main species. The presence of A. rufipalpis, A. gallicus, A. sordidus and A. litigiosus could not be proved so far.

Keywords: Wireworm, Agriotes spp., monitoring, Austria

Introduction

In the eastern parts of Austria different wireworms represented within the genus Agriotes (Coleoptera, Elateridae) are harmful pests. Dispersal and species composition of Elateridae species in Austria is unknown. In 2004 we monitored Agriotes spp. in agriculturally important areas. The aims of the study were to examine which species of the genus Agriotes are present in Lower Austria, the flight patterns of the adults and the spatial and temporal distribution of different larval instars.

Material and methods

Location A potato field (90 m x 300 m) in Untermallebarn (16° 10' 11''east, 48° 27' 51''north) near Hollabrunn (60 km north of Vienna) was investigated. The field has been under organic cultivation since 1998. It is situated in the eastern part of Austria which is influenced by the pannonical climate with hot summers and low annual precipitation but moderate coldness in winter. In 2000 and 2001 Lathyrus sp., between 2001 and 2002 a mixture of leguminous plants, followed by sugar beet (Beta vulgaris), wheat (Triticum aestivum) and 2004 potatoes (Solanum tuberosum) were cultivated. The trial site is surrounded by a country road in the south and north, a sweet cumin (Foeniculum vulgare) field in the east and a sugar beet (Beta vulgaris) field in the west.

123 124

Bait trapping 48 bait traps were positioned net-shaped in distances of 20 m x 30 m on the sample section. The traps were dug in the furrow at the same level with the potato and covered with plastic lids. The bait traps (CHABERT and BLOT, 1992) were provided with holes in the bottom and filled with vermiculit, 30 ml maize and 30 ml wheat. This mixture was moistened a few hours before taken to the field. Every 18 to 21 days the traps were refilled. The content was hand- sorted and afterwards kept in Tullgren-funnels for further extractions. The wireworms were counted and will be identified in following investigations.

Pheromone trapping To monitor adults sex pheromone traps (YATLOR funnel traps) were placed in a line in the centre of the field 40 m apart (FURLAN et al., 2001). Trap A was baited concurrently with the lures for A. ustulatus, A. lineatus, A. brevis, trap B with the lures for A. sputator, A. litigiosus, A. obscurus; trap C with the lures for A. rufipalpis, A. gallicus and A. sordidus. Each trap was brought out in four replications. Cap Position: ...... low for A. brevis, medium for A. lineatus and high for A. ustulatus in the same trap;low for A. obscurus, medium for A. sputator, high for A. litigiosus on the same trap. Replacement of the caps:. never for A. brevis, every 45 days for the other ones. Inspections: ...... at least once per week Installation of lures: ...... March 20 A. obscurus, May 15 A. litigiosus, June 5 A. ustulatus, April 15 other species.

Results and discussion

Bait trapping Sixty-six wireworms were captured by bait traps (species determinations are ongoing at the moment).From the end of April 2004 until the beginning of July 2004 low wireworm density could be observed, but the frequency of wireworms increased in the end of the season (Figure 1). From the end of July to the beginning of August 83% of the total amount of wireworms were caught.

40 40

20 16 captured wireworms

10 4 4 2 0 0 7/5/2004 28/5/2004 17/5/2004 5/7/2004 23/7/2004 10/8/2004 date Figure 1. Sum of wireworms/date captured by bait traps (wireworms in soil surrounding traps are not included). 125

Two areas of the field showed the highest infestation: the edge and the centre. Although in literature is mentioned that bait trapping tends to be less effective when alternative food sources are present (PARKER and HOWARD, 2001), it was possible to determine the increase of larval population over one vegetation period by using this method. Trap catches may be influenced by seasonal soil temperature variations (CHABERT and BLOT, 1992) and other climatical influences.

Pheromone trapping A total of 460 beetles were captured during the course of the experiment (this number includes only individuals, which were captured by their own pheromone –not those in traps with a non specific pheromone). A. ustulatus (36%), A. lineatus and A. brevis were captured most frequently. A. sputator and A. obscurus could also be detected. Adults of A. rufipalpis, A. gallicus, A. sordidus and A. litigiosus were not found. A. ustulatus was being swarming in summer from the end of June until the end of the second week in August as shown in Figure 2. The flight peak of A. ustulatus was observed in the first half of July which complies with data by FURLAN (1996). 84,3% of individuals found were swarming in July.

20,00

s 15,00

s u

10,00

. A

5,00

0,00

4 4 4 4 4 4 4 4 4 4 4 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0

......

4 5 5 5 5 6 6 6 7 7 7 8 8

0 0 0 0 0 0 0 0 0 0 0 0 0

......

4 3 2 1 0 8 7 6 5 4 3 1 0

2 0 1 2 3 0 1 2 0 1 2 0 1 date

Figure 2. Swarming pattern of A. ustulatus captured by pheromone traps in 2004. Data average of four replicates. Bars show mean of trap captures every third day. Curve show LLR-smoothing (LLR= local linear regression).

According to the results of adult catches in 2004 it could be expected that A. ustulatus, A. lineatus and A. brevis are the most important pests from an agriculture point of view in this part of Austria. The ongoing identification of wireworms captured in the soil is needed for final conclusions. 126

Acknowledgements

We thank Anton Riedl for allowing us to conduct research on his field. Thanks also go to Johann Jung who has helped with the processing of the samples.

References

Chabert, A. & Blot, Y. 1992: Estimation des populations larvaires de taupins par un piège attractif. – Phytoma 436: 26-30. Furlan, L. 1996: The biology of Agriotes ustulatus Schäller (Col., Elateridae). I. Adults and oviposition. – Journal of Applied Entomology 120: 269-274. Furlan, L., Tóth, M., Yatsinin, V. & Ujvary, I. 2001: The project to implement IPM strategies against Agriotes species in Europe: what has been done and what is still to be done. – Proceedings of XXI IWGO Conference, Legnaro Italia, 27 ottobre – 3 Novembre 2001, 253-262. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the U.K. – Agricultural and Forest Entomology 3: 85- 98.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 127-132

A stable isotope analysis of wireworms puts new light on their dietary choices in arable land

Christian Pázmándi, Michael Traugott Centre for Mountain Agriculture and Institute of Zoology and Limnology, University of Innsbruck, Technikerstraße 13, A-6020 Innsbruck, Austria

Abstract: Wireworms, the larvae of click beetles (Coleoptera: Elateridae), are facultative but severe pests feeding on agricultural crops. Other potential food sources are weeds, litter and soil organic matter, but detailed studies on their feeding ecology are missing. Thus, we investigated their feeding patterns with stable isotope analysis. Laboratory experiments clarified the relationship between the stable isotope composition of the wireworms and their diet. Field data point to Agriotes obscurus as feeding on plant roots only. Hemicrepidius niger is most likely omnivorous as a species, but the individuals seem to specialize on one type of diet.

Keywords: Agriotes obscurus, Hemicrepidius niger, diet, stable isotope analysis, pest

Introduction

Wireworms are the soil living larvae of click beetles (Coleoptera: Elateridae) and a worldwide pest (Parker and Howard, 2001), attacking maize and potatoes and other crops (Hill, 1987). Further observations have been done on wireworms feeding on detritus and decaying plant material (Gunn & Cherett, 1993) and weeds (Maceljski, 1968). Soil organic matter has been mentioned as a possible diet as well (Langenbuch, 1932, Schaerffenberg, 1942). Some species also feed on animals (Schimmel, 1989). The most important pest species all belong to the genus Agriotes: A. obscurus, A. lineatus and A. sputator are a major problem in the U.K. (Parker & Howard, 2001) and in North America since their introduction from Europe (Vernon et al., 2000). The situation in Central Europe is presumably alike. Any risk assessment of potential wireworm damage to agricultural crops, as well as any control strategy, has to be based on a detailed analysis of the dietary choices in the specific environment they occur in. Key environmental factors influencing their food selection might be climate, soil characteristics and cropping as well as cultivation history. Traditional approaches, like feeding observations and gut dissections, as well as recently developed molecular approaches (Symondson, 2002, Traugott, 2003, Juen and Traugott, in press) both have their merits but also their limitations. Feeding observations are simple and cheap, but offer only snapshots at best (Bearhop et al., 2004), and can hardly be used for soil living animals like wireworms. Gut dissections are even less appropriate, as wireworms consume their diet in a liquid state (Langenbuch, 1932), leaving no discernible parts in the gut. DNA-based approaches are powerful and highly specific, identifying even small amounts of prey at the species level. However, they work only for food still present in the gut, but not for food which has subsequently been assimilated into the body (Juen & Traugott, in press). In this paper, we present a stable isotope analysis of food selection in wireworms. This approach utilizes the fact that many chemical elements occur in at least two isotopes, like 12C and 13C for carbon or 14N and 15N for nitrogen. Their standardized ratio is expressed by the delta 13C and delta 15N values, measured in ‰. As the isotope composition of the diet is reflected in the consumer’s tissue (Scheu, 2002), a cumulative view of an animal’s feeding

127 128

history can be obtained (Scheu & Falca, 2000). For A. obscurus, the feeding record of the last four months can be tracked (Pázmándi & Traugott, in prep.). The difference between the carbon isotopic composition of the consumer and its diet is small, showing an elevation of about 0.5 ‰ in the consumer (McCutchan et al., 2003), which is only significant in large samples. In nitrogen, consumer and diet show a clear trophic shift in their isotopic composition, with an average elevation of 2.3 ‰ in the consumer (McCutchan et al., 2003).

Our analysis proceeded in two steps: 1.) Laboratory experiments determined the magnitude of the trophic shift from the diet to the wireworms’ tissue. 2.) The results of these experiments enable us to interpret the isotopic data gathered in the field, from plants, litter, manure, soil organic matter, and wireworms.

Material and methods

Laboratory experiments Laboratory-reared larvae of A. obscurus were kept individually in plastic tubes (42 ml), filled with a moist soil-peat substrate at 18°C. They were fed with germinating wheat up to the beginning of the sixth or seventh instar. Seven larvae were taken from the experiments, and their seventh and eighth abdominal segments were cut off together, dried and weighed into tin capsules. Six samples of the diet were also dried and weighed into tin capsules. Acetanilid was used as the standard. The isotopic composition of carbon and nitrogen of all samples was determined at the Competence Centre for Stable Isotopes at the University of Göttingen (Germany).

Field data The Agricultural School in Rotholz (Austria) cultivates fields in organic practice for more than a decade. We sampled three of them. The fields “Dauergrünland” and “Kappenhofwiese” were permanent grass fields with a humus content (Pázmándi & Traugott, in prep.) of 4.1 % and 6.8 %, respectively. “Au Ost” was an agricultural field with a humus content of 3.6 % which had been cultivated with a mixture of grass and clover for the last fours years. In spring 2004, between late March and mid-April, soil, litter, manure and the most abundant plants (roots and shoots) were sampled. Additional soil cores (about 0.3 x 0.3 x 0.3 m) were taken and the wireworms extracted with a modified Kempson extractor (Meyer, 1980, 1995). All wireworms were identified by morphological characters to species level and developmental stage following the keys of Klausnitzer (1994) and Kaupp & Wurst (1997). Plant roots, litter, manure, soil samples, and the seventh and eighth abdominal segments of 15 sixth, seventh or eighth instar wireworms were dried, prepared for isotope analysis as described above and analysed at the Competence Centre for Stable Isotopes at the University of Göttingen (Germany) as well.

Results and discussion

Laboratory experiments Table 1 shows that there is no significant trophic shift from the wheat fed to the tissue of A. obscurus for delta 13C. The slight increase of 0.29 ± SE 0.28 ‰ was statistically insignificant (t-test, df = 11, t = -1.04, P = 0.32). The existence of a trophic shift for the isotopic composition of carbon is a matter of debate (see McCutchan et al., 2003), and clearly detectable only in large samples, amenable to more statistical rigor. The trophic shift for delta 15N between the diet and A. obscurus is 2.6 ± SE 0.15 ‰ and statistically significant (t-test, df = 11, t = -16.77, P < 0.001). McCutchan et al. (2003) report a 129

trophic shift for delta 15N of 2.4 ± SE 0.42 ‰ between vascular plants and animals, which fits to the data obtained in our study.

Table 1. Delta 13C and delta 15N values in ‰ (ξ ±SE) for wheat, A. obscurus fed with wheat, and the trophic shift between them.

delta 13C delta 15N Wheat (n = 6) -25.6 ±0.16 2.6 ± 0.09 A. obscurus (n = 7) -25.3 ± 0.22 5.1 ± 0.12 Trophic shift 0.29 ± 0.28 2.6 ± 0.15

Field data The most abundant species overall was H. niger (n = 19), followed by A. obscurus (n = 11). We also found Adrastus montanus (n = 4), Adrastus pallens (n = 1) Athous haemorrhoidalis (n = 1), Athous subfusucus (n = 1), Agriotes lineatus (n = 1) and Agrypnus murinus (n = 1). Each field is presented below separately: most delta 13C and delta 15N values roughly matched among the three fields, but those of the soil showed large differences in the delta 13C values.

N 6 15

delta 4

-2 -32-30-28-26-24-22-20

13 delta C

Figure 1. Delta 13C and delta 15N values of plant roots (empty diamonds), litter (triangle pointing upwards) and manure (triangle pointing downwards), soil (light squares), Hemicrepidius niger (filled dark circles) and Agriotes obscurus (dark circles with light centre) from the field “Kappenhofwiese”.

Fig. 1 shows that in the field “Kappenhofwiese” all individuals of H. niger did not feed on soil organic matter, with one possible exception. Extrapolating from the trophic shift as determined for A. obscurus, soil feeders should have delta 13C values slightly more positive than, or equal to, the soil samples, and the delta 15N values should be higher by about 2.6 ‰. Only one individual was within this range, with a trophic shift of 2.84 ‰. Manure and litter could be excluded as a diet by the same reasoning. Plant roots, which clustered together in the 130

lower left hand corner of fig. 1, disqualified as food as well for all but one individual, which could have fed on the roots of Dactylis glomerata and/or Plantago media. Three individuals could be classified as carnivores, as it would take not one but two trophic shifts for 15N to connect them to plant roots of matching delta 15N values. Herbivore prey could be the bridge, being one trophic shift above the plants and one trophic shift below H. niger. All three individuals of A. obscurus were clearly herbivores: many plant roots fit as dietary candidates, but soil, litter and manure do not.

8 N 15 6 delta 4

-2 -32 -30 -28 -26 -24 -22 -14 -12

13 delta C

Figure 2. Delta 13C and delta 15N values of plant roots (empty diamonds), litter (triangle pointing upwards) and manure (triangle pointing downwards), soil (light squares), Hemicrepidius niger (filled dark circles) and Agrypnus murinus (dark hexagonal with light centre) from the field “Au Ost”.

Fig. 2, showing the situation for the field “Au Ost”, confirms carnivory for H. niger. Soil, litter, manure and plant roots were out of the dietary range. The one individual of A. murinus had no food candidate sampled in here as well, with its delta 15N value to close or to far from all potential feeding substrates. Carnivory for A. murinus has been stated by Klausnitzer (1994) and Schimmel (1989). Given the relatively low delta 15N value of the sampled individual, it probably fed on herbivores or detritivores, but not on other carnivores. It is interesting to note that the soil sampled had delta 13C values within the range of C4- plants, which have delta 13C values around – 13 ‰ (Larcher, 1994). Maize, for instance, has a mean delta 13C value of – 11.1 ‰ (Pázmándi & Traugott, pers. obs.). Most likely, maize litter brought in by the wind from an adjacent maize field found its way into the soil, altering its 13C value. One individual of Veronica filiformis had roots with a remarkably high delta 15N value of 10.92 ‰. The other four root samples of V. filiformis, from the fields “Au Ost” and “Kappen- hofwiese”, had delta 15N values between 1.95 ‰ and 4.16 ‰, in the range of other plant roots. A. obscurus was validated as a herbivore by the individual sampled in the field “Dauergrünland” (Fig. 3) as well. The two sampled individuals of H. niger gave diverging clues about their feeding patterns, similar to the situation in the field “Kappenhofwiese”. One individual had a delta 15N value high enough to make it a carnivore. For the other one, the 131

roots of Plantago media fitted as food. The same was true for the one individual sampled of A. haemorrhoidalis, a close relative of H. niger (Klausnitzer, 1994).

N 6 15

delta 4

-2 -32 -30 -28 -26 -24 -22 -20 delta 13C

Figure 3. Delta 13C and delta 15N values of plant roots (empty diamonds), litter (triangle pointing upwards) and manure (triangle pointing downwards), soil (light squares), Hemicrepidius niger (filled dark circles), Agriotes obscurus (dark circle with light centre) and Athous haemorrhoidalis (filled dark hexagon) from the field “Dauergrünland”.

Our stable isotope analysis points to H. niger as an omnivore, classifying it as a type B generalist (Bearhop et al., 2004). Those generalists feed on a mixed diet at the level of the population only: different individuals feed on different types of diet, but a single individual sticks with its feeding habits. Most individuals of H. niger in this study were likely carnivores, two were likely herbivores. Schimmel (1989) classifies H. niger as phytophagous: the relative size of this phytophagous subpopulation is an upcoming subject (Pázmándi & Traugott, in prep.). Summarizing, the feeding patterns of wireworms can be tracked with stable isotope analysis. The next step is to correlate their feeding patterns with environmental parameters in samples from many locations in Central Europe and to filter out the cues for their dietary choices (Pázmándi & Traugott, in prep.).

Acknowledgements

We would like to thank Hannes Haas of the Agricultural School in Rotholz (Austria) for giving us access to their fields and providing all the necessary information, and Anita Juen of our laboratory for assisting with data analysis. This study was supported by a grant from the Austrian Science Fund (FWF, project number P16676) and the regional state of Tyrol.

References

Bearhop, S., Adams, C.E., Waldron, S., Fuller, R.A. & MacLeod, H. 2004: Determining trophic niche width: a novel approach using stable isotope analysis. – J. Anim. Ecol. 73: 1007-1012. 132

Eggers, T. & Jones, T.H. 2000: You are what you eat … or are you? – Tr. Ecol. Evol. 15: 265-266. Gunn, A. & Cherett, J.M. 1993: The exploitation of food resources by soil- and macro- invertebrates. – Pedobiologia 37: 303-320. Hill, D.S. 1987: Agricultural insect pests of temperate regions and their control. – Cambridge University Press, U.K.. Juen, A. & Traugott, M. in press: Detecting predation and scavenging by DNA gut-content analysis: a case study using a soil insect predator-prey system. – Oecologia. Kaupp, A. & Wurst, C. 1997: Nachträge und Ergänzungen 42. Familie Elateridae. – In: Klausnitzer, B. (ed.): Die Larven der Käfer Mitteleuropas. 4. Band. Gustav Fischer Verlag, Jena, Germany: 330-344. Klausnitzer, B. 1994: 42. Familie Elateridae. – In: Klausnitzer, B. (ed.): Die Larven der Käfer Mitteleuropas. 2. Band. Myxophaga/Polyphaga. Gustav Fischer Verlag, Jena, Germany: 118-189. Langenbuch, R. 1932: Beiträge zur Kenntnis der Biologie von Agriotes lineatus L. und Agriotes obscurus L.. – Z. angew. Entomol. 19: 278-300. Larcher, W. 1994: Ökophysiologie der Pflanzen. – Ulmer Verlag, Stuttgart, Germany. Maceljski, M. 1968: Zur Kenntnis der Wechselbeziehungen zwischen Bodenschädlingen, Unkräutern und deren Bekämpfungsmaßnahmen. – Anz. Schädlingskd. 41: 81-84. McCutchan Jr., J.H., Lewis Jr., W.M., Kendall, C. & McGrath, C.C. 2003: Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur. – Oikos 102: 378-390. Meyer, E. 1980: Aktivitätsdichte, Abundanz und Biomasse der Makrofauna. – In: Ökologische Untersuchungen an Wirbellosen des zentralalpinen Hochgebirges (Obergurgl, Tirol). Vol. IV. Janetschek (ed.). Veröffentlichungen der Universität Innsbruck, Austria: 1-54. Meyer, E. 1995: Endogeic Macrofauna. – In: Methods in soil biology. Schinner, Öhlinger, Kandeler & Margesin (eds.). Springer Verlag, Berlin & Heidelberg, Germany: 346-354. Parker, W.E. & Howard, J.J. 2001: The biology and management of wireworms (Agriotes spp.) on potato with particular reference to the U.K.. – Agric. Forest Entomol. 3: 85-98. Schaerffenberg, B. 1942: Der Einfluss von Humusgehalt und Feuchtigkeit des Bodens auf die Fraßtätigkeit der Elateridenlarven. – Anz. Schädlingskd. 18: 133-136. Scheu, S. 2002: The soil food web: structure and perspectives. – Eur. J. Soil Biol. 38: 11-20. Scheu, S. & Falca, M. 2000: The soil food web of two beech forests (Fagus sylvatica) of contrasting humus type: stable isotope analysis of a macro- and a mesofauna-dominated community. – Oecologia 123: 285-296. Schimmel, R. 1989: Monographie der rheinland-pfälzischen Schnellkäfer (Insecta: Coleo- ptera: Elateridae). – Pollichia, Bad Dürkheim, Germany. Symondson, W.O.C. 2002: Molecular identification of prey in predator diets. – Mol. Ecol. 11: 627-641. Traugott, M. 2003: The prey spectrum of larval and adult Cantharis species in arable land: An electrophoretic approach. – Pedobiologia 47: 161-169. Vernon, R.S., Kabaluk, T. & Behringer, A. 2000: Movement of Agriotes obscurus (Coleo- ptera: Elateridae) in strawberry (Rosaceae) plantings with wheat (Gramineae) as a trap crop. – Can. Entomol. 132: 231-241. Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 133-142

Pheromone composition of European click beetle pests (Coleoptera, Elateridae): common components – selective lures

Miklós Tóth1, Lorenzo Furlan2 1 Plant Protection Institute, HAS, Budapest, Herman O. u. 15, H-1022 Hungary 2 Dipartimento di Agronomia Ambientale, Produzioni Vegetali, Entomologia – Università degli Studi di Padova, via Romea, 16 – 3502 Legnaro (PD), Italy

Abstract: Pheromone compositions of Agriotes brevis, A. litigiosus, A. lineatus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator and A. ustulatus (Coleoptera, Elateridae) have been studied. These species include the most important pest click beetles in Europe. Some components proved to be common pheromone components in several species, while other compounds were unique for only one species. Highly attractive lures have been developed on the basis of the above analyses. We studied the performance of these lures in many countries of Western and Central Europe with special regard to selectivity. In the course of the above, Europe-wide trapping tests using the pheromone baits discovered or optimized by our team we were successful in showing out the geographical occurrrence of the 8 most important agricultural pest click beetle species. The application of pheromone traps shows perspectives in the detection, monitoring and establishment of damage thresholds for these species, or in some cases in the direct control through mass trapping.

Keywords: pheromone traps, Agriotes brevis, A. litigiosus, A. lineatus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, Coleoptera, Elateridae, European distribution

Introduction

Wireworms, the larvae of click beetles (Coleoptera, Elateridae), rank among the most important soil-dwelling agricultural pests worldwide. In most countries insecticides are applied to soil on a schedule, without actual risk assessment of wireworm damage, mostly because of the clumsiness and labor-intensiveness of conventional methods of population sampling and density estimation for these pests. In Italy, for example, of the total area treated with soil insecticides, only a small percentage is actually in economic danger of wireworm attack (Furlan, 1989, Furlan et al., 2002). Trapping the adults could assist in making long-term forecast decisions (more than a year) on the need for soil insecticide treatments in the area in question. Similar to other groups of insect pests, sex pheromone baited traps would be ideal monitoring tools (Furlan et al., 1997). Click beetles form a well distinct, more or less uniform group within Coleoptera, both by taxonomy, and by other life habits. Evidence for the existence of long-range sex pheromones within this taxonomic group have been demonstrated for a number of species from different continents (Borg-Karlson et al., 1988, Ivaschenko and Adamenko, 1980, Kamm et al., 1983, Yatsynin et al., 1980). Most studies on this subject deal with Euroasian spp. and originate from scientists from the former Soviet Union (Kudryavtsev et al., 1993, Siirde et al., 1993, Yatsynin et al., 1996). We have recently developed pheromone baits and traps for catching males of all important pest click beetles in Central and Western Europe. Traps and baits were optimized in tests conducted at several sites mainly in Hungary, Italy and Switzerland. The most effective

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pheromone combinations for each species were tested in a Europe-wide comparative effort. In the present paper we summarize results of these studies.

Material and methods

Connected to the genitals of female click beetles there is a bulbous gland-like structure, which emits its content into the ovipositor. According to general opinion this organ stores the pheromone produced by the insect (Oleschenko et al., 1976, Ivaschenko and Adamenko, 1980). Female sex pheromone gland extracts from reared or collected individuals of all species were prepared as described by carefully piercing the pheromone gland by a fine glass capillary and collecting the liquid inside into the capillary (Oleschenko et al., 1976, Ivaschenko and Adamenko, 1980). The samples obtained were dissolved in hexane, and were analysed by capillary gas chromatography – mass spectrometry. The identified structures were synthetized and their biological activity was studied in electrophysiological and field trapping tests. Experiments aimed at the optimization of bait composition, and of trap design were conducted first of all in Italy, Hungary and Switzerland. Traps baited with the optimized baits of each species were sent to many countries of Europe, where local cooperators concurently conducted field trapping tests.

Results and discussion

Agriotes brevis Concerning the pheromone composition of this species we did not find data in earlier literature. Our analyses showed that the pheromone extract was dominated by two components: geranyl butanoate and (E,E)-farnesyl butanoate. These same two compounds proved to be active in the field and the presence of both components was necessary for attraction of males to traps (Tóth et al., 2002a). The optimized bait containing both components in equal amounts captured large numbers of A. brevis in Italy at all sites tested (Fig. 1). Among other countries the brevis bait was specific at sites with good A. brevis populations. Presence of this species was reliably detected by our traps in Slovenia, Austria, and Bulgaria (near Sofia). In Hungary, Romania and Croatia the bait was catching A. sputator probably due to the geranyl butanoate content (see also results and discussion for A. sputator). It is of high interest that at a site with both A. brevis and A. sputator present (Bulgaria, Sofia) very few catches of A. sputator were recorded in brevis baited traps. The presence of geranyl butanoate may explain also catches of A. proximus Schwarz in Portugal (where no A. brevis was caught), as at this site A. proximus catches were observed only in case of such baits which contained geranyl butanoate (alone or in combination; baits for A. sputator and A. lineatus). This phenomenon needs further scrutiny, as the main pheromone component of Russian populations of A. proximus has been identified as (E,E)-farnesyl acetate, and several other farnesyl and geranyl esters (among them also geranyl butanoate) were present as minor or trace components (Yatsynin et al., 1996). In the field the 99:1 mixture of (E,E)-farnesyl acetate and neryl isovalerate was attractive (Yatsynin et al., 1980). Although this latter blend was not tested by us, it is noteworthy, that in our field tests in Portugal traps baited with (E,E)-farnesyl acetate did not catch a single A. proximus. Research for the discovery of the active pheromone composition for Western European populations of A. proximus is underway. Catches of lower numbers of A. acuminatus Stephens at Piemonte (Italy) may also be attributable to the geranyl butanoate content of the bait. (see also discussion about A. sputator). 135

Fig. 1. Click beetle spp. captured in traps baited with the synthetic pheromone of A. brevis in different countries of Europe. Bait composition: geranyl butanoate / (E,E)-farnesyl butanoate in a ratio of 1:1.

Agriotes lineatus L. The main component of the pheromone gland extract was found to be geranyl octanoate in our analyses (Tóth et al., 2003). This compound has been previously described as the main pheromone component in A. lineatus by several authors (Borg-Karlson et al., 1988, Kudryavtsev et al., 1993, Siirde et al., 1993). In our preliminary field activity test in Hungary in 1994 traps baited with geranyl octanoate caught a total of 30 A. lineatus. In the same test the mixture of (E,E)-farnesyl acetate and neryl isovalerate, described earlier as attracting A. lineatus populations in the West Ukraine (Kudryavtsev et al., 1993, Siirde et al., 1993) was inactive in Hungary. Instead of A. lineatus, this bait attracted 93 males of A. ustulatus (see detailed discussion later). In a further preliminary test in Switzerland in 1997 the bait containing 10% geranyl butanoate added to geranyl octanoate (the main pheromone component) caught a total of 273 beetles vs zero in traps baited with only the octanoate. The presence of geranyl butanoate had been reported in this species, (Yatsynin et al., 1991, 1996), but no data on the activity of the binary mixture vs geranyl octanoate alone was published. Based on these results we used a 10:1 mixture in the Europe-wide comparative trials. Large numbers of A. lineatus were captured in almost all countries: United Kingdom, Germany, Austria, Switzerland, Italy, Slovenia, Croatia, Romania, Bulgaria, Greece, Spain, France and also at both sites in Hungary (Fig. 2). The lineatus bait was fairly specific all over Europe, with low catches of A. sputator and A. obscurus at some sites (these species share one component with the lineatus bait, resp. – not shown on figure). In Portugal instead of A. lineatus, again catches of A. proximus were observed (see discussion about A. brevis). Apart from Europe, our baits were successful in capturing A. lineatus also in Canada, where this species had been introduced probably from England (Vernon and Tóth, unpublished). 136

Fig. 2. Click beetle spp. captured in traps baited with the synthetic pheromone of A. lineatus in different countries of Europe. Bait composition: geranyl octanoate / geranyl butanoate in a ratio of 10:1.

Agriotes litigiosus Rossi Russian authors reported geranyl isovalerate as the main pheromone of this species (Yatsynin et al., 1980, Kudryavtsev et al., 1993). However, later scrutiny revealed that these authors had worked with the species A. litigiosus var. tauricus Heyd. (V.G. Yatsynin, personal communication). Our results showed that the same compound occured as the main pheromone component in A. litigiosus populations originating from Italy (Toth et al., 2003). There was no apparent difference between the pheromone composition of "dark" (= var. laichartingi) and "red" [= fenotypus (fen.) typicus] morphological forms of A. litigiosus. The two varieties, which are usually geographically separated, present differences in adult colour and larval morphology. According to observations conducted in Italy and Switzerland swarming patterns are different too (L. Furlan, personal communication). The addition of (E,E)-farnesyl isovalerate or (E)-8-hydroxygeranyl 1,8-diisovalerate, two compounds which proved to be synergistic in A. litigiosus var. tauricus (Yatsynin and Rubanova, 1983) did not influence catches in any of the morphological forms of A. litigiosus (Table 4). Therefore traps baited with geranyl isovalerate alone were used in the Europe-wide trapping tests. The target species A. litigiosus was caught in all Italian test sites, in Austria and Greece (Fig 3). At sites more to the north or to the west no catches were recorded. On a single occasion some specimens of A. ustulatus were captured in traps in Croatia; however, since this result was not repeated, probably it was a result of cross contamination with pheomone samples during handing of the traps.

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Fig. 3. Click beetle spp. captured in traps baited with the synthetic pheromone of A. litigiosus in different countries of Europe. Bait composition: geranyl isovalerate.

Agriotes obscurus L. Our analyses showed geranyl hexanoate and geranyl octanoate as dominant pheromone components in a ratio of 1:4 (Tóth et al., 2003), supporting earlier reports on the presence of these two compounds in A. obscurus (Borg-Karlson et al., 1988, Yatsynin et al., 1996). In contrast to earlier reports on the attractivity of geranyl hexanoate on its own (Kudryavtsev et al., 1993, Siirde et al., 1993), in our tests the presence of both compounds was necessary for attracting adults. No significant difference was observed between 2:1, 1:1 and 1:2 mixture ratios. Our present results support earlier findings in Russia (Yatsynin et al., 1996). Traps baited with the 1:1 above mixture captured large numbers of A. obscurus especially in northern countries, or at sites with humid, cool climate, i.e. in the United Kingdom, Germany, Switzerland, Ticino in Italyh, Slovenia, Croatia, and Romania (Fig. 4). This bait was also very effective in Canada, where the species had been introduced probably from England (Vernon and Tóth, unpublished). Instead of A. obscurus however, another species, A. sordidus Illiger was caught in other parts of Italy, Spain and France, and A. rufipalpis Brullé in Bulgaria and Greece (Fig. 4). The geranyl hexanoate content of the bait may be an explanation of this phenomenon, as this compound is a potent sex attractant for both later spp. (see later).

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Fig 4. Click beetle spp. captured in traps baited with the synthetic pheromone of A. obscurus in different countries of Europe. Bait composition: geranyl octanoate / geranyl hexanoate in a ratio of 1:1.

Fig 5. Click beetle spp. captured in traps baited with the synthetic pheromone of A. rufipalpis in different countries of Europe. Bait composition: geranyl hexanoate. 139

A. rufipalpis Brullé At the beginning of our studies here was no previously published information on the pheromone composition of this species. In our studies no reliable analysis of pheromone gland extracts could be conducted, as we failed to collect female A. rufipalpis in large enough numbers. However, geranyl hexanoate was found to be attractive towards males of the species in the field (Tóth et al., 2002b). Traps baited with this compound captured well in Austria and Serbia (Fig. 5). Especially high numbers were caught in Greece, Romania and Hungary (several sites), which suggests that the pests status of this species may be more important in these latter countries, than previously thought.

A. sordidus Illiger When testing the A. rufipalpis attractant geranyl hexanoate, large numbers of A. sordidus were captured in Italy (Tóth et al., 2002b). No previous information on the pheromone composition of this species was found in the literature. Analysis of gland extracts showed major peaks at the retention times of geranyl hexanoate and (E,E)-farnesyl hexanoate (Tóth et al, 2003). Later field tests revealed that the presence of the farnesyl compound did not influence catches by the geranyl ester, which compound can be used successfully alone for catching A. sordidus. Traps baited with this compound captured large numbers of males in all parts of Italy, France and Spain (Fig. 5). A. sordidus and A. rufipalpis share geranyl hexanoate as main pheromone component. Based on our results it appears that A. sordidus is present only in the Western Mediterranean, while A. rufipalpis is widespread in the Eastern Mediterranean and Central Europe. In Slovenia, where the two areas may overlap, neither species was captured. In Switzerland traps baited with gerany hexanoate captured A. gallicus Lacordaire, in Bulgaria, although in low numbers, Cidnopus pilosus Leske (Fig. 5). The pheromone composition of neither species has been known before. Neither of them is regarded as a pest.

A. sputator L. In previous reports on the pheromone of A. sputator geranyl butanoate was reported as the main component (Siirde et al., 1993, Yatsynin et al., 1986). Indeed, results of our gland extract analyses showed that the extract was dominated by a very large peak of geranyl butanoate (Tóth et al., 2003). In field activity tests in Hungary, geranyl butanoate on its own attracted large numbers of males and the addition of neryl butanoate, earlier claimed to be synergistic (Siirde et al., 1993), had no effect on captures. The addition of (E,E)-farnesyl hexanoate, alone or together with geranyl propionate also had no effect on catches by geranyl butanoate, although these compounds had been reported earlier to be present in pheromone extracts (Yatsynin et al., 1996). Consequently we used geranyl butanoate on its own as a bait for the Europe-wide trapping tests. Most beetles were caught in countries to the north and in Central Europe – United Kingdom, Germany, Switzerland, Croatia, Serbia, Romania, Bulgaria, Austria, Slovenia and Hungary (Fig. 6). Our bait was excellent in capturing A. sputator also in Canada, where the species had been introduced from England (Vernon and Tóth, unpublished). At one of the Italian sites we recorded catches of A. acuminatus. Probably geranyl butanoate is a sex attractant for this species, as it was also captured in traps with the A. brevis bait (which also contains this compound), also in Italy (Fig. 1). There is no mention of the pheromone composition of this species in the literature. The species is not regarded as an important agricultural pest. 140

Fig 6. Click beetle spp. captured in traps baited with the synthetic pheromone of A. sputator in different countries of Europe. Bait composition: geranyl butanoate

Fig 7. Click beetle spp. captured in traps baited with the synthetic pheromone of A. ustulatus in different countries of Europe. Bait composition: (E,E)-farnesyl acetate.

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In the tests in Portugal, where no A. sputator was caught, the traps regularly captured A. proximus. This result inevitably points to the importance of geranyl butanoate in the pheromonal communication of this species (see also A. lineatus).

A. ustulatus Schaller In pheromone extracts of A. ustulatus females originating from Italy our analyses showed (E,E)-farnesyl acetate to be the predominating component in the pheromone gland extract (Tóth et al., 2003), confirming earlier results for populations in Russia (Kudryavtsev et al., 1993, Siirde et al., 1993, Yatsynin et al., 1996). There were virtually no differences in pheromone composition of extracts from the "black", or "red" morphological phenotypes, which are always present in any population, irrespective of source. Consequently we used (E,E)-farnesyl acetate on its own in the bait for our Europe-wide trapping efforts. High captures were recorded in Germany, Switzerland, the northeastern part of Italy, Austria, Slovenia, Croatia, Serbia, Romania, Bulgaria and Hungary (Fig 7). No captures were recorded in the United Kingdom, Portugal, Spain, France, Greece, and other parts of Italy. the species seems to be missing in the Mediterranean.

Acknowledgements

The authors are indebted to all cooperators who participated in the above studies at the different localities. This research was partially supported by grants OTKA T017693 and T029126, and also by grant NKFP OM-00116/2001.

References

Borg-Karlson, A.K., Agren, L., Dobson, H. & Bergström, G. 1988: Identification and electro- antennographic activity of sex-specific geranyl esters in an abdominal gland of female Agriotes obscurus (L.) and A. lineatus (L.) (Coleoptera: Elateridae). – Experientia 44: 531-534. Furlan, L. 1989: Analisi delle possibilità di riduzione dell’impiego di geosidisinfestanti nella coltura del mais nel Veneto. – L’Informatore Agrario 17: 107-115. Furlan, L., Tóth, M. & Ujváry, I. 1997: The suitability of sex pheromone traps for implementing IPM strategies against Agriotes populations (Coleoptera: Elateridae). – Proceedings of XIX IWGO Conference, Guimaraes, August 30 – September 5: 173-182. Furlan, L., Di Bernardo, A. & Boriani, M. 2002: Proteggere il seme di mais solo quando serve. – L’Informatore Agrario 8:131-140. Ivaschenko, I.I. & Adamenko, E.A. 1980: Site of pheromone production in females of the click beetle Selatosomus latus (Coleoptera, Elateridae).– Zool. Zh. 59: 225-228 (in Russian). Kamm, J.A., Davis, H.G. & McDonough, L.M. 1983: Attractants for several genera and species of wireworms (Coleoptera:Elateridae). – Coleopt. Bull. 37: 16-18. Kudryavtsev, I., Siirde, K., Lääts, K., Ismailov, V. & Pristavko, V. 1993: Determination of distribution of harmful click beetle species (Coleoptera, Elateridae) by synthetic sex pheromones. – J. Chem. Ecol. 19: 1607-1611. Oleschenko, I.N., Ivashchenko, I.I. & Adamenko, E.A. 1976: Biological activity of sex pheromones of female click beetles.– Selskokhozyaistvennaya Biologiya 11: 256-258 (in Russian). 142

Siirde, K., Lääts, K., Erm, A., Kogerman, A., Kudryavtsev, I., Ismailov, V. & Pristavko, V. 1993: Structure-activity relationships of synthetic pheromone components in sex communication of click beetles (Coleoptera, Elateridae). – J. Chem. Ecol. 19: 1597-1606. Tóth, M., Imrei, Z., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Subchev, M., Tolasch, T. & Francke, W. 2002a: Identification of the sex pheromone composition of the click beetle Agriotes brevis Candeze (Coleoptera: Elateridae). – J. Chem. Ecol. 28: 1641-1652. Tóth, M., Furlan, L., Szarukán, I. & Ujváry, I. 2002b: Geranyl hexanoate attracting males of click beetles Agriotes rufipalpis Brullé and A. sordidus Illiger (Coleoptera: Elateridae). – J. Appl. Ent. 126: 312-314. Tóth, M., Furlan, L., Yatsynin, V.G., Ujváry, I., Szarukán, I., Imrei, Z., Tolasch, T., Francke, W. & Jossi, W. 2003: Identification of pheromones and optimization of bait composition for click beetle pests in Central and Western Europe (Coleoptera: Elateridae). – Pest Manag. Sci. 59: 1-9. Yatsynin, V.G. & Rubanova, E.V. 1983: Studies on the chemical structure and biological activity of the pheromone of Agriotes tauricus Heyd. (Coleoptera: Elateridae).– Zasch. Zern. Kult. Vred. Bol. Uslov. Int. Zeml. (Krasnodar) 26: 106-114 (in Russian). Yatsynin, V.G., Oleschenko, I.N., Pubanova, E.V. & Ismailov, V.Y. 1980: Identification of active components of the sex pheromones of click beetles Agriotes gurgistanus, A. litigiosus and A. lineatus. –Khim. Sel'sk. Kho. Moscow, Khimiya: 33-35 (in Russian). Yatsynin, V.G., Karpenko, N.N. & Orlov, V.N. 1986: Sex pheromone of the click beetle Agriotes sputator L. (Coleoptera: Elateridae). –Khim. Komm. Zhivot., Edition Moskva, Nauka: 53-57 (in Russian). Yatsynin, V.G., Rubanova, E.V., Orlov, V.N., Lebedeva, K.V. & Bocharova, N.I. 1991: Pheromones of the click beetles Agriotes tadzhikistanicus, A. lineatus, A. meticulosus, A. caspicus (Coleoptera, Elateridae). –Prob. Khim. Komm. Zhiv., Moscow, Nauka: 101-106 (in Russian). Yatsynin, V.G., Rubanova, E.V. & Okhrimenko, N.V. 1996: Identification of female- produced sex pheromones and their geographical differences in pheromone gland extract composition from click beetles (Col., Elateridae). – J. Appl. Ent. 120: 463-466.

Diabrotica

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 145-146

The Monitoring Program for the Western Corn Rootworm (Diabrotica virgifera virgifera LeC.) in Austria 2004

Peter C. Cate Austrian Agency for Health and Food Safety (AGES), Institute for Plant Health, Spargelfeldstr. 191, 1220 Vienna, Austria. Email: [email protected]

Abstract: The 2004 monitoring program for the Western Corn Rootworm (WCR) (Diabrotica virgifera virgifera LeC.) in Austria showed a further westward expansion of this pest. At present it occupies 8000 km2 in the eastern part of Austria, now being distributed in all or parts of the provinces of Burgenland, Niederösterreich, Wien and Steiermark. No specimens were discovered in other parts of the country.

Key words: Diabrotica virgifera virgifera, monitoring, Austria

Introduction

The Western Corn Rootworm (WCR) (Diabrotica virgifera virgifera LeC.) was first discovered in Europe near Belgrade airport in 1992. Since then it has spread continuously through East and Southeast Europe. It was first detected in Austria on 10th July, 2002 in the eastern region of Seewinkel in Burgenland province, near the border to Hungary and Slovakia. It has also been introduced to a number of western European countries by air or land traffic.

Material and methods

The monitoring program for WCR was installed in eastern Austria in 1999, but since the discovery of the first adults it has been expanded to the whole country, and the number and density of monitoring stations increased correspondingly. In 2004 a total of 667 monitoring stations in all provinces were set up, mostly concentrated in the eastern provinces of Burgenland, Niederösterreich and Steiermark. Figure 1 shows the number of traps per province in Austria in 2004. Csalomon® PAL pheromone traps were used at all stations. Depending on maize development, traps were installed at the end of June/beginning of July and monitored until the end of September/beginning of October. The traps were controlled weekly and renewed at the end of July and the end of August. Trap location was determined by GPS. The traps were installed and controlled by the provincial plant protection services, all data then being sent to the federal plant protection service at the AGES, where they were compiled on a country-wide basis and where weekly distribution maps were drawn up. This information was then re- distributed to the provincial offices and the Ministry of Agriculture, Forestry, Conservation and Water Resources.

Results and discussion

Of the 667 traps installed in Austria, beetles were recorded in 326 traps. The grand total of beetles captured was 11156, whereby 9341 were caught in Burgenland province, 1642 in

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Niederösterreich, 140 in Steiermark and 33 in Wien (table 1). In 2004 the influx of WCR along the entire eastern border of the country continued undiminished. Distribution now ranges up to approx. 75 km into Austrian territory, whereby new infections were primarily recorded in the northern and southern areas. In the North the range of the pest increased by approx. 40 kilometers inland, compared to 2003. In the South the increase in range was about 20 kilometers. No beetles were recorded in other parts of the country. In addition to the traps displaced for monitoring purposes AGES installed an additional 147 pheromone and floral traps as well as emergence traps and photo-eclectors for research purposes. In all of these traps 77575 beetles were caught. Further 7269 beetles were caught in traps set up by a commercial agrochemical firm on experimental fields. All of these traps were located in the district of Neusiedl/See in the province of Burgenland, where the highest population density in Austria occurs.

Gmund

206 Krems 7

Linz Wien Wels

Oberösterreich Baden Steyr Niederösterreich 26 Eisenstad Wiener Neustadt Salzburg 22 Kufstein Eisenerz Bregenz 226 Bruck Burgen- Salzburg land Feldkirch 5 Steiermark 9 Innsbruck 160 Bludenz Tirol Landeck Voralberg Graz Tirol Lienz Kärnten 6 Klagenfurt Villach

Figure 1: Monitoring program for WCR in Austria: number of traps per province in 2004

Table 1: Results of the 2004 Monitoring Programme in Austria

Results of the 2004 Monitoring Programme in Austria number of traps number of province number of traps with beetles beetles Burgenland 226 183 9341 Niederösterreich 206 96 1642 Steiermark 160 43 140 Wien 7 4 33 Oberösterreich 26 0 0 Salzburg 5 0 0 Kärnten 6 0 0 Tirol 9 0 0 Vorarlberg 22 0 0 Austria 667 326 11156

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 147-154

Trap types for capturing Diabrotica virgifera virgifera (Coleoptera, Chrysomelidae) developed by the Plant Protection Institute, HAS, (Budapest, Hungary): performance characteristics

Miklós Tóth Plant Protection Institute, HAS, Budapest, Herman O. u. 15, H-1022 Hungary

Abstract: Following the first appearance of the western corn rootworm (Diabrotica v. virgifera) (Coleoptera, Chrysomelidae) in Europe in 1993, several trap types have been developed by our Institute for detection and monitoring purposes. Some of these trap types are baited with the synthetic sex pheromone (therefore they attract and capture only males), while others with a floral attractant (which attracts females and to a lesser extent also males). Each trap type can be recommended for optimal use in different situations. A detailed evaluation of the performance characteristics of these trap types is given.

Keywords: pheromone trap, floral attractant, trap types, trap performance, Diabrotica v. virgifera, Coleoptera,, Chrysomelidae,

Introduction

When the western corn rootworm (WCR) was first detected in Europe (Camprag and Baca, 1995), we felt that very soon a highly sensitive detection device would be needed to follow the spread of this dangerous pest throughout the continent. Since pheromone traps are ideal for the purpose, and the structure of the sex pheromone of WCR was already known (Guss et al., 1982), in our lab we synthetized the sex pheromone, and started the development of trap designs which, when baited with the synthetic sex pheromone, are reasonably efficient in capturing WCR. One drawback of pheromone-baited traps is that they will attract and catch only one sex (in case of WCR the males). However, the capture of females would have definite advantages in an agricultural monitoring system, so the development of another trap capable of catching also females was asked for by many agricultural experts. In case of the WCR flower-derived attractants have been described which attract both females and males (see for a review Metcalf, 1994; Metcalf et al., 1998). We chose for the development of a female-attractive trap 4-methoxy-cinnamaldehyde (MCA) and indole as a floral bait, as these compounds proved to be highly attractive to WCR in tests in the US (Metcalf et al., 1995). In the present paper an overview of our trap development results in the past decade is given.

Material and methods

The sex pheromone 8-methyl-2-decyl propanoate (racemic) was synthetized by described methods (Tóth et al., 2003). The compound was formulated in rubber dispensers (see for details Tóth et al., 1996, 2003) For the floral lure indole (Sigma-Aldrich Kft, Budapest, Hungary) and 4-methoxi- cinnamaldehyde (Bedoukian Inc, Danbury, USA) were purchased commercially and were

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>95% pure as stated by the suppliers. The floral compounds were formulated in sealed polyethylene bag dispensers (see for details Tóth et al., 2003). Field tests were conducted in Yugoslavia (mainly during the first years after the appearance of WCR in Europe) or Hungary, by internationally established methods for such experiments (for detailed description see Tóth et al., 1996, 2003)

Results and discussion

It became clear from the very beginning that conventional sticky “Delta” traps were not very efficient (probably due to difficulties WCR beetles may encounter when trying to home in into the relatively small opening for getting into the trap), and much higher catches can be recorded in trap designs with an outside, open sticky surface (Fig. 1).

Fig. 1. Preliminary comparison of trap designs sticky delta and sticky panel baited with synthetic sex pheromone vs. unbaited for catching WCR (data from Tóth et al., 1996).

In the course of later optimizations, the PAL (= abbreviation of the Hungarian word ”cloak”) trap design was developed (Fig 2), which proved to be highly efficient, and also it could be assembled and set up attached to a maize stem in the field with ease (Tóth et al., 2003). Since the PAL trap is baited with the sex pheromone, it attracts only males, which are then captured on the outside open sticky surface of the trap. We decided to make the trap transparent, so that random captures of non-target insects be kept to a minimum. Information on the spread and occurrence of WCR in European countries has largely been collected by using PAL traps in the past decade. The EU-research project DIABROTICA (QLK5-CT-1999-01110) recommends to use PAL traps baited with pheromone as the standard detection tool for WCR in Europe. 149

Fig. 2. Comparison of several trap designs baited with synthetic sex pheromone vs. unbaited for catching WCR (data from Tóth et al., 2003).

Fig. 3. Effect of yellow colour on WCR captures in sticky “cloak” traps baited with the floral bait (data from Tóth et al., 2003).

In the case of WCR it was also known that certain floral compounds isolated from pumpkin flowers exerted strong attraction towards both sexes of adult beetles (see for a review Metcalf, 1994; Metcalf et al., 1998). Based on this our lab produced a synthetic floral bait targeted for females, as a supplement to the pheromone-baited traps. The floral bait was tested in transparent and yellow PAL-shaped sticky traps, and it appeared that the presence of yellow colour as visual cue was more important for females than for males, increasing female catches significantly (Fig. 3) (Tóth et al., 2003). Our results 150

confirmed the earlier suggestion that colour plus chemical stimuli had greater impact on female beetles (Hesler and Sutter 1993). Therefore, our female-targeted new trap codenamed PALs was produced with a sticky surface in yellow colour. The PALs trap is baited with the floral lure. Although in most situations not as sensitive as the PAL trap with the pheromone, the great advantage of the PALs trap is that it catches predominantly females, and to a lesser extent also males (Tóth et al., 2003). The basic requirement for a sampling tool (i.e. trap) used for the study of quantitative aspects (i.e. estimation of population density, threshold catch levels, etc.) is that it should sample constantly the same proportion of the population over time (= its efficiency should remain constant). (Wall, 1989) Although very sensitive in detection, sticky traps have the inherent deficiency that their efficiency will constantly change over time, which makes them unsuitable for the study of such quantitative aspects. The development of non-saturating, non-sticky traps may be an answer. This is why we set out to develop a high capacity funnel trap for WCR. Our efforts resulted in the VARs+ trap (= abbreviation of the word “funnel” in Hungarian) (Tóth et al., 2000) (Fig. 4).

Fig 4. Cross section diagram of VARs+ funnel trap (after Tóth et al., 2000)

The VARs+ trap can be baited with both the pheromone and floral baits together, thus catching both male and female WCR at high sensitivity and very high selectivity (Tóth et al., 2000). However, for best performance, a killing agent (i.e. small piece of household anti-moth strip with vapour action) should be added to both the upper and lower catch containers of the VARs+ trap, otherwise insects already in the trap can eventually find their way out and escape (Fig. 5). 151

Fig. 5. Catches of WCR in VARs+ traps baited with both peromonal and floral baits at different heights, with or without insecticide added to catch containers.

Experience showed that for detection and monitoring purposes traps are best set up in maize fields (preferably where maize had been cultivated for several years), at least 5-10 m inside the field, below the level of the top of vegetation. However, in a population density study one must bear in mind that sometimes there are very pronounced differences in population density in different areas of the same field. It appears that traps set out at the height of maize cobs capture more than traps at soil level (Figs. 5-6).

Fig. 6. Influence of trap height on catches of WCR in VARs+ traps, baited with both pheromonal and floral baits.

In exhasutive season-long parallel tests the performance of our WCR trap types was evaluated (Imrei et al., 2002a,b). Yellow sticky traps (without chemical bait) were also included in the tests since these are also used for trapping WCR in some areas. 152

Most males were caught in PAL and VARs+ traps (Fig 7). Female catches were highest in PALs and VARs+ traps. Yellow sticky traps (without bait) captured negligible numbers of beetles

Fig. 7. Comparison of performance of sticky and funnel trap types for catching WCR.

Fig. 8. Comparison of percentages of females caught in different sticky and funnel trap types for catching WCR. 153

Table 1. Performance characteristics of WCR trap types (based on results in Imrei et al, 2002a,b, Tóth et al, 2003)

Trap type PAL PALs VARs+ yellow sticky pheromone & no chemical Bait type pheromone floral floral bait females & males & females & Sex caught males males females males Sex ratio (vs. practically higher % of higher % of close to natural natural) 100% males females females Detection highly sensitive sensitive highly sensitive not sensitive with very Monitoring reliable reliable reliable high populations high (>10000 Capacity limited limited limited beetles) Non-target very few - high many very many very many insects caught selectivity

Table 2. Usage characteristics of WCR trap types (based on results in Imrei et al, 2002a,b, Tóth et al, 2003)

Trap type PAL PALs VARs+ yellow sticky pheromone & Bait type pheromone floral no chemical bait floral Assembling easy easy complicated simple and design Maintenance dirty (sticky) dirty (sticky) clean dirty (sticky)

Killing agent sticky material sticky material insecticide sticky material Costs (used for cheaper cheaper more expensive not applicable detection) Costs (used for season-long more expensive more expensive cheaper more expensive monitoring)

When comparing ratio of females in the catch, females in highest ratio were caught in the PALs traps (Fig. 8). PAL traps caught almost exclusively males. The sex ratio in VARs+ traps resembled most closely the natural sex ratio of the population at the test site. All of these trap types are offered to growers as members of the CSALOMON® trap family through the non-profit extension service of the Plant Protection Institute HAS (Budapest, Hungary). Attempts to further improve and simplify the VARs+ funnel trap design are underway and will be reported on in the near future. 154

Performance and usage characteristics of the different trap types are summarized in Table 1-2. It is worth to note that there is no single “best” trap type. Some of the trap types may be advantageous from one viewpoint, others from other viewpoints. The optimal trap type for a given purpose should be selected based on the circumstances and objectives of the study in question.

Acknowledgements

The author is greatly indebted to all cooperators who participated in the above studies at the different localities. Special thanks are due to Drs. I. Sivcev (Zemun, Yugoslavia). This research was partially supported by the EU-research project DIABROTICA (QLK5-CT-1999- 01110) and by grants OTKA T017693, T029126 of HAS.

References

Camprag, D. & Baca, F. 1995: Diabrotica virgifera (Coleoptera, Chrysomelidae); a new pest of maize in Yugoslavia. – Pestic.Sci. 45: 291-292. Guss, P.L., Tumlinson, J.H., Sonnet, P.E. & Proveaux, A.T. 1982: Identification of a female- produced sex pheromone of the western corn rootworm Diabrotica virgifera virgifera. – J. Chem. Ecol. 8: 545-556. Hesler, L.S. & Sutter, G.R. 1993: Effect of trap color, volatile attractants, and type of toxic bait dispenser on captures of adult corn rootworm beetles (Coleoptera: Chrysomelidae). – Environ. Entomol. 22: 743-750. Imrei, Z., Tóth, M., Vörös, G., Szarukán, I., Gazdag, T. & Szeredi, A. 2002a: Comparison of performance of different trap types for monitoring of Diabrotica virgifera virgifera. – Proc. XXI IWGO Conf. VIII Diabrotica Subgr. Meeting, Oct. 27 - Nov. 3, 2001, Legnaro-Padua-Venice: 39-45. Imrei, Z., Tóth, M., Vörös, G., Szarukán, I., Gazdag, T., & Szeredi, A. 2002b: Performance evaluation of different trap types for monitoring of Diabrotica virgifera virgifera. – Növényvédelem 38: 279-287 (in Hung.). Metcalf, R.L. 1994: Chemical ecology of Diabroticites. – In: P.H. Jolivet, M.L. Cox and E. Petitpierre (eds.): Novel aspects of the biology of Chrysomelidae. Kluwer Academic Publishers, The Hague, The Netherlands: 153-169. Metcalf, R.L., Lampman, R.L. & Deem-Dickson, L. 1995: Indole as an olfactory synergist for volatile kairomones for Diabroticite beetles. – J. Chem. Ecol. 21: 1149-1162. Metcalf, R.L., Lampman, R.L. & Lewis, P.A. 1998: Comparative kairomonal chemical ecology of Diabroticite beetles (Coleoptera: Chrysomelidae: Galerucinae: Luperini: Diabroticina) in a reconstituted tallgrass prairie ecosystem. – J. Econ. Ent. 91: 881-890. Tóth, M., Tóth, V., Ujváry, I., Sivcev, I., Manojlovic, B. & Ilovai, Z. 1996: Sex pheromones also for beetles? The development of a pheromone trap for the western corn rootworm (Diabrotica v. virgifera LeConte) (Coleoptera: Chrysomelidae) – the first beetle sex pheromone trap in Hungary. – Növényvédelem 32: 447-452 (in Hung.). Tóth, M., Imrei, Z. & Szöcs, G. 2000: Non-sticky, non-saturable, high capacity new pheromone traps for Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and Helico- verpa (Heliothis) armigera (Lepidoptera: Noctuidae). – Integr. Term. Kert. Szántóf. Kult. 21: 44-49 (in Hung.). Tóth, M., Sivcev, I., Ujváry, I., Tomasek, I., Imrei, Z., Horváth, P. & Szarukán, I. 2003: Development of trapping tools for detection and monitoring of Diabrotica v. virgifera in Europe. – Acta Phytopath. Entomol. Hung. 38: 307-322. Wall, C. 1989: Monitoring and spray timing. – In: A.R. Jutsum & R.F.S. Gordon (eds): Insect Pheromones in Plant Protection. Wiley & Sons: 39-66.

Miscellaneous

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 157-161

The impact of the fungal BCA Metarhizium anisopliae on soil fungi and animals

Martin Kirchmair1, Lars Huber2, Elke Leither2, Hermann Strasser1 1 Institute of Microbiology, Leopold-Franzens University Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria; e-mail: [email protected] 2 Institute of Zoology, Johannes Gutenberg-University of Mainz, 55099 Mainz, Germany; e-mail: [email protected]

Abstract: In a vineyard near Geisenheim, Germany, the efficacy of soil treatment with Metarhizium anisopliae colonised barley against grape phylloxera was evaluated. It could be shown that M. anisopliae is effective against grape phylloxera. With respect to an European registration, the development of biological control agents (BCA) must also be accompanied by a responsible assessment of the risks that may be associated with their application. Therefore, the M. anisopliae densities in soil (CFU g-1 dry soil) were compared with data on the diversity and abundance of other soil fungi. An additional goal was to study non-target effects on soil fauna. To assess the impact of Metarhizium colonised barley on soil fungi, soil suspensions were plated on potato-dextrose-agar (PDA) and incubated for one week at 25 °C. The colonies were counted and identified at genus level. The effects on the edaphon were studied through soil samples taken with a core borer. A dynamic extraction of the soil fauna was applied. Lumbricidae were extracted with a mustard suspension. No differences in the abundances of the different species of Acari, Collembola or Lumbricida could be found between the variants: (i) no treatment, (ii) sterilised barley, and (iii) M. anisopliae colonised barley. No influence on Harpalus affinis, the only observed member of Carabidae, was observed. The evaluation of the PDA dilution plates revealed no changes in the composition of cultivable soil fungi. Although we found no influence on non-target organisms, a future goal is to determine potential effects of Metarhizium BCAs on invertebrates and microbial cenoses, respectively, at different locations.

Keywords: biocontrol, entomopathogen, Hyphomycetes, Metarhizium anisopliae, grape phylloxera, Daktulosphaira vitifoliae, non-target effects

Introduction

Entomopathogenic fungi are effective agents for the control of subterranean insect pests and provide farmers with a promising alternative to chemical pesticides. For instance, Beauveria brongniartii is successfully used as a biocontrol agent (BCA) against larvae of the common cockchafer (Melolontha melolontha L.) in Austria, Italy, Switzerland and France; Metarhizium anisopliae (Metschn.) Sorok. is in trial as BCA against grape phylloxera, Daktulosphaira vitifoliae (Fitch) in Germany (Kirchmair et al. 2004; Huber et al. 2004). The development of microorganisms as control agents also requires a responsible assessment of the risks that may be associated with their application. For instance, the fate of BCAs regarding their potential dispersal and establishment in the environment as well as non-target effects are issues of concern. In this study, field trials to assess risks associated with the application of M. anisopliae colonised barley have been set up as part of an efficacy study in vineyards against grape phylloxera. These studies address important issues such as the impacts which the introduction of large amounts of fungal entomopathogens may have on soil fungi and on the edaphon.

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Material and methods

Field trial layouts A full randomised block design (EPPO, 2004) was used for the M. anisopliae risk assessment trial in a vineyard near Geisenheim, Hessen, Germany. Untreated plots and plots treated with sterile barley kernels (33 kg ha-1) served as controls. M. anisopliae colonised barley was applied in an amount of 50 kg ha-1 in May 2003.

Quantification of fungal BCAs in the soil Soil samples (depth 0-10 cm and 10-20 cm) were taken with a core borer on August 2003. Samples from each layer were mixed, air-dried, and sieved through a 2 mm sieve. Ten gram sub-samples from each depth (three replicates) were added to 40 mL 0.1 % (w/v) Tween80®, shaken at 150 rpm for 30 min, and then treated in an ultrasonic bath for 30 s. Agar plates selective for Metarhizium (Strasser et al., 1996), supplemented with 22 g L-1 glucose monohydrate) were inoculated with 50 µL of these soil suspensions or dilutions thereof (four replicates per sub sample) and were then incubated for 14 days at 25 °C and 60 % relative humidity (RH). Colonies formed by Metarhizium are given as CFU g-1 soil dry weight.

Diversity of soil fungi Potato-Dextrose-Agar plates supplemented with Streptomycin (100 mg L-1), Tetracycline (50 mg L-1) and Dichloran (2 mg L-1 as 0.2 % w/v ethanolic solution) were inoculated with 50 µL of the extract obtained through the procedure described in the previous paragraph (four replicates per sub sample). The plates were incubated at 25 °C and 60 % RH. After one week the colonies were counted and assigned to the genera and taxonomical groups identified by morphological characters.

Extraction the edaphon For studying effects on the edaphon soil samples were taken with a core borer. For a dynamic extraction of the soil fauna the method of Kempson et al (1963) was applied. Lumbricidae were extracted with a mustard suspension according to Gunn (1992). Data processing and statistical analyses MS Excel 2000 and Statistica 6.1 were used for data processing and statistical testing. Multivariate datasets (fungal diversity) were analysed with Discriminant Function Analysis (DFA).

Results and discussion

In the tramline of treated plots a M. anisopliae density of 104-105 CFU g-1 dry soil was detected. No relevant density of Metarhizium could be found in the soil before application. Acari (mites) are the most common soil invertebrates in the trial site followed by Colembola (springtails). No significant changes of the abundances of the edaphon (Figure 1) as well as abundances of different collembolan species (Figure 2) could be observed between the different variants. Application of sterilised barley as well as of Metarhizium colonised barley seems to have no effect on cenoses of soil arthropods. The only observed carabid species – Harpalus affinis – was not affected by Metarhizium application. Also the abundances of Lumbricidae showed no differences between the variants. For the evaluation of a possible influence of the Metarhizium application on the cenoses of soil fungi, discriminant function analysis was applied of the data obtained from the PDA dilution plates. With this statistical method possible shifts of fungal diversity and quantitative changes in the abundances of different genera can be recognized. The graph of the DFA shows a high spread within and between the variants (Figure 3). No discrete groupings could be observed. This picture is 159

supported by low eigenvalues and relatively low values for Wilk’s Lambda. Summarizing these results, no influence of Metarhizium application could be detected on the cenoses of soil fungi and soil invertebrates. None of the data from these in situ studies indicates environmental risks posed by the application of M. anisopliae to the soil. It should be mentioned that our data refer to observations on only one growing season and verifications are needed. Nevertheless, hitherto the gathered data sound promising: Despite high BCA densities in the soil – mostly above recommended control thresholds of 5 x 103 cfu g-1 dry wt soil (unpublished data) – neither the indigenous soil fungi nor the invertebrate soil fauna were negatively affected by the BCA itself. Similar observations have been made by Dromph (2001) and Hozzank et al. (2003) for B. brongniartii and M. anisopliae, respectively. But more experiments at different locations over more than one season are indispensable. Risk assessment data are a major leap to list fungal entomopathogens in Annex I of Council Directive 91/414/EEC in the near future. However, obstacles still have to be overcome, because every member state is authorized to assess BCAs by taking into account local climate, cropping pattern and diet. From today’s perspective this will delay the use of reliable control agents.

Figure 1: Abundances of the edaphon (mean values and standard deviations); Acronymes: C: Control, B: Plots treated with sterilised barley, M: Plots treated with Metarhizium anisolpliae colonised barley. ■: Acari; ■: Collembola, □: Others (Pseudoscorpiones, Isopoda, Pauropoda, Symphyla, Diplopoda, Chilopoda, Diplura, Psocoptera, Coleoptera, Diptera)

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Figure 2: Abundances of Collembola (mean values and standard deviations); Acronymes: ■: Control, ■: Plots treated with sterilised barley, □: Plots treated with Metarhizium anisolpliae colonised barley. Hyp-man: Hypogastrura manubrialis, Will-den: Willemia denisi, Xen-spe: Xenylla species, Anu-gra: Anurida granaria, Pro-arm: Protaphorura armata, Mes-kra: Mesaphorura krausbaueri, Par-cal: Paratullbergia callipygos, Ste-den: Stenaphorura denisi, Psa-alb: Pseudosinella alba, Wiw-nig; Willowsia nigromaculata, Het-nit: Heteromurus nitidus, Fol-par: Folsomides parvulus, Isa-not: Isotoma notabilis, Iso-min: Isotomiella minor, Pse-alt. Pseudanurophorus alticolus, Onc-cra: Oncopodura crassicornis, Arr-cae: Arrhopalites caecus, Sph-pum: Sphaerida pumilis.

Figure 3: Discriminant Function Analysis of abundances of different fungal genera (Alternaria sp., Aspergillus sp., Cladosporium sp., Fusarium sp., Gliocladium sp., Paecilomyces sp., Penicillium sp., Trichoderma sp., Ulocladium sp., Zygomycetes, Yeasts and “Others”). 161

Acknowledgements

The authors wish to thank H. Matzke (Schweizer Samen AG, Switzerland) for supplying us with the Ma 500 colonised barley. We are indebted to R. Pöder, T. Längle, M. Porten, G. Eisenbeis for helpful discussions, M. Hammes for his help in the field and the laboratory and E. H. Rühl for the access to greenhouses at the Geisenheim Research Centre, Germany. This work was supported by the Forschungsring des Deutschen Weinbaus (FDW), the Bundesanstalt für Landwirtschaft und Ernährung (Project No. BLE 03OE001) the Feldbausch Foundation, Department of Biology, University of Mainz and the. Heinrich-Birk-Gesellschaft. HS thanks the European Union (EU) for partial funding of the research (grant QLK1-CT- 2001-01391).

References

Dromph, K.M. 2001: Dispersal of entomopathogenic fungi by collembolans. – Soil Biol. & Biochem. 33: 2047-2051. EPPO 2004: EPPO Standards PP1. 2nd Edition. Volume 1: General introduction. – The European and Mediterranean Plant Protection Organization. Paris. Hozzank, A., Keller, S., Daniel, O. & Schweizer, C. 2003: Impact of Beauveria brongniartii and Metarhizium anisopliae (Hyphomycetes) on Lumbricus terrestris. – IOBC/wprs Bull. 26(1): 31-34. Gunn, A. 1992: The use of mustard to estimate earthworm populations. – Pedobiologia 36: 65-67. Huber, L., Leither, E., Eisenbeis, G., Porten, M. & Kirchmair, M. 2004: Metarhizium anisopliae – ein Bodenpilz zur biologischen Kontrolle der Reblaus. – Proceedings of the 1st International Symposium for Organic Wine Growing, INTERVITIS INTERFRUCTA (International Technology Trade Fair for Wine, Fruit and Fruit Juice), 11.-15.05.2004, Stuttgart, Germany: 12-23 Kempson, D., Lloyd, M. & Geglhardi, J. 1963: A new extraktor for woodland litter. – Pedo- biologia 3: 1-21. Kirchmair, M., Huber, L., Rainer, J. & Strasser, H. 2004: Metarhizium anisopliae, a potential biological control agent against grape phylloxera. – Biocontrol 49: 295-303. Strasser, H., Forer A. & Schinner F. 1996: Development of media for the selective isolation and maintenance of virulence of Beauveria brongniartii. – In: T. Jackson & T. Glare (eds.). Microbial Control of Soil Dwelling Pests. AgResearch, Lincoln: 125-130. 162 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 163-167

Biocontrol potential of entomopathogenic nematodes against nut and orchard pests

Stefan Kuske1, Claudia Daniel2, Eric Wyss2, Jean-Paul Sarraquigne3, Mauro Jermini4, Marco Conedera,5 Jürg M. Grunder1 1 Agroscope FAW Wädenswil, P.O. Box 185, CH-8820 Wädenswil, Switzerland; 2 FiBL, Ackerstrasse, Postfach, CH-5070 Frick, Switzerland; 3 Association Nationale des Producteurs de Noisette, Lamouthe 47290 Cancon, France; 4 Agroscope RAC Changins, Centro di Cadenazzo, CH-6594 Contone, Switzerland; 5 WSL, Sottostazione Sud delle Alpi, via Belsoggiorno 22, CH-6504 Bellinzona, Switzerland; 6 E-nema, Klausdorfer Str. 28-36, D-24223 Raisdorf, Germany

Abstract: Semi-field and field experiments were carried out to evaluate the biological control potential of entomopathogenic nematodes (EPNs) against grubs in hazelnut, chestnut and cherry orchards. Mortality of hazelnut weevils in interred containers treated with 2.2x106 infective juveniles (IJ) m-2 of Heterorhabditis bacteriophora was 75.4%. H. indica caused 65.2% and S. feltiae 43.3% pest mortality, but numbers were not significantly different from the untreated control (33.4%). Mortality of the chestnut weevil was slightly increased in S. carpocapsae (2x106 IJ m-2) treated containers (44.6%) compared to untreated control containers (39.2%), but differences were either not significant. Soil applications against the European cherry fruit fly did not lead to any pest control effect and we suggest that there is little potential for EPNs to control this key insect pest of sweet cherries.

Keywords: biological control, entomopathogenic nematodes, Heterorhabditis bacteriophora, H. indica, Steinernema carpocapsae, S. feltiae, Balaninus nucum, Curculio elephas, Cydia splendana, Rhagoletis cerasi.

Introduction

Entomopathogenic nematodes (EPNs) are often used to control soil-dwelling insect pests in high value crops and turf (Shapiro-Ilan et al., 2002). They are mainly applied in cases where no alternative control measures are available, in systems where chemical compounds fail, or in systems where resistance to insecticides has developed (Ehlers, 2003). There are several agricultural crops with key pests that spend at least parts of their life cycle in the soil and where EPN applications could offer a solution for environmentally sound pest control. Within the framework of the COST-Action 850 efforts were made to evaluate the biological control potential of EPNs against hazelnut, chestnut and sweet cherry pests in France and Switzerland. In all three systems the key insect pests damage fruits or nuts before entering the soil for diapause in the larval or pupal instar. These circumstances make these pests appropriate targets for EPNs. The European cherry fruit fly Rhagoletis cerasi (Dipt., Tephritidae) is the most important insect pest of sweet cherries in Switzerland. Damage on organically produced fruits often exceeds levels of market tolerance and organic farmers are lacking efficient control measures. However, recent laboratory studies indicated promising results when EPNs where applied against the larval instar of the European cherry fruit fly (Gokce et al. 2003, Koeppler et al. 2003) and the Western cherry fruit fly R. indifferens (Yee and Lacey 2003). We hypothesised that positive control effects of EPN soil applications could occur not only against R. cerasi

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but also when used against the hazelnut weevil Balaninus nucum (Col., Curculionidae) and the chestnut pest Curculio elephas (Col., Curculionidae). In the present study we investigated the biological control potential of EPN soil applications against these three species. Semi- field or field trials were carried through with commercial EPN products in hazelnut, chestnut and cherry orchards. The control potential of EPNs was estimated by assessing larval mortality or adult emergence in semi-field and field experiments respectively. Data presented in this study are preliminary results from ongoing projects.

Material and methods

EPNs All EPNs used in these experiments derived from commercial products that were provided by the companies or distributors (see Table 1), except the Heterorhabditis indica strain which was not available as commercial product yet. All EPN strains considered in semi-field and field experiments had previously been tested for infectivity under laboratory conditions and showed promising results (Table 3).

Pest larvae All pest larvae used in the experiments derived from natural populations and were collected when escaping damaged nuts or fruits for entering into the soil.

Table 1. EPN species, product name and nematode supplier used in this study.

Nematode species Product name Supplier Steinernema carpocapsae Carponem Andermatt Biocontrol, Grossdietwil (Switzerland) S. feltiae I Traunem Andermatt Biocontrol, Grossdietwil (Switzerland) S. feltiae II nema PLUS E-nema, Raisdorf (Germany) Heterorhabditis nema TOP E-nema, Raisdorf (Germany) bacteriophora H. indica no commercial E-nema, Raisdorf (Germany) product H. megidis DMR Nematoden Andermatt Biocontrol, Grossdietwil (Switzerland)

Study sites, experimental layouts & EPN applications Micro-plot trials were carried out in South-western France against the hazelnut weevil, and in Southern Switzerland against chestnut pests. Field experiments were carried out in North- western Switzerland against the European cherry fruit fly. Details of the study sites and the experimental set-ups are summarised in Table 2. Micro-plot trials consisted of field plots in which PVC containers were inserted into the soil in a completely randomised block design. Containers were artificially infested with up to 43 pest larvae per replicate. EPNs were applied in 0.1-0.2 l of water as a curative treatment to each container, except for the open field trials in the cherry orchard, where they were applied in 6 litres of water in a hand held sprinkling can. Following application the same amount of water as for the EPN application was used to wash the nematodes into the soil. 165

Experimental plots were artificially infested with pest larvae that naturally burrowed into the soil for diapause. Mortality of pest larvae in interred containers or adult emergence on field plots was monitored after larval exposure to commercially available EPN products. Larval mortality was assessed by washing out the soil filled containers and collecting dead and intact pest larvae. Adult emergence was assessed with emergence traps (photo-eclectors).

Table 2. Overview of semi-field and field experiments with EPNs against orchard pests.

Target pests Experimental Hazelnut weevil Chestnut weevil European cherry fruit set-up Balaninus nucum Curculio elephas fly Rhagoletis cerasi Location of Cancon, South- Cadenazzo, Ticino, Aesch BL, Switzerland study site western France Switzerland Year 2003 2003 2003 Application August October June period Species S. feltiae II S. carpocapsae S. feltiae I H. bacteriophora S. carpocapsae H. indica H. megidis Container 0.5 m, 46 litres 0.4 m, 3 litres Open field, 1m2 mini- depth & parcels capacity Dosage 2.2 x 106 IJ/m2 2 x 106 IJ/m2 2 x 106 IJ/m2 Irrigation Yes (Micro-sprinkler) No (Rain) No (Rain) Mean number of pest 53 43 38 larvae/replicate Replicates 3 6 5

Results and discussion

Soil applications of EPNs against the hazelnut weevil B. nucum showed a tendency to increase mean pest mortality in EPN treated containers compared to control containers. H. bacteriophora applications led to 75.4±6.5% (n=3), H. indica to 65.2% (n=1), and S. feltiae to 43.3±14.8% (n=3) mortality, whereas mortality was 33.4±36.4% (n=3) in the control. Differences between means were not statistically significant (ANOVA: F=1.944, df=3, p>0.05; see also Table 3). One of the reasons for not significant differences in mean mortality was probably the low number of replicates and the high variability in the control treatments. Moreover, the relatively high water input into the PVC-containers due to the sprinkler irrigation system, may have increased natural mortality of B. nucum larvae. Nevertheless, these results indicate that EPNs could contribute to the biological control of the hazelnut weevil. Additional experiments under semi-field and field conditions are still needed to confirm our expectations. Mean mortality (±SD) of C. elephas larvae was similar in S. carpocapsae treated containers (44.6±14.2%) and in control containers (39.2±12.7%) (ANOVA: F=0.485, df=1, p>0.05; Table 3). One reason for lack of control may lay in the selection of a S. carpocapsae 166

based EPN product in this experiment. Although this species caused high mortality in laboratory bioassays (see Table 3.) it may not be suitable to control C. elephas. S. carpocapsae is known as a typical ambusher species that does not travel far and spends most of the observation period nictating (Campbell and Gaugler 1993). We therefore assume that S. carpocapsae did not follow up the host to increasing soil depths and that C. elephas escaped parasitism by S. carpocapsae when burying into the soil immediately after emerging from damaged chestnuts. Therefore, additional experiments under semi-field or field conditions should consider cruiser species as well, and EPN strains that are still active under relatively cold soil temperatures, since chestnut weevil larvae enter the soil in late autumn and early winter.

Table 3. Nematode induced mortality of target pests in laboratory, semi-field and field trials.

Hazelnut weevil Chestnut weevil European cherry fruit fly Nematode species Laboratory1 Semi- Laboratory1 Semi- Laboratory1 Field field field Steinernema – carpocapsae S. feltiae – Heterorhabditis – – megidis H. bacteriophora – – – – H. indica – – – – –

Control potential: ≤ 33% = low, =34-66% = medium, ≥ 67%= high, – = no data available 1 Laboratory data derive from preliminary experiments using same EPN strains as for semi-field and field trials.

EPN soil applications against the European cherry fruit fly proved to be ineffective for all nematode treatments included in this study. Adult emergence from EPN treated plots was similar as from untreated control plots. Emergence was 11.1% on S. feltiae, 12.6% on S. carpocapsae, and 21,3% on H. megidis treated plots, whereas 18,9% of the number of exposed larvae developed into adults on the control plots. Thus, both our preliminary lab experiments (see Table 3), as well as the EPN soil applications in the field, showed only little or no impact on R. cerasi. These results are partly in contrast with results of Koeppler et al. (2003), who found up to 80% mortality of R. cerasi, and with results of Gokce et al. (2003) who found up to 84% mortality of R. cerasi when using S. carpocapsae in laboratory bioassays, respectively. One reason for the lower impact of EPNs against R. cerasi in our study – compared to the studies cited above – may lay in the fact that for experimentation we did not collect pest larvae from dissected fruits, but only considered larvae that escaped from cherries on their own before entering the soil. However, additional laboratory and field experiments are needed to confirm our data. The findings presented in this study are preliminary results from three ongoing projects. The data indicate that the selection of the most virulent EPN strains as well as the improvement of application technique and application time are necessary to increase pest control by EPNs in hazelnut, chestnut, and cherry orchards. However, soil applications of EPNs against the European cherry fruit fly proved to be ineffective and may be difficult to improve in a way to achieve control levels of economic importance. 167

Acknowledgements

We are grateful to Arne Peters, Brigitte Baur and Bernard Blum for their contributions to this work. We also like to thank the company Andermatt Biocontrol AG for supplying nematodes, and the COST-Action 850 for funding this study.

References

Bovey, P., Linder, A. & Müller, O. 1975: Recherches sur les insectes des châtaignes au Tessin (Suisse). – Schweizerische Zeitschrift für Forstwesen 126(11): 781-820. Campbell, J.F. and R. Gaugler, R. 1993: Nictation behavior and its ecological implications in the host search strategies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae). – Behaviour 126: 155-169. Ehlers, R.-U. 2003: Biocontrol nematodes. – In: H.M.T. Hokkanen & A.E. Hajek (eds.). Environmental Impacts of Microbial Insecticides. Kluwer Academic Publishers, The Netherlands: 177-220. Gokce, A. et al. 2003: Infectivity of three entomopathogenic nematodes to European Cherry fruit fly. – 9th European Meeting of the IOBC/WPRS Working Group Insect Pathogens and Entomopathogenic Nematodes, held in Salzau, Germany 23-29 May 2003: 34. Koeppler, K., Peters, A. & Vogt, H.: Initial results in the application of entomopathogenic nematodes against the European Cherry Fruit Fly Rhagoletis cerasi L.. – 9th European Meeting of the IOBC/WPRS Working Group Insect Pathogens and Entomopathogenic Nematodes, held in Salzau, Germany 23-29 May 2003: 57. Shapiro-Ilan, D.I. et al. 2002: Factors affecting commercial success: Case studies in cotton, turf and citrus. – In: R. Gaugler (ed.). Entomopathogenic Nematology. CABI Publishing, Oxon, UK: 333-355. Yee, W.L. & Lacey, L.A. 2003: Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes. – Biological Control 27(3): 349-356. 168 Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 169-173

Occurrence and harmfulness of Brachyderes incanus L. (Coleoptera: Curculionidae) to young Scots pine (Pinus sylvestris L.) trees planted on post-fire areas

Henryk Malinowski, Alicja Sierpinska Forest Research Institute, Department of Forest Protection, Sekocin - Las; 05-090 Raszyn, Poland, e-mails: [email protected]; [email protected]

Abstract: The aim of this work was to evaluate the occurrence and harmfulness of Brachyderes incanus L. (Coleoptera, Curculionidae) to young Scots pine (Pinus sylvestris L.) trees planted on areas formerly burned during the huge forest fire in 1992. The B. incanus outbreak got started 4 to 5 years after the fire (3 to 4 years after the planting of young trees) and was connected with the diminishing of macronutrients contents in a soil. In the course of gradation, the population density of B. incanus is increasing and decreasing fastidiously. During 2 generations (2 years), the number of beetles has increased from 1 to 2 individuals/ tree to 50 individuals/ tree. Although more than 95 % of one-year- old needles on 2 highest verticils were damaged, the infested trees have not died. The reason of the fact was, that damages were no longer than 35 % of needle length and needles damaged in such a degree has not fallen down immediately, but successively until the developing of a new generation of needles. The feeding of 50 B. incanus beetles/ tree caused the 30 % decrease in annual increments in height of young pine trees. Two years after the outbreak, the infested pine trees did not reach the height of trees in the control.

Keywords: Brachyderes incanus outbreak, harmfulness, young Scots pine plantations, post-fire areas

Introduction

For many years Brachyderes incanus L. (Coleoptera, Curculionidae) was regarded in Poland as the pest of a small economic importance. It appears in young and old stands, however a mass appearance is observed in 6 to 10 years old pine forests. In 1999 – 2001 it occurred on large areas, reaching about 10 000 ha of Scots pine plantations in 2000. The most favourable conditions for development of the insect's outbreak occur on reforested post-fire areas. As a result of 1992 forest fires, great areas in a different regions of Poland had to be reforested (mostly with Scots pine): in Rudy Raciborskie, Rudziniec and Kędzierzyn Forest Districts - 9 000 ha, in Potrzebowice Forest District – 5 000 ha, in Cierpiszewo and Szprotawa Forest Districts – 6 000 ha and in Grodziec Forest District – 700 ha. It can be assumed that B. incanus outbreak involved more than 20 000 ha of post-fire areas. The insect has one generation per year. It is overwintering in the litter as a beetle. The spring feeding and laying eggs to the soil lasts since the end of March till the beginning of June. Depending of the temperature, the embryonation can take 2 – 6 weeks. B. incanus larvae (June, July, the beginning of August) feed young and old pines roots, also roots of grass, coniferous and deciduous bushes and trees. Despite of the fact, that B. incanus larvae cause similar (but much less) injuries like Melolontha melolontha larvae, they are not regarded as dangerous as beetles are. Larvae pupate in August and the new generation of beetles appears in the end of August, in September or in the beginning of October. They usually feed until the first frost. B. incanus beetles feed pine needles, but in the period of a mass appearance they can also feed spruce and larch needles and even a bark of young birch, oak and beech

169 170

branches. Beetles can walk up to the tree crown and down to the soil many times during the season. In our studies we have concentrated on observations of beetles harmfulness to young pine trees. The aim of our studies was to evaluate the effect of B. incanus outbreak on young Scots pine trees development on forest areas burned in 1992.

Material and methods

In spring 2000 observation plots were established on B. incanus outbreak area in Grodziec Forest District, after alarming reports of State Forest administration concerning the B. incanus situation on post-fire areas. In autumn 1999 they noted 50 – 80 beetles/tree. Plots were established on areas with the most defoliated trees and on areas with the slightest defoliated trees (control). There were no trees without any defoliation caused by B. incanus beetles. Between the spring 2000 and the autumn 2002, there were 6 observation periods. Every one lasted 6 – 9 weeks, with several terms of catching beetles. Beetles were catched on square linen sheets fixed permanently under 10 typical trees per observation plot. Needles damages were estimated twice a year: in June – damages caused by beetles of the whole, one generation (before and after winter hibernation) and in October – damages caused by young beetles (before winter hibernation). Estimated values were: percent of damaged needles from branches of the 2 highest verticils and percent of the needles lengths damaged by the beetles feeding. To check the effect of B. incanus outbreak on the growth and the annual increment of pine trees, measurements of the pine trees height and annual increments in height of pine trees were done. The number of died trees/ plot was checked after the outbreak. The occurrence of other insect species and fungal pathogens of pine was monitored during the studies.

Results and discussion

Population density of B. incanus on post-fire area in Grodziec Forest District, 1999 – 2002 Immediately after 1992 forest fire, the combustion of plant materials and organic substances has lead to the increase of some macronutrients in soil. Four or five years after fire, the contents of macronutrients diminished significantly as a result of their uptake by newly planted trees or penetration to the deeper layers of soils profile. Also a lack of soil microorganisms degrading organic material falling down to the soil surface was the reason of changes mentioned above. The contents of macronutrients in pine needles were related to their contents in soil and indicated poorer supply for trees on burnt areas (Olejarski, 1999; Zwolinski et al., 2004; Malinowski et al., 2004). It created favourable conditions for developing of B. incanus outbreak. In spring 1999, according to information of State Forests administration, the number of beetles after hibernation reached a level of 50 till 100 individuals/ tree and the new generation of beetles, which emerged in August, reached a level of 50 till 80 individuals/ tree. The B. incanus population density in terms of the mean beetles catching is presented in Figure 1. The highest numbers of individuals/ tree/ one observation day (50 beetles) were noted on observation plots in spring and autumn 2000. Between autumn 2000 and spring 2001 the number of catched beetles decreased 5 times and in spring 2002 reached the same level as on control plots. During the studies several individuals per catching period were found on Scots pine trees from control plots.

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No. of beetles/ tree before hibernation 60 50 40 30 20 10 0 11. 17. 22. 25. 4. 7. 11. 13. 18. 28. 5. 6. 8. 14. 15. 23. August September October 2000 2001

No. of beetles/ tree after hibernation 60 50 40 30 20 10 0 9. 3. 7. 2. 7. 8. 1. 24. 2 10. 11. 16. 26. 27. 30. 1 14. 17. 21. 24. 31. 12. March April May June

2000 2001 2002

Fig. 1. The catching of B. incanus beetles from infested Scots pine trees: before hibernation (the higher figure) and after hibernation (the lower figure)

B. incanus beetles feed mainly needles of the 2 highest verticils. Beetles after winter hibernation feed old needles. They never destroy buds. The new generation of beetles, before hibernation, firstly feeds old needles and than new ones, totally developed. The result of such a feeding behaviour is a low level of tree's mortality, even during outbreaks.

The number of B. incanus beetles and the level of needles damages The effect of the number of B. incanus beetles on the percent of damaged needles and damages of needles lengths is presented in Figure 2. The percent of damaged needles of the 2 highest verticils was the same for the beetles generation of 1999/2000 and 2000/2001, despite of the fact, that maximal number beetles/ tree/ one observation day was lower. Also differences in damages of needles lengths caused by beetle generation of 1999/2000 and 2000/2001 were not big. Surprising was the relatively high percent of needles damaged by the 2001/2002 generation. Beetles in maximal amounts 2 – 4 individuals/ tree/ one observation day caused injuries of almost 40 % of needles, however needles lengths damages were on the control level. Smaller injuries of needles lengths resulted in extended period of needles presence on the trees. When checked in spring 2003, 2 generations of needles were present on formerly highly infested with B. incanus pine trees.

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Maximal no. of beetles/ tree: 1999/ 2000, I 50 - 80

1999/ 2000, C 3 - 4

2000/ 2001, I 12 - 49

2000/ 2001, C 3 - 7

2001/ 2002, I 2 - 4

2001/ 2002, C 1 - 2 10080 60 40 20 0 20 40 60 80 100

Damaged needles [%] Needles length damages [%]

Fig. 2. The effect of numbers of B. incanus beetles on the percent of damaged needles and damages of needles length.

The effect of B. incanus outbreak on the growth of young pine trees

Height of pine trees [cm] 300

250

200

150

100

0 1996 1997 1998 1999 2000 2001 2002 2003 Infested trees Control trees Fig. 3. The mean height of Scots pine trees from infested with B. incanus and control plots.

Until 1998 heights of trees on infested and control plots in Grodziec FD were similar. In the following years, the growth of trees on infested plots was significantly reduced - trees from infested plots were by one fourth (70 cm) lower than control trees. Two years after B. incanus outbreak infested trees did not reach the height of trees in the control. The negative impact of B. incanus outbreak on young pine trees is also seen, when we take into consideration the annual increment in height, which was reduced by one third on infested trees in comparison to control trees (Fig. 4).

Other effects of B. incanus outbreak on young pine trees During the outbreak 6 died trees (among 300) were found on highly infested with B. incanus plot and 4 died trees (among 300) - on the control plot. The percent of trees with different defects was equal 32 for infested trees, and 4 for control trees. Defects resulted mainly from the producing of tree stems from side buds. The reason of the fact was the destroying of

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terminal buds by Rhyacionia duplana (Hbn.), which preferred as a food buds from trees damaged with B. incanus. R. buoliana (Den. and Schiff.), Coccus turionella (L.), Exoteleia dodecella (L.) and Pissodes spp. were also feeding sporadically the pine trees formerly damaged by B. incanus. Fungal pathogens such as Armillaria sp., Melampsora pinitorqua Rostr. occurred sporadically.

Annual increment in height [cm] 60

0 1996 1997 1998 1999 2000 2001 2002 2003 Infested trees Control trees

Fig. 4. The mean annual increment in height of Scots pine trees on infested and control plots.

Acknowledgments

We are grateful to Zbigniew Wierzbowski (State Forests) for providing some of the information reported in this paper.

References

Malinowski, H., Wierzbowski, Z. & Tarwacki, G. 2004: [Effect of Brachyderes incanus L. outbreak on the development of Scots pine (Pinus sylvestris L.) plantations on post-fire areas]. – Lesne Prace Badawcze [Forest Research Papers] 4: in press (in Polish). Olejarski, I. 1999: [Influence of soil cultivation methods on the state of reforestation on large post-fire areas]. – Doctors dissertation, FRI Warsaw: 52 pp. (in Polish). Zwolinski, J., Matuszczyk I. & Hawrys Z. 2004: [Chemical properties of soils and Scots pine needles and microbiological activity of soils on forest areas burnt in forest districts of Rudy Raciborskie and Potrzebowice in 1992]. – Lesne Prace Badawcze [Forest Research Papers] 1: 119-133 (in Polish).

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Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 175-178

Is differentiated host plant preference of Agriotes sp. and Melolontha sp. mediated by root volatiles?

Sonja Weissteiner, Stefan Schütz Institute for Forest Zoology and Conservation, Buesgenweg 3, D-37077 Göttingen, Germany

Abstract: Effects of different root volatiles on the behaviour of soil dwelling larvae were investigated. Choice tests were performed with larvae of Melolontha and Agriotes in order to determine the role of different volatiles on the orientation of the larvae moving in the underground. The investigated organisms had to choose between carrots (Daucus carota ssp. sativus) and potatoes (tubers and roots, Solanum tuberosum). In this experiment the organisms show a clear preference for carrots. GC-MS (Gas Chromatography-Mass Spectrometry) analysis of volatile compounds released by undamaged and damaged roots shows different feeding induced volatile pattern if chewed by Melolontha or Agriotes larvae.

Keywords: soil dwelling beetle larvae, choice test, root volatiles

Introduction

Plants and insects live and function in a complex multitrophic environment. Most multitrophic studies, however, almost exclusively focussed on aboveground interactions (Dicke 1994, Schütz & Hummel 1997, Schütz et al. 1997a, Apel et al. 1999, Schütz et al. 1999, Turlings & Fritzsche 1999, Dicke & Bruin 2001a, Dicke & Bruin 2001b). There are a lot of speculations about belowground living insects and their way of living, but until now there was very little experimental investigation (Horber 1954, Hauss & Schütte 1976, Hasler 1986). A rather unknown topic is the orientation behaviour of soil living organisms. One of the current hypotheses indicate that the orientation of belowground living insects is partly guided by a CO2-gradient (Hasler 1986) which is caused by plant root respiration. This means that CO2 for soil inhabiting polyphagous larvae could function as an non specific lure to find their potential host plants. In addition, volatile secondary plant substances released by the roots might be utilized by the larvae as an important additional clue for their orientation toward host plants. Furthermore, non volatile secondary plant substances which are released by roots as root exsudates, can act as feeding stimulants. Odourant compounds were identified which are released by plant roots and which may be able to attract or repel belowground living insects.

Material and methods

Insect provenience and growth conditions Organically cultivated carrots (Daucus carota ssp. sativus) and potatoes (tubers and roots, Solanum tuberosum) were used for the study. Larvae of Melolontha sp. were collected in a forest near Darmstadt, larvae of Agriotes sp. originate from outdoor experiments carried out near Mainz and Braunschweig.

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Experimental design During the experiment Melolontha larvae were kept individually in black 10 l- plastic buckets together with carrots and potato plants whereas Agriotes larvae were kept in groups of five larvae per bucket.

Belowground feeding experiment After one week the roots were visually inspected for signs of feeding damage. The experiment was prolonged for those larvae who did not show any clear decision for one of the plants. After three weeks all but one of the larvae had fed at least on one type of the two kinds of roots available.

Sampling of root-volatiles At the end of each experiment, samples for GC-MS analysis were collected from the bare roots for two hours using the cloosed-loop-stripping-analysis (CLSA) method (Boland et al. 1984).

Results and discussion

Choice test The results show clear feeding preferences: both Melolontha and Agriotes highly favoured carrots, if they had the opportunity to decide between carrots (c) and potatoes (p).

Melolontha sp. (N =10, χ² p<0.001) Agriotes sp. (N=12, χ ² p<0.05 ) c p c p 90% 10% 75% 25%

Table 1: Percentages of damaged carrots (c) and potatoes (p) caused by larvae of Melolontha and Agriotes. In the test with the larvae of Agriotes we found feeding signs on carrots and potatoes in 30 % of the buckets.

Gas-Chromatography/Mass-spectrometry(GC-MS) of carrots and potatoes Volatile pattern of carrots and potatoes are quite different. Moreover, feeding damage on carrots caused by the different insect species led to different damage induced root-volatile pattern (Figure 1). First electrophysiological experiments demonstrate that antennae of Melolontha larvae are able to detect some of these compounds (Weissteiner & Schütz 2004). So, considering 1) the differences in volatile pattern from roots of different plant species, 2) the possibility of damage induced compounds specific to the insect species feeding on the plant root, and 3) the fact that antennae of Melolontha larvae are able to detect at least a part of these compounds, it seems highly probable that these insects use volatile organic compounds released by plant roots to perform their root choice demonstrated in behavioural assays. Moreover, volatile emissions by plant roots specific to the feeding insects might be the basis for aggregation behaviour or density regulation of larvae causing these emissions.

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5 7·10 1 Æ α-pinene 5 Æ τ -terpinene 6 2 Æ β-pinene 6 Æ terpinolene 6·105 a 3 Æ o-cymene 7 Æ caryophyllene 5 5·10 4 Æ D-limonene 8 Æ farnesene

4·105 9 Æ β-Myrcene 3·105 10 Æ 2-ethyl-hexan-1-ol Abundance 11 Æ 1,4-p-menthadien-7-ol 5 2·10 3 12 Æ 1,3,8-p-menthatriene 7 4 1·105 13 Æ borneol 5 12 8 14 Æ 3-ethenyl-1,2-dimethyl-cyclohexa-1,4- 0·105 diene 8 10 12 14 16 18 20 22 15 Æ bornylacetat Retention time (min) 16 Æ 2,4-bis(1,1-dimethylethyl)-phenol 17 Æ bis(1-methylethylidene)-cyclobutene 3,5·105 18 Æ α-curcumene 6 3,0·105 19 Æ (E)-γ -bisabolene 10 b 2,5·105

2,0·105

5 1,5·10 9 Abundance 1,0·105

15 5 0,5·10 11 12 7 16 1 13 14 0,0·105 8 10 12 14 16 18 20 Retention time (min)

9·105 10 8·105

5 7·10 6 c 6·105 5·105 4·105 7 15 Abundance 3·105

5 19 2·10 9 1·105 11 12 17 18 16 0·105 8 10 12 14 16 18 20 22 Retention time (min)

Figure 1. Chromatograms of root volatiles from Daucus carota ssp. sativus a) roots of a non damaged Daucus carota ssp. sativus b) roots of Daucus carota ssp. sativus damaged by feeding of Agriotes sp. c) roots of Daucus carota ssp. sativus damaged by feeding of Melolontha sp.

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Acknowledgements

We thank Stefan Rath and Jörg Berger for their help in the field, Katrin Katzur who supplied the larvae of Agriotes sp. and Matthias Schulz for helpful discussions.

References

Apel, K.-H., Kätzel, R., Lüttschwager, D., Schmitz, H. & Schütz, S. 1999: Untersuchungen zu möglichen Mechanismen der Wirtsfindung durch Phaenops cyanea F. (Col., Buprestidae). – Mitt. Dtsch. Ges. allg. angew. Ent. 12: 23-27. Boland, W., Ney, P., Jaenicke, L. & Gassmann, G. 1984: A "closed-loop-stripping" technique as a versatile tool for metabolic studies of volatiles. – In: Analysis of Volatiles. Schreier P. (ed.), Walter de Gruyter & Co, Berlin: 371-380. Dicke, M. 1994: Local and systemic production of volatile herbivore-induced terpenoids – their role in plant-carnivore mutualism. – J. Plant Physiol. 143: 465-472. Dicke, M. & Bruin, J. 2001a: Chemical information transfer between damaged and undamaged plants – preface. – Biochem. Syst. Ecol. 29: 979-980. Dicke, M. & Bruin, J. 2001b: Chemical information transfer between plants: back to the future. – Biochem. Syst. Ecol. 29: 981-994. Hasler, T. 1986: Abundanz- und Dispersionsdynamik von Melolontha vulgaris U. in Intensiv- obstanlagen. – Diss. ETH Zürich: 128 S. Hauss, R. & Schütte, F. 1978: Über die Eiablage des Maikäfers (Melolontha melolontha L.) in Abhängigkeit von den Wirtspflanzen des Engerlings. – Z. ang. Ent. 86: 167-174. Horber, E. 1954: Maßnahmen zur Verhütung von Engerlingsschäden und Bekämpfung der Engerlinge. – Mitt. Schweiz. Landw. 2: 18-36. Schütz, S. & Hummel, H.E. 1997: Einfluss erhöhter atmosphärischer Ozon-Konzentrationen auf die Interaktion von Kartoffelpflanzen (Solanum tuberosum Lin., Sorte: Granola) und Blattläusen (Myzus persicae Sulzer). – Mitt. Dtsch. Ges. allg. angew. Ent. 11: 297-301. Schütz, S., Weißbecker, B., Klein, A. & Hummel, H.E. 1997a: Host plant selection of the Colorado Potato beetle as influenced by damage induced volatiles of the Potato plant. – Naturwissenschaften 84: 212-217. Schütz, S., Weißbecker, B., Hummel, H.E., Apel, K.-H., Schmitz, H. & Bleckmann, H. 1999: Insect antennae as a smoke detector. – Nature 398: 298-299. Turlings, T.C.J. & Fritzsche, M.E. 1999: Attraction of parasitic wasps by caterpillar-damaged plants. – In: Proc. Novartis foundation Symp. 223, Insect-plant interactions and induced plant defence. Wiley, Chichester: 21-38. Weissteiner, S. & Schütz, S. 2004: The role of insect olfaction in belowground-aboveground interaction. – In: Book of Abstracts, 12th Symposium on Insect-Plant Relationships in Berlin, 7.-12.08.2004: 146.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 179-184

Persistence of the insect pathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin on soil surface and on oilseed rape leaves

Christina Pilz1, Siegfried Keller2, Rudolf Wegensteiner1 1 Departement of Forest and Soil Sciences; University of Natural Resources and Applied Life Sciences, Vienna; 2 Agroscope FAL Reckenholz, Zürich

Abstract: The persistence of M. anisopliae spores (FAL-strain 715) was tested under controlled (greenhouse) and natural (field) conditions. The aim was to develop a suitable methodology to detect spores on leaves and on the soil surface in order to study the persistence of the spores under field and greenhouse conditions and different storage conditions. In the greenhouse a spore suspension of M. anisopliae with a concentration of 2 x 107 spores/ml was sprayed to planted oilseed rape plants, where the seeds have been treated with a molluscizid or have been left untreated. Leaf samples were taken one-, sixteen-, twenty-four -hours and one week after the application. One hour after the application with spore suspension one part of the samples was kept at -18°C and another part at + 4°C to study the influence of the storage temperature on spore survival. No significant differences were found between the treatments of the seeds and the two storage temperatures, although storage at -18°C tended to have lower the number of colony forming units per g fresh mass leaves than storage at 4°C. In the field experiment two different procedures were done. They differed in the concentration of the spore suspension (2 x 107 and 2 x 106 spores/ml) and the application time and -quantity. In the field leaf samples and samples of the soil surface were taken at the same intervals as in the greenhouse. The results show that spores of M. anisopliae are persistent on the soil surface over one week without significant density reduction. In contrast, the persistence of spores on leaves decreased rapidly under controlled as well as under field conditions. Within 24 hours most of the spores on the oilseed rape leaves disappeared or lost viability.

Keywords: Metarhizium anisopliae, persistence, Brassica napus, oil seed rape

Introduction

Metarhizium anisopliae is a natural enemy of different insect pests. Earlier experiments showed that it can be used for integrated pest management control (Butt et al., 2001). Before using M. anisopliae as a biological control agent in practice, it is important to check the persistence of spores under different conditions. M. anisopliae spores should survive as long as necessary to infect the target insects, but they should not infect any non-target insects. In our experiments the persistence of M. anisopliae spores on the soil surface and on oilseed rape leaves was tested under greenhouse and field conditions.

Material and methods

In the Greenhouse temperature was 22°C (± 1°C) and relative humidity: 60-70% with a photoperiod: L: D = 12:12. In the field within the period of the experiment (from 15th to 29th of April 2004) the average temperature was 11°C (min: 4°C max: 16°C), the size of the field plots (n= 24) was 4.75 x 2.64 m.

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Spore suspension Conidia of M. anisopliae spores were directly streaked on selective medium plates. After 12 days of incubation in an air- conditioned chamber (22°C; ± 1°C) conidia were harvested: for one Petri dish 10ml 0.05% Tween 80 were pipetted onto the plate and the conidia were scraped off with a pipette. Afterwards the spore suspension was collected with a pipette and transferred into a beaker. The suspension was homogenized with a reciprocal shaker for 40 minutes to separate the “chain-like” conidia branches. The number of spores per millilitre was counted with a haemocytometer and finally the suspension was diluted with 0.05% Tween 80 (in tap water) as required.

Application of spore suspension and sampling In the Greenhouse a spore suspension (M. anisopliae FAL-strain 715) with a concentration of 2x107 spores/ml (in 0.05%Tween 80) was sprayed on four weeks old oilseed rape plants having 4-6 leaves/plant. Two types of seedlings were used: treated with a molluscicide and untreated. In the greenhouse differences between molluscicide-treated and untreated oil seed rape seeds as well as the stability of spores under cooled or frozen conditions on leaves were tested. Leaf samples (all fully developed leaves) were taken before application and 1, 17, 24 and 168 hours after the treatment. One hour after the application with spore suspension samples were transferred to the cold storage room at +4°C or into the freezer at -18°C. Field experiment: Two different spore concentrations (2x106 spores/ml and 2x107 spores/ml) applied once and twice (first on 15.04.2004 and second on 22.04.2004) were tested. At the first application time the rape plants were immediately before flowering (growth stage BBCH 57). For testing the persistence of M. anisopliae spores on the soil surface soil samples were collected from the plots with the higher spore concentration only. Leaf samples were taken before application and 1, 16, 24, 72, and 168 hours after the treatment. Therefore 3 – 4 leaves from the middle-part of the main stalk were cut off with scissors. Soil samples (0-4 cm depth) were taken from the field at the same intervals as the leaf samples; the soil samples were collected by pressing plastic cups (d: 4.5 cm; h: 6 cm) 4 cm into the soil. The samples were kept at 4°C until processing.

Methods to detect M. anisopliae spores Selective medium: A semi-selective medium adapted from Strasser et al. (1996) was used: 10g peptone from meat pancreatically digested, 20 g glucose and 18 g agar, all dissolved in 1 litre distilled water and autoclaved at 120 °C for 20 minutes. At a temperature of 60 °C, 0.6 g streptomycin, 0.05 g tetracycline and 0.05 g cyclohexamide (dissolved in sterile water) and 0.1 ml dodine were added. Leaf samples: 3-7 g leaves (fresh mass) were homogenized with 100ml tap water in a standard mixer for 15 sec. The mixture was filtered through a nylon.tissue with a mesh distance of 0.5 mm placed in a “Buechner”-funnel. Before plating 100 µl of the suspension with a Drigalsky spatula on selective medium the suspension was shaked for 10 sec. Three replications per sample were conducted. After 10 days incubation at 22°C ± 1°C all colony forming units of M. anisopliae were counted. M. anisopliae spores were identified by their typically green coloured spores. Soil surface: For preparation of soil samples the methodology from Keller et al., (1999) was used: 20g fresh soil was weighted into an “Erlenmeyer” flask. 100ml tap water, containing 1.8 g/l tetra-Sodiumdiphospate-Decahydrat (=Natriumpyrophosphat) was added, to favour disaggregation of the soil. The flasks were shaken for 3 hours on a shaker at 120rpm. Directly before plating them on selective medium they were shortly shaken again and after 15 sec. of sedimentation 100 µl soil suspension was distributed with a Drigalsky spatula on selective

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medium. Three replications per sample were conducted. The incubation and evaluation was the same as with leaf samples. Galleria bait method (Zimmermann, 1986): Four G. mellonella larvae were added to plastic cups (diameter: 4, 5 cm; height: 6 cm) filled with 60g soil sieved trough a 5 mm wire-mesh. During the first 5 days of incubation at 22°C ± 1°C the cups were turned daily to keep the larvae moving in the soil. After three weeks the dead larvae were removed, infection was checked and infection rates were calculated.

Statistical methods For statistical analyses the program SPSS 11, 5 and Microsoft Office Excel 2003 were used. The differences between untreated and molluscicide-treated seeds as well as differences between cooled and frozen leaves were calculated with a paired t-test. The persistence of M. anisopliae spores on leaves and on the soil surface was calculated with the variance analysis of repeat measurements (MANOVA).

Results

Greenhouse: No significant differences between the persistence of spores on leaves from treated and untreated seeds were found (p ≥ 0.05) (figure 1). Furthermore no significant differences in the persistence of the spores on leaves between leaf samples stored cooled or frozen (p ≥ 0.05) were determined, although cooled samples showed a higher number of colony forming units (CFU)/g per leaf fresh mass (LFM) (474 000 CFU/g LFM) compared to frozen samples (327 000 CFU/g LFM).

100000

80000

60000 untreated 40000 treated 20000

0 CFU/g fresh mass leaves mass fresh CFU/g 0 1 17 24 168 hours after treatment

Figure 1: Persistence of M. anisopliae spores on oilseed rape leaves in the greenhouse (number of colony forming units per g leaf fresh mass) on leaves from treated and untreated seeds before application and 1-, 17-, 24- and 168 hours after treatment.

Field: Differences were found between the two spore concentrations on leaves: In the higher concentration the number of CFU per g LFM was significantly higher compared to the lower spore concentration (figure 2).

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Data about the stability of spores on leaves showed a very short viability under greenhouse and under field conditions. The number of CFU per g LFM decreased dramatically within 24 hours (figure 1 and 2).

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15000 2x10*6 sp/ml 2x10*7 sp/ml 10000

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Figure 2: Persistence of M. anisopliae on leaves in the field after spraying two different spore concentrations.

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2000 first treatment 1500 second treatment

1000 cfu/g dry mass soil dry cfu/g 500

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Figure 3: Persistence of M. anisopliae spores in soil samples after a first and second treatment with spore suspension (2x107sp/ml).

Selective medium: Spores of M. anisopliae survived on the soil surface for two weeks and significant higher numbers of CFU per g soil fresh mass were counted after a second treatment (figure 3).

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Galleria bait method: Soil samples were collected before application and 1-, 16 -, 24 -, 72 and 168 hours after the treatment. After three weeks incubation about 50% of the recovered G. mellonella larvae were infected with M. anisopliae (figure 4).

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40,00 infection rate%

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Figure 4: M. anisopliae infection rates of G. mellonella in soil samples before application and 1, 16, 24, 72 and 168 hours after the treatment with spore suspension.

Discussion

The persistence of M. anisopliae spores was different on leaves and on the soil surface. In our experiments, spores on leaves were inactivated within a few hours in contrast to spores on soil surface. One reason for the decrease of the number of infected G. mellonella larvae might be the rainfall between 16 and 24 hours after the first treatment. Therefore spores from the leaves probably have been washed off to the soil surface and this could be the reason that in soil samples taken 24 hours after the treatment the percentage of infected G. mellonella larvae was higher than in samples taken 16 hours after the treatment. The most important influencing factors are probably solar radiation, relative humidity, rainfall, leaf expansion, temperature, soil-covering index, edaphic factors, plant volatiles and plant morphology (Zimmermann, 1982; Fargues et al., 1996; Inyang et al., 2000; Butt, 2002; Strasser & Erschbamer, 2003). Many experiments measuring the persistence of M. anisopliae spores in the soil have been conducted (Inyang et al., 2000; Vestergaard et al., 2000; Butt et al., 2001). They confirm our results concerning the persistence of M. anisopliae spores on the soil surface. According toVänninen et al. (2000) M. anisopliae spores are able to survive in the upper soil layer for three years. Using M. anisopliae as a biological control agent could be an advantage, because of the relative long persistence on the soil surface and the short viability on leaves, e.g. adult pest insects could be infected by spores on leaves and larvae could be infected during their development in the soil. The fungi should have a sufficient long persistence for infection of the target host, but not too long to prevent infections of beneficial or indifferent arthropods. Therefore, coinci-

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dence of host presence and application time is very important. Next steps of research on this topic should be on application time to optimize the coincidence of the fungal spores and the target host, frequency of spraying, formulation of spore suspensions, optimal inoculation dose and selection of host specific fungus strains.

Acknowledgements:

We thank the Center for International Relations of the BOKU-University, Vienna, and the Center of scholarship of the Federal Ministry for Education, Science and Culture for funding the study trip to Switzerland and the Swiss Federal Research Station for Agro-ecology, Zurich, for making the experiments possible and for the support during the tests.

References:

Butt, T.M. 2002: Use of entomogenous fungi for the control of insect pests. – The Mycota XI, Agricultural Applications: 111-134. Butt, T.M., Jackson, C.W. & Magan, N. (2001): Fungi as Biocontrol Agents: Progress, Problems and Potential. – CAB International. Fargues, J., Goettel, M.S., Smits, N., Ouedraogo, A., Vidal, C., Lacey, L.A., Lomer, C.J. & Rougier, M. 1996: Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes. – Mycopathologia 153(3): 171-181. Inyang, E.N., McCartney, H.A., Oyejola, B., Ibrahim, L., Pye, B.J., Archer, S.A. & Butt, T.M. 2000: Effect of formulation, application and rain on the persistence of the entomogenous fungus Metarhizium anisopliae on oilseed rape. – Mycological Research 104: 653-661. Keller, S., David-Henriet, A.-I. & Schweizer, C. 2000: Insect pathogenic soil fungi from Melolontha melolontha control sites in the canton Thurgau. – IOBC/wprs Bull. 23(8): 73- 78. Strasser, H., Forer, A. & Schinner, F. 1996: Development of media for the selective isolation and maintenance of virulence of Beauveria brongniartii. – Proc. 3 rd Internat. Workshop Microbial Control of Soil Dwelling Pests: 125-130 Strasser, H. & Erschbamer, M. 2003: Effect of temperature on conidia germination and vegetative growth of Metarhizium anisopliae. – IOBC/wprs. Bulletin 26(1): 117-120 Vänninen, I., Tyni-Juslin, J. & Hokkanen, H. 2000: Persistence of augmented Metarhizium anisopliae and Beauveria bassiana in Finnish agricultural soils. – BioControl 45: 201- 222. Vestergaard, S. & Eilenberg J. 2000: Persistence of released Metarhizium anisopliae in soil and prevalence in ground and rove beetles. – IOBC/wprs Bulletin 23(2): 181-185. Zimmermann,G. 1982: Effect of high temperatures and artificial sunlight on the viability of conidia of Metarhizium anisopliae. – Journal of Invertebrate Pathology 40: 36-40. Zimmermann,G. 1986: The “Galleria bait method” for detection of entomopathogenic fungi in soil. – Journal of Applied Entomology 102: 312-215

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 185-188

The natural distribution of the entomopathogenic soil fungus Metarhizium anisopliae in different regions and habitat types in Switzerland

Sónia Rodrigues 1,2, Ralf Peveling2, Peter Nagel2, Siegfried Keller1 1 Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, CH-8046 Zürich, 2 Institute of Environmental Sciences – Biogeography, St. Johanns-Vorstadt 10, CH-4056 Basel, Switzerland

Abstract: The natural distribution of the entomopathogenic soil fungus Metarhizium anisopliae was examined in the Swiss regions of Baselland and Zurcher Oberland. These regions differ from each other with respect to altitude, climate and soil characteristics. In each region, four different habitat types were investigated: cropland, meadowland, coniferous and broad-leaved forest. The objective was to monitor the frequency of occurrence of M. anisopliae in relation to habitat type, using the Galleria bait method. The fungus was found in all habitats in both regions, but showed significantly different distribution patterns and frequencies. It was generally more abundant in Zurcher Oberland than in Baselland. In both regions, meadows showed the highest frequencies, followed by cropland. In contrast, M. anisopliae was rare in forests.

Keywords: Metarhizium anisopliae, natural occurrence, Galleria bait method

Introduction

The hyphomycete soil fungus Metarhizium anisopliae infects a wide range of soil-dwelling insects and can be an important natural control agent regulating populations. Thus, mycoinsecticides on the basis of spores of M. anisopliae have been developed to control hypogeal pests such as grubs and wireworms but also pests that do not have soil-dwelling larval or adult stages (e.g., locusts). The distribution and frequency of occurrence of M. anisopliae is strongly related to physicochemical soil properties and insect host densities. Therefore, knowledge about the natural distribution of M. anisopliae in soils can provide important evidence of its potential as a biocontrol agent for regional insect pests (Hajek & Leger 1994). Large-scale studies on the natural occurrence and distribution of entomopathogenic fungi have been conducted, inter alia, in Poland (Mietkiewski et al. 1994), Finland (Vänninen 1996), Italy (Tkaczuk & Renella, 2003), Canada (Bidochka et al. 1998) and Tasmania (Rath et al. 1992). On a micro-scale, Meyling & Eilenberg (2005) studied the frequency of occurrence and distribution of Beauveria bassiana and M. flavoviride within a single field in Denmark. In Switzerland, Keller et al. (2003) examined the distribution of entomopathogenic fungi in different parts of the country. However, these studies did not include the northwestern part of Switzerland (Jura region), nor did they include forests. In the present study, we examine the frequency of occurrence of M. anisopliae in different soils from the regions Baselland (Jura, BL) and Zurcher Oberland (ZH). Our study includes four ecosystems (habitat types), (1) nutrient-poor meadows, (2) cultivated land, (3) broad-leaved and (4) coniferous forest.

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Material and methods

All habitat types were represented in both regions. Altogether, we sampled soils from 80 sites (10 sites per habitat type and region). At each site, 10 sub-samples were collected at a depth of 10–15 cm. To isolate M. anisopliae from the soil samples, we used the Galleria bait method (Zimmermann 1986). After removing roots and gravel, soil samples were first sifted through a 5 mm sieve. Thereafter, plastic boxes (6 cm high, diameter 4,5 cm) were filled with 60 g of soil, and four G. mellonella late instar larvae were introduced. The larvae were incubated at 20°C in dark conditions. During the first five days, the boxes were turned once daily to keep the of the bait larvae movin in the soil. After 14 to 18 days, the larvae were examined and classified as (1) healthy, (2) infected with M. anisopliae (sporulating cadavers) or (3) dead for other reasons (non-sporulating cadavers) (Kessler et al. 2003).

Results

Metarhizium anisopliae was found at 77.5% of all sites (both regions pooled). The fungus was detected in all soils from nutrient-poor meadows but not in all soils from cultivated land or forest. Samples from Zurcher Oberland had higher frequencies of occurrence than Baselland. The difference was significant at P < 0.01 (nested ANOVA on arcsine-transformed data).

100%

75%

50%

Frequency of occurence 25%

0% Baselland Zurcher Oberland

Figure 1. Mean frequency of occurrence (standard error) of M. anisopliae in soil samples from Baselland and Zurcher Oberland, using the Galleria bait method.

A comparison among habitat types and regions revealed that the frequency of occurrence of M. anisopliae was highest in soils from meadows, both in Baselland and Zurcher Oberland, followed by cropland in ZH (Fig. 2). Coniferous forests, cropland in Baselland and broad- leaved forests in Baselland had the lowest frequencies.

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100%

aaabc ababc

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Broad-leaved forest Coniferous forest 50% Cropland Meadow

Frequency of occurence 25%

0% BL ZH Figure 2. Mean frequency of occurrence (standard error) of M. anisopliae in different habitat types. Means not sharing the same letter are significantly different at P < 0.05 or lower (One-way ANOVA of arcsine transformed data, followed by NK multiple comparison of means).

Discussion

In the present study, M. anisopliae was detected at almost all sites, confirming the cosmopolitan character of this fungus. However, the study showed different distribution patterns and frequencies of occurrence among regions and habitats, with higher frequencies in Zurcher Oberland than in Baselland. This seems to be related to different environmental conditions. The two regions vary with respect to altitude, climate and soil properties. The sites in Zurcher Oberland are located in the pre-alpine zone which is characterised by low input land use systems. These systems are likely to enhance the diversity and density of soil-dwelling insects, including potential hosts of M. anisopliae. Studies are under way to analyse the relationship between fungal densities and environmental factors. The differences between habitat types can be explained by a combination of biotic and abiotic conditions. In this study, meadows showed higher frequencies than all other habitats. Similar observations were made in other parts of Switzerland where densities of M. anisopliae were higher in meadows than in arable land (Keller et al. 2003). Low densities of M. anisopliae in arable land seem to be related to the scarcity of hosts due to soil cultivation and the input of fertilizers and pesticides. Forests showed the lowest frequencies of occurrence of M. anisopliae. This confirms results from Bidochka et al. (1998) who found that M. anisopliae was more abundant in agricultural than in forest soils, whereas the contrary was true for Beauveria bassiana. The density and diversity of soil organisms in (semi-) natural habitats such as forests differs profoundly from that in arable land. This might change interactions between potential host species and affect the growth of and competition among entomopathogenic fungi.

References

Bidochka, M.J., Kasperski, J.E.& Wild, G.A.M. 1998: Occurrence of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana in soils from temperate and near- northern habitats. – Can. J. Bot. 76: 1198-1204.

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Hajek, A.E. & Leger, R. J. St. 1994: Interactions between fungal pathogens and insect hosts. – Annu. Rev. Entomol. 39: 293-322. Keller, S., Kessler, P.& Schweizer, C. 2003: Distribution of insect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metarhizium anisopliae. – BioControl 48: 307-319. Kessler, P., Matzke, H., & Keller, S. 2003: The effect of application time and soil factors on the occurrence of Beauveria brongniartii applied as a biological control agent in soil. – Journal of Invertebrate Pathology 84: 15-23. Meyling, N.V. & Eilenberg J. 2005: Natural occurrence and spatial distribution of Beauveria bassiana and Metarhizium flavoviride in a Danish agro-ecosystem. Insect Pathogens and Entomoparasitic Nematodes – IOBC/wprs Bulletin (in press). Mietkiewski, R., Klukowski, Z. & Balazi, S. 1994: Entomopathogenic fungi isolated from soil of mid-forest meadows of Sudety mountains. – Roczniki Nauk Rolniczych, Seria E, 24(1/2): 33-38. Rath, A.C., Koen, T.B. & Yip, H.Y. 1992: The influence of abiotic factors on the distribution and abundance of Metarhizium anisopliae in Tasmanian pasture soils. – Mycol. Res. 96(5): 378-384. Tkaczuk, C. & Renella, G. 2003: Occurrence of entomopathogenic fungi in soils from Central Italy under different managements. – IOBC/wprs Bulletin 26(1): 99-102. Vänninen, I. 1996: Distribution and occurrence of four entomopathogenic fungi in Finland: effect of geographical location, habitat type and soil type. – Mycol. Res. 100(1): 93-101. Zimmermann, G. 1986: The “Galleria bait method” for detection of entomopathogenic fungi in soil. – Zeitschrift für Angewandte Entomologie 102: 213-215.

Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 189-192

What have BIPESCO and RAFBCA achieved that could help with risk assessment and registration?

Hermann Strasser, Barbara Pernfuss Leopold-Franzens University, Institute of Microbiology, Technikerstrasse 25, A-6020 Innsbruck, Austria

Abstract: BIPESCO (FAIR6-CT-98-4105; http://bipesco.uibk.ac.at) was an EU-funded project to develop entomogenous fungi for the control of subterranean insect pests like scarabs and weevils. The project was analysed by ADAS (http://www.adas.co.uk) concerning its impact in Area 4 of Framework 4, and has been put into the small group of projects judged to be success stories. RAFBCA (QLK1-CT2001-01391, http://www.rafbca.com) was the follow up project because BIPESCO members believed that the new methodologies needed refinement and validating. The RAFBCA consortium identified realistic risk assessment strategies as important to ensure public safety and to propose guidelines for a clear, cost effective registration procedure. Both projects had strong relevance to SMEs as the end-users, because the consortia gave support for five fungal biocontrol agents to overcome the registration hurdle by completing the dossiers for notification. Future activities are scheduled which will focus on improving sustainable, quality-based crop production systems by applying fungal biological control agents. The overall aim will be to give scientific support to policy by reviewing the current legislation, guidelines and guidance documents at member state level and at EU level and compare this with similar legislation in other countries where introduction of new biocontrol agents has proven to be more successful.

Keywords: anamorphic fungi, fungal biocontrol agent, risk assessment, policy oriented research

Biological Pest Control (BIPESCO)

BIPESCO, acronym for Biological Pest Control, was an EU-funded project to develop entomogenous fungi for the control of subterranean insect pests like scarabs and weevils. European scientists and four industrial partners from seven different European countries participated in this multidisciplinary, multifaceted project. Particular attention has been focused on methods for improving production and field efficacy of fungal BCAs (Biological Control Agents). The data generated could help accelerate registration of BCAs based on promising isolates of Beauveria and Metarhizium (Strasser, 2004). The non-confidential information was published and/or in press in more than 30 international, refereed scientific papers. Additionally, more than 100 BIPESCO contributions provided information that helped end users (e.g. policy makers, registration authorities, industry) and the public in making more informed decisions regarding the use and the risks, if any, that fungal BCAs may poses to plant, human and animal health (Strasser & Butt, 2005). Methods and strategies were suggested which could standardise the risk assessment of fungal biological control agents (Butt et al., 2001). The BIPESCO consortium believed that the new methodologies developed needed refinement and validating. One initiative was an EU RTD-project (QLK1-CT-2001-01391) with the acronym RAFBCA (http://www.rafbca.com).

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Risk Assessment of Fungal Biological Control Agents (RAFBCA)

The RAFBCA project has been funded under the Fifth Framework Programme of the European Commission, Quality of Life and Management of Living Resources Programme (QoL), Key Action 1 - Food, Nutrition and Health, (Contract n°QLK1-CT- 2001-01391). Ten partners from nine countries have cooperated in this project from November 2001 to October 2004. They have generated data that could help address key registration questions for BCAs. The overall aim of this unique project has been to establish whether metabolites produced by fungal BCAs enter the food chain and if they pose a risk to human and animal health.

Identification and detection of metabolites The team has identified efficacious strains of mycoinsecticides (Beauveria brongniartii, Metarhizium anisopliae, Verticillium lecanii), mycoparasites (Gliocladium spp., Trichoderma harzianum), and a mycoherbicide (Stagonospora convolvuli) as well as their major metabolites (oosporein, destruxins, gliotoxin, peptaibols, elsinochrome A). They have generated data that provide a better understanding of the type and quantity of the selected major metabolites as well as their distribution and regulation. All the partners have been involved in field and glasshouse trials under commercial/ semi-commercial conditions to determine the fate of fungal BCAs and their metabolites in the food chain (e.g. lettuce, cucumber, tomato, potato, maize) and plant growing media. They have shown that BCA metabolites do not enter the food chain or pose a risk to consumer and animal health, nor pose environmental problems.

Development of tools and methodologies The RAFBCA team has developed methods and tools to conduct a targeted risk assessment - making this more reliable and industry more competitive. Standard Operating Protocols will be made available to a wide audience. Some of the methods and tools include: (i) Molecular probes to monitor several important fungal BCAs in the environment. (ii) Highly sensitive SF-9 insect cell-line - more sensitive than mammalian cell-lines to a wide range of fungal metabolites. (iii) Highly sensitive single cell organism (Paramecium caudatum) and invertebrates (Artemia salina, Daphnia magna) - very sensitive to metabolites and crude extracts of fungal BCAs. (iv) Extraction methods that give good recovery and repeatability, (v) Evaluation of the Ames and Vitotox tests - pure metabolites and crude extracts from BCAs were not genotoxic or mutagenic.

Case studies – mycoherbicide and mycoinsecticides Field bindweed and hedge bindweed are considered among the twelve economically most important weeds. Stagonospora convolvuli LA39, an effective biocontrol agent of both bindweed species produces elsinochrome A as a major metabolite. Data obtained from greenhouse- as well as from field-trials demonstrate that elsinochrome A is rarely present in the applied product and if so, in amounts which are far too small to pose any risk to the environment or the consumer. Further, elsinochrome A is neither produced on the crop nor on the bindweed, therefore the risk that this toxic compound enters the food chain is negligible. The entomopathogenic fungi Metarhizium anisopliae and Verticillium lecanii successfully control a wide range of soil and foliar pests. Destruxins, major metabolites of both species, were shown not to enter tomato, cucumber or radish fruit in large-scale greenhouse trials when the BCAs were applied at normal and 10 fold higher dose than recommended. Results indicate that the metabolites pose no risk to growers, consumers and the environment. The use of the entomopathogenous fungus Beauveria brongniartii for Melolontha melolontha (European cockchafer) control is recommended and poses no risk to potatoes.

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Oosporein, the major metabolite secreted by B. brongniartii, does not enter potato plants. Physical-chemical characterisation of oosporein shows that oosporein poses no risk to human health and the environment.

Significance and impact of both EU projects

Benefits of SMEs, consumers and growers It has been demonstrated that fungal BCAs do not pose a risk to human health and the environment. This strengthens the argument for their use as safe alternatives to chemical pesticides and could support an international agreement for the development of pest control strategies, which reduce or eliminate the use of harmful chemical pesticides. This will, ultimately, encourage commercial development of the BCAs and through increased sales increase wealth and create jobs to meet increased demand for these products.

Contributions to EC policy BIPESCO and RAFBCA impacts on Directive 91/414/EEC and Directive 2001/36/EEC (data required on microbial BCAs in Annexes II and III Part B) by showing that the evaluation of fungal biocontrol agents and their major metabolites during registration of BCAs could be simplified. Both consortia has generated new data that can be used to develop a new risk assessment strategy that could help accelerate risk assessment of fungal metabolites and reduce registration costs. They have devised strategies that could lead to a more balanced system for risk assessment and registration -and enable the EC to compete with the USA and other countries. BIPESCO and RAFBCA have data that could help end users (policy makers, registration authorities, industry) and the public in making more informed decisions about fungal BCAs.

Dissemination The consortium has disseminated results through flyers, numerous international scientific journals, and via oral and poster presentations at national and international symposia. The BIPESCO team has (co-) organised four highly successful International Symposia: (i) the Melolontha-Tagung 2000, 23rd February, 2000, Auer, Italy. (ii) “Bioactive Fungal Metabolites – Impact and Exploitation”; held 22nd–27th April, 2001 at the University of Wales, Swansea; and the Closing Meeting of BIPESCO FAIR6 CT-98-4105" , 24th January 2002, University of Vienna, Austria. Furthermore, the BIPESCO team was helping to the "Third Meeting of the Melolontha Subgroup IOBC wprs Working Group "Integrated Control of Soil Pests" which is to be held 24th-26th September 2001 in Aosta, Italy. The RAFBCA team has organised three highly successful workshops: (i) Helsinki, August 2004, in collaboration with the SIP, IBMA, and IOBC. (ii) Brussels, September 2004, in collaboration with the IOBC and IBMA and (iii) Innsbruck, October 2004, in collaboration with the IOBC. The BIPESCO website (http://bipesco.uibk.ac.at) and RAFBCA website (http:// www.rafbca.com) have been visited by numerous groups from all over the world.

Future activities A new activity must be the next step: BIPESCO and RAFBCA members prepare themselves to follow up with a policy oriented research project funded by the 6th Framework Programme of the Europoan Union (Call identifier: FP6-2004-SSP-4). The aim of RAFBCA II will be to review current legislation, guidelines and guidance documents at Member State and EU level and compare this with similar legislation in other countries where the introduction of new biopesticides has proven to be more successful.

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It is scheduled that our future research activities will focus more on improving sustainable, quality-based crop and animal production systems (including non-food products and uses) and developing techno-economic references to support the EU legislation.

Acknowledgements

This presentation was supported by the European Commission by the Quality of Life and Management of Living Resources Programme (QoL), Key Action 1 on Food, Nutrition and Health (RAFBCA: QLK1-CT-2001-01391).

References

Butt, T.M., Jackson, C. & Magan, N. 2001: Fungal Biocontrol Agents: Progress, Problems & Potential. – CABI Wallingford: 390 pp. Strasser, H. 2004: Biocontrol of important soil dwelling pests by improving the efficacy of insect pathogenic fungi. – Laimburg J. 1(2): 236-241. Strasser, H., Butt, T.M. 2005: The EU BIPESCO project – latest results on safety of fungal biocontrol products. – IOBC/wprs Bulletin (in press).