IOBC/wprs Bulletin Vol. 28(2) 2005

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) 

Copyright: IOBC/WPRS 2005 

The Publication Commission of the IOBC/WPRS: 

Horst Bathon 

Federal Biological Research Center 

for Agriculture and Forestry (BBA) 

Institute for Biological Control 

Heinrichstr. 243 

D-64287 Darmstadt (Germany) 

Tel +49 6151 407-225, Fax +49 6151 407-290 

e-mail: h.bathon@bba.de 

ISBN 92-9067-174-0 

Luc Tirry 

University of Gent 

Laboratory of Agrozoology 

Department of Crop Protection 

Coupure Links 653 

B-9000 Gent (Belgium) 

Tel +32-9-2646152, Fax +32-9-2646239 

e-mail: luc.tirry@ugent.be

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

List of Participants 

BASSO Barbara 

National Research Council 

University of Milano 

Via Celoria, 26 

20133 Milano 

Italy 

Tel: + 39 02 50314718 

Fax: + 39 02 50314764 

E-mail: barbara.basso@unimi.it 

BENKER Ullrich 

Bayerische Landesanstalt für Landwirtschaft 

Institut für Pflanzenschutz 

Lange Point 10 

85354 Freising 

Germany 

Tel: + 49 8161 71 5720 

Fax: + 49 8161 71 5753 

E-mail: ullrich.benker@lfl.bayern.de 

BLACKSHAW Rod 

School of Biological Sciences 

University of Plymouth 

Drake Circus 

PL4 8AA Plymouth 

United Kingdom 

Tel: + 44 1626 325600 

E-mail: rblackshaw@plymouth.ac.uk 

BRENNER Hermann 

LBBZ Arenenberg 

Fachstelle Pflanzenschutz und Ökologie 

Arenenberg 

8268 Salenstein 

Switzerland 

Tel: + 41 71 663 31 40 

Fax: + 41 71 664 28 67 

E-mail: hermann.brenner@kttg.ch 

BRUNNER Nina 

Ludwig Boltzmann Institut 

für biologischen Landbau 

Felbigergasse 93 /11 

1140 Vienna 

Austria 

Tel: + 43 (0) 1 0650 8262545 

E-mail: sauerampfer@hotmail.com 

CATE Peter C. 

Österreichische Agentur für Gesundheit 

und Ernährungssicherheit Ges.m.b.H. (AGES) 

Spargelfeldstr. 191 

1226 Vienna 

Austria 

Tel: + 43 (0)1 73216 5223 

Fax: + 43 (0)1 73216 5216 

E-mail: peter.cate@ages.at 

CLERES Sabine 

lbu-Labor für Boden- und Umweltanalytik 

der ERIC SCHWEIZER SAMEN AG 

Postfach 150 

3602 Thun 

Switzerland 

Tel: + 41 (0) 33 227 57 30 

Fax: + 41 (0) 33 227 57 39 

E-mail: sabine.cleres@schweizerseeds.ch 

DALLA VIA Josef 

Land- und Forstwirtschaftliches Versuchszentrum 

Laimburg 

Laimburg 6 

39040 Auer 

Italy 

Tel: + 39 0471 969510 

Fax: + 39 0471 969599 

E-mail: josef.dallavia@provinz.bz.it 

EILENBERG Jørgen 

Royal Veterinary and Agricultural University 

Department of Ecology 

Thorvaldsensvej 40 

1871 Frederiksberg 

Denmark 

Tel: + 45 35 282692 

Fax: + 45 35 282670 

E-mail: joergen.eilenberg@ecol.kvl.dk 

ENKERLI Jürg 

Swiss Federal Research Station 

for Agroecology and Agriculture 

Reckenholzstrasse 191 

8046 Zürich 

Switzerland 

Tel: + 41 1 377 7206 

Fax: + 41 1 377 7201 

E-mail: juerg.enkerli@fal.admin.ch 

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ERICSSON Jerry 

University of British Columbia 

Department of Plant Science 

301 – 1249 Granville Street 

V6Z 1M5 Vancouver 

Canada 

Tel: + 604 694 0791 

Fax: + 604 850 2150 

E-mail: ericsson@zoology.ubc.ca 

ESTER Albert 

Applied Plant Research, Arable farming 

and field production of vegetables 

Edelhertweg 1 

8219 PH Lelystad 

The Netherlands 

Tel: + 31 320 291633 

Fax: + 31 320 230479 

E-mail: albert.ester@wur.nl 

FRÖSCHLE Manfred 

Landesanstalt für Pflanzenschutz 

Reinsburgstr. 107 

70197 Stuttgart 

Germany 

Tel: + 49 711 6642445 

Fax: + 49 711 6642499 

E-mail: manfred.froeschle@lfp.bwl.de 

FURLAN Lorenzo 

Group of Entomolgy, Departement of 

Agronomy, University of Padova 

Via Carozzani 18 

30027 San Donà di Piave 

Italy 

Tel: + 39 0335 7162739 

Fax: + 39 0421 596659 

E-mail: lorenzo.furlan@inwind.it 

GUENTER Martin 

Andermatt Biocontrol AG 

Stalermatten 6 

6146 Grossdietwil 

Switzerland 

Tel: + 41 62 917 50 05 

E-mail: guenter@biocontrol.ch 

HADAPAD Ashok 

University of Hohenheim 

Institute of Phytomedicine, Dept. of Entomology 

Otto-Sander Str. 5 

70593 Stuttgart 

Germany 

Tel: + 49 711 459 3220 

Fax: + 49 711 459 2408 

E-mail: ahadapad@uni-hohenheim.de 

HOZZANK Alexandra 

InfoXgen 

Königsbrunnerstrasse 8 

2202 Enzersfeld 

Austria 

Tel: + 43 (0)2262 672214 31 

Fax: + 43 (0)2262 672214 33 

E-mail: a.hozzank@agrovet.at 

JUEN Anita 

Centre for Mountain Agriculture 

University of Innsbruck 

Technikerstrasse13 

6020 Innsbruck 

Austria 

Tel: + 43 (0)512 507 5695 

E-mail: anita.juen@uibk.ac.at 

KABALUK Todd 

Agriculture and Agri-Food Canada 

c/o Pacific Agri-Food Research Centre 

Box 1000 Agassiz 

British Columbia 

Canada VOM 1AO 

E-mail: kabalukt@agr.gc.ca 

KATZUR Katrin 

Biologische Bundesanstalt für Land- und 

Forstwirtschaft 

Messeweg 11-12 

38104 Braunschweig 

Germany 

Tel: + 49 531 2994576 

Fax: + 49 531 2993008 

E-mail: k.katzur@bba.de 

KELLER Siegfried 

Federal Research Station for Agroecology 


8046 Zürich 

Switzerland 

Tel: + 41 1 377 7211 

Fax: + 41 1 377 7201 

E-mail: siegfried.keller@fal.admin.ch

KOLLER Robert 



Institut für biologischen Pflanzenschutz 

Heinrichstraße 243 

64287 Darmstadt 

Germany 

Tel: + 49 (0) 6151 407 0 

E-mail: RobKoller@gmx.de 

KOUTNY Andreas 

Landeslandwirtschaftskammer Tirol 

Fachabteilung: Pflanzenbau - Landtechnik 

Brixnerstrasse 1 


Austria 

Tel: + 43 (0) 512 5929 230 

E-mail: andreas.koutny@lk-tirol.at 

KRENN Andreas 

F. Joh. Kwizda GmbH 

Dr. Karl Lueger-Ring 6 

1010 Vienna 

Austria 

Tel: + 43 (0)1 53468 235 

Fax: + 43 (0)1 53468 280 

E-mail: j.raffalt@kwizda-agro.at 

KROMP Bernhard 

Ludwig Bolzmann Institute for 

Biological Agriculture & Applied Ecology 

Rinnboeckstr. 15 

1110 Vienna 

Austria 

Tel: + 43 1 712 98 99 

E-mail: bernhard.kromp@univie.ac.at 

KRON-MORELLI Roberto 

Agrifutur srl 

Via Campagnole 8 

25020 Alfianello (BS) 

Italy 

Tel: + 39 030 9934776 

Fax: + 39 030 9934777 

E-mail: rkm@numerica.it 

LAENGLE Tobias 

Institut für Mikrobiologie 

Leopold-Franzens-Universität 

Technikerstrasse 25 


Austria 

Tel: + 43 (0) 512 507 6010 

Fax: + 43 (0) 512 507 2929 

E-mail: tobias.laengle@uibk.ac.at 

LANDL Marion 

Institut für Pflanzenschutz 

Universität für Bodenkultur 

Peter Jordan Strasse 

1180 Vienna 

Austria 

Tel: + 43 (0) 1 47654 3355 

E-mail: marion.landl@boku.ac.at 

MUŠKA František 

State Phytosanitary Admin. SRS-OPOR 

Zemedelska 1a 

61300 Brno 

Czech Republic 

Tel: + 420 5 45137057 

Fax: + 420 5 45211078 

E-mail: muska34@volny.cz 

NEUHOFF Daniel 

Institute of Organic Agriculture 

University of Bonn 

Katzenburgweg 3 

53115 Bonn 

Tel: + 49 228 735616 

Fax: + 49 228 735617 

E-mail: d.neuhoff@uni-bonn.de 

PAFFRATH Andreas 

Landwirtschaftskammer Nordrhein-Westfalen 

Endenicher Allee 60 

53115 Bonn 

Germany 

Tel: + 49 228 7031537 

Fax: + 49 228 7038537 

E-mail: andreas.paffrath@lwk.nrw.de 

PARKER William 

ADAS 

Woodthorne 

WV6 8TQ Wolverhampton 


Tel: + 44 1746 712815 

E-mail: bill.parker@adas.co.uk 

van der PAS Rick 

Koppert Biological Systems 

Veilingweg 17 

2651 BE Berkel en Rodenrijs 

The Netherlands 

Tel: + 31 10 514 0444 

Fax: + 31 10 512 1005 

E-mail: hvdsteen@koppert.nl 

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PÁZMÁNDI Christian 





Austria 

Tel: + 43 0512 507 5689 

E-mail: christian.pazmandi@uibk.ac.at 

PERNFUSS Barbara 


Leopold-Franzens Universität Innsbruck 



Austria 

Tel: + 43 (0)512 507 6012 

Fax: + 43 (0)512 507 2929 

E-mail: barbara.pernfuss@uibk.ac.at 

PETERS Arne 

e-nema GmbH 

Klausdorfer Str. 28-36 

24223 Raisdorf 

Germany 

Tel: + 49 4307 82950 

Fax: + 49 4307 829514 

E-mail: a.peters@e-nema.de 

PILZ Christina 

Universität für Bodenkultur 

Spittelauerlände 19-21/5/5/16 

1090 Vienna 

Austria 

Tel: + 43 69911370292 

E-mail: pilzchristina@hotmail.com 

POZENEL Anka 

Kmetijsko gozdarski zavod Nova Gorica 

Pri hrastu 18 

5000 Nova Goriza 

Slovenia 

Tel: + 386 5 3861787 

E-mail: anka.pozenel@go.kgzs.si 

RAFFALT Josef 

F. Joh. Kwizda GmbH 

Dr. Karl Lueger-Ring 6 

1010 Vienna 

Austria 

Tel: + 43 (0)1 53468 235 

Fax: + 43 (0)1 53468 280 

E-mail: j.raffalt@kwizda-agro.at 

RODRIGUES Sonia 

Agroscope FAL Reckenholz 


8046 Zürich 

Switzerland 

Tel: + 41 1 377 7467 

E-mail: sonia.rodrigues@stud.unibas.ch 

ROT Mojca 

Kmetijsko gozdarski zavod Nova Gorica 

Pri hrastu 18 

5000 Nova Goriza 

Slovenia 

Tel: + 386 5 3351211 

Fax: + 386 5 30 27 312 

E-mail: mojca.rot@kvz-ng.si 

SCHEPL Ute 

Landwirtschaftskammer Nordrhein-Westfalen 

Endenicher Allee 60 

53115 Bonn 

Germany 

Tel: + 49 2287031598 

Fax: + 49 2287038598 

E-mail: ute.schepl@lwk.nrw.de 

SCHNETTER Wolfgang 

Erlenweg 10 

69429 Waldbrunn-Schollbrunn 

Germany 

E-mail: wolfgang.schnetter@urz.uniheidelberg.de 

SCHWEIGKOFLER Wolfgang 

Research Centre for Agriculture 

and Forestry - Laimburg 

39040 Auer 

Italy 

Tel: + 39 0471 969643 

E-mail: wolfgangschweigkofler@hotmail.com 

SHAH Syed Farooq Abbas 

School of Biological Sciences 

University of Wales, Swansea 

Singleton Park 

SA2 8PP Swansea 


Tel: + 44 1792 513652 

E-mail: f.a.shah@swansea.ac.uk

SIERPINSKA Alicja 

Forest Research Institute in Warsaw 

05-090 Raszyn 

Sekocin-Las 

Poland 

Tel: + 48 22 7150 547 / 544 

Fax: + 48 22 7150 557 

E-mail: a.sierpinska@ibles.waw.pl 

STRASSER Hermann 


Leopold-Franzens-Universität 



Austria 

Tel: + 43 (0)512 507 6008 

E-mail: hermann.strasser@uibk.ac.at 

TOEPFER Stefan 

CABI Bioscience CH 

c/o Plant Health Service 

of Csongrad County in Hungary 

Rarosi ut 110 PO 99 

6800 Hodmezovasarhely 

Hungary 

Tel: + 36 62 535740 

Fax: + 36 62 246036 

E-mail: stoepfer@gmx.net 

TOTH Miklos 

Plant protection Institute HAS 

Herman O. u. 15 

1022 Budapest 

Hungary 

Tel: + 361 3918639 

Fax: + 361 3918655 

E-mail: h2371tot@ella.hu 

TRAUGOTT Michael 





Austria 

Tel: + 43 (0)512 507 5696 

E-mail: michael.traugott@uibk.ac.at 

VERNON Robert S. 

Agriculture and Agri-Food Canada 

Pacific Agri-Food Research Center 

PO Box 1000 

Agassiz, British Columbia 

Canada 

E-mail: vernonbs@agr.gc.ca 

WEISSTEINER Sonja 

Institut für Forstzoologie und Waldschutz 

Buesgenweg 3 

37077 Göttingen 

Germany 

Tel: + 49 551 393609 

Fax: + 49 551 392089 

E-mail: sweisst@gwdg.de 

ZELGER Roland 

Research Center for Agriculture 

and Forestry Research, Laimburg 

39040 Auer 

Italy 

Tel: + 39 0471 969601 

Fax: + 39 0471 969599 

E-mail: roland.zelger@provinz.bz.it 

ZIMMERMANN Gisbert 



Institute for Biological Control 

Heinrichstrasse 243 

64287 Darmstadt 

Germany 

Fax: + 49 6151 407 290 

E-mail: g.zimmermann@bba.de 

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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 Switzerland, 

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 

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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

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 

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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 Toepfer 1 , Jozsef Kiss 1 , Gyorgy Turoczi 1 , Judit Komaromi 1 , Ulrich Kuhlmann 2 

1 

St. Istvan University, Plant Protection Department, Pater K. Street 1, HU - 2100 Gödöllö, 

mailto: stoepfer@gmx.net 

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 km 2 (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 Diabroticainfested 

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

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 

3

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 10 6 spores per cm 2 ; 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).

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. 

5

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.

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 Rootworm TM 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.). 

7



pp. 9-12 

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

control strategies 

Siegfried Keller 1 , Gisbert Zimmermann 2 

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. 


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 

colonised (ha) 

Melolontha 

melolontha 

Size of area 

with economic 

damage (ha) 

Control strategies used 

155 000 70 000 insecticides, Beauveria 

brongniartii, mechanical 

means (nets) 

42 700 7 950 insecticides, B. brongniartii 

M. 

hippocastani 

Amphimallon 

solstitiale 

1 100 45 insecticides 

A. majale 100 60 Metarhizium anisopliae 

(trials) 

Phylloperta 31 000 30 000 insecticides, nematodes, 

horticola 

mechanical 

Hoplia spp. 3 3 nematodes 

wireworms 

(Elateridae) 

9 600 2 000 insecticides 

Noctuidae 35 25 insecticides 

Tipulidae 50 50 insecticides 

Curculionidae 

Others: 

27 125 22 670 insecticides, nematodes, 

mechanical 

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

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 

area 

colonised 

(ha) 

Melolontha 

melolontha 

Melolontha 

hippocastani 

Size of area 

with 

economic 

damage (ha) 

Estimated 

amount of 

damage 

(EURO) 

Tendency 

of population 

deve- 

lopment 

Austria >30 000 30 000 20 000 slightly 

increasing 

Belgium present no data increasing 

Control 

strategies 

B.br. 

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. 

Czech 

Republic 

12 000 250 800 000 strongly 

increasing 

insecticides, 

B. bassiana 

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. 

11

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. 


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.

Non-target effects of insect pathogenic fungi 

Nicolai V. Meyling 1 , Jørgen Eilenberg 1 1, 2 

, Charlotte Nielsen 

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 

13 

Integrated Protection in Oilseed Crops 


pp. 13-18 

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.

14 

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 melanogrammum 

in a Danish greenery plantation. The effects were studied as prevalences in nontarget 

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. 

15

16 

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. 


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. 

17

18 

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



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), 

21

22 

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.


The first count of the grubs in the infested site was on the 29 th 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 m 2 the infestation was at a very high level. Among all plots the Beauveria plot 

was the most infested one. After five weeks, on the 3 rd 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 31 st 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. 

Grubs per m 2 

300 

250 

200 

150 

100 

50 

0 

Monitoring on 29th of April 

1st count on 3rd of June 

2nd count on 31st of August 

Var 1 Var 2 Var 3 Var 4 Var 5 Var 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 3 rd 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 31 st 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 

23

24 

detected on the 3 rd of June looked at that time very healthy. Maybe the Confidor has a longterm 

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? 


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

Isolation of Beauveria brongniartii from soil: 

Are the available isolation tools neutral? 



pp. 25-29 

Jürg Enkerli 1 , Priska Moosbauer 1,2 , Franco Widmer 1 , Silvia Dorn 2 , Siegfried Keller 1 

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. 


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 fiftyone 

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 (10 7 /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

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 10 7 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 10 5 /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 postinoculation 

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 

technique 

Microsatellite loci and allele sizes [bp] 

Bb1F4 Bb2A3 Bb2F8 Bb4H9 Bb5F4 Bb8D6 

Genotype 

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 


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). 

27


Average survival time [d] 

70 

60 

50 

40 

30 

20 

10 

0 

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 

technique 

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. 

No. of isolates Genotype Original isolation 

technique 

Abundance of genotype 

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

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. 

29



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 20 th 

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). 


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. 


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. 

km 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

Forest borders damaged by cockchafers, 

Uranean flight 

1955 

1958 

heavy 

medium 

weak 

total 

1961 

1964 

1967 

1970 

1973 

1976 

1979 

1982 

Year 

1985 

1988 

1991 

1994 

1997 

2000 

2003 

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.-.

km 

160 

140 

120 

100 

80 

60 

40 

20 

0 

Forest borders damaged by cockchafers, 

Bernese flight 

1960 

1963 

1966 

1969 

1972 

1975 

1978 

1981 

1984 

1987 

1990 

1993 

1996 

1999 

2002 

Year 

haevy 

medium 

weak 

total 

Figure 2: Development of the Melolontha population in the Bernese flight area from 1960-2002. No 

data were recorded 1963-1969 and 1981. 

mean number of 

damages 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

Number of damages done by white grubs. 

Mean of three years 

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 

33

34 

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. 

Conclusions 

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 

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. 

35



pp. 37-44 

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 1 , Kerstin Jung 1 , Stefan Scheu 2 , Gisbert Zimmermann 1 , Joachim Ruther 3 

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)-3hexen-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. 


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 

trap of Berlin 

0.2 66 1224 crossed 

BBA Institute for 

Biological 

Control 

1.1 88 7500 crossed 

FU Free University 

of Berlin 

2.0 71 3200 crossed 

HF Hessian 

Forestry 

Department 

2.8 47 7200 flat

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, 1x10 9 

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 

39

40 

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

Mean captues +/- SE per trap & day 

70 

60 

50 

40 

30 

20 

10 

0 

a 

b 

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). 

Mean captures +/- SE per trap & day 

160 

140 

120 

100 

80 

60 

40 

20 

0 

a 

high Hoch middle Mittel low Niedrig 

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). 

b 

a 

c 

a 

41

42 

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

120 

100 

80 

60 

40 

20 

0 

* 

Control Treatment 

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 10 3 -10 4 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. 


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. 

43

44 

Strasser, H., Forer, A. & Schinner, F. 1996: Development of media for the selective isolation 

and maintenance of virulence of Beauveria brongniartii. – Proc. 3 rd 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 Waldmaikäfer. 

– Nachrichtenbl. Deutsch. Pflanzenschutzd. 50: 249-256.

45 

“New” white grubs



pp. 47-50 

Control of the garden chafer Phyllopertha horticola with GRANMET-P, 

a new product made of Metarhizium anisopliae 

Barbara Pernfuss 1 , Roland Zelger 2 , Roberto Kron-Morelli 3 , Hermann Strasser 1 

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 m 2 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 10 5 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, EPPOstandards, 

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). 


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 m 2 ), 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.

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. 


A quantity of 500 Phyllopertha horticola larvae per m 2 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. 

M. anisopliae [cfu per g soil dry weight] 

1000000 

100000 

10000 

1000 

100 

10 

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). 

49

50 

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 10 4 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 10 2 spores g -1 dry weight. In treated plots and fairways 

the density of spores remained stable or increased. One year post treatment 6 x 10 3 spores g -1 

soil dry weight were re-isolated when GRANMET-P was applied once, and 1.7 x 10 5 spores g -1 

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. 


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 Phyllopertha 

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.



pp. 51-55 

Timing of nematode application to control white grubs (Scarabaeidae) 

Arne Peters 1 , Henk Vlug 2 

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 22 nd gave better control of P. horticola than those applied at 

August 9 th . 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 nonsusceptible 

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, Amphimallon 

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. 


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 

22 nd and on August 9 th . 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, 18 th 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 18 th , the second treatment on September 

10 th . Evaluation was done on September 25 th 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 golfcourse, 

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 1 st 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 20 th , 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. 


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. 

Beetle 

Egg 

1st instar 

2nd instar 

3rd instar 

Pupa 

May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May 

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. 

Number of grubs per sample (n=8) 

4 

3 

2 

1 

0 

-1 

±1.96*Std. Err. 

±1.00*Std. Err. 

Mean 

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 

18 th (unpublished results, Plant Protection Service Schwerin, Germany). 

Number of grubs per sample (n=8) 

11 

9 

7 

5 

3 

1 

-1 

untreated 

±1.96*Std. Err. 

±1.00*Std. Err. 

Mean 

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). 

53

54 

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. 

Beetle 

Egg 

1st instar 

2nd instar 

3rd instar 

Pupa 

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 

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 3 rd instars were treated in the field (Fig. 

5). 

On a sports field near Bremen a treatment with H. bacteriophora in March 1999 against 

2 nd instar larvae stopped further grub damage (data not published). In sufficiently humid

summers, like 2004, treatments in July against 3 rd 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: 9 th 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. 

55



pp. 57-61 

Metarhizium anisopliae for white grub control in Nepal 

Yubak Dhoj GC 1 , Siegfried Keller 2 

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 10 7 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 10 9 spores/ml, 

10 5 spores/ml, 10 3 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 


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 10 7 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 (10 9 , 10 5 , 10 2, 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).


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 

species 

Geographic 

origin 

Number of 

isolates White 

grubs 

Isolated from 

Soil/GBM Soil/SM 

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 22-24 0 C at a concentration of 1*10 7 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) 

SEM 

CV% 

6.953 

2.357 

10.81 

10.92 

2.713 

23.14 

Figures in column followed by same letter are not significantly different at p

60 

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 22-24 0 C 

Strain Concentration % mortality % mycosis 

10 9 spores/ml 79.3a 52.00a 

10 5 spores/ml 72.7ab 45.33a 

10 2 M2 

spores/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

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. 


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 Bodenbehandlungen 

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. 


and maintenance of virulence of Beauveria brongniartii. – Proc. 3 rd 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. 

61



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 10 8 to 2.2 x 10 9 conidia/cm 2 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 10 7 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/m 2 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. 

63

64 


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 10 6 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 10 6 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/cm 2 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 10 7 

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. 

Isolate 

Host 

Conidia size (µm) 

Length Width 

TG50 (95%) 

Sporulation 

x10 8 conidia 

per cm 2 * 

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

66 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

20 

0 

14.36 

a 

Bbr 23 

c 

Bbr 23 

a 

ae 

16.78 

Bbr 30 

cd 

Bbr 30 

b 

bd 

d 

Bbr 41 

b 

Bbr 41 

b 

a a 

9.07 

Bbr 50 

c 

Bbr 50 

c 

ac 

20.41 

Bbr 59 

cd 

Bbr 59 

Fig. 1. Pathogenicity of B. brongniartii isolates on 3 rd instar larvae of M. melolontha (Fig.1A) and H. 

serrata (Fig. 1B) at 2 x 10 7 conidia/ml. (Means ± SE with the same index are not significantly 

different (Tukey‘s HSD multiple comparison (p

The mean mortality, mycosis and LT50 induced by each isolate at a concentration of 

2 x 10 7 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

68 

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. 


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 extracellular 

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. 1 st 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

ongniartii. IFOAM 2000: the world grows organic. – Proceedings 13 th 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. 

69

71 

Wireworms



pp. 73-79 

European wireworms (Agriotes spp.) in North America: 

Distribution, damage, monitoring, and alternative integrated pest 

management strategies 

Robert S. Vernon 1 , Wim Van Herk 1 and Jeff Tolman 2 

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, 

73

74 

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 24 o C. 

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

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-cm 2 ) 

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-cm 2 , 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 longterm 

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. 

75

76 

% 

CATEGORIZATION OF MORBIDITY 

FIPRONIL (0.001%) 

ALIVE WRITHING LEG & MOUTH MOUTH DEAD 

100 

80 

60 

40 

20 

0 

1 DAT 7 DAT 35 DAT 91 DAT 

% 

CATEGORIZATION OF MORBIDITY 

CLOTHIANIDIN (0.1%) 

ALIVE WRITHING LEG & MOUTH MOUTH DEAD 

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. 

100 

80 

60 

40 

20 

0 

1 DAT 7 DAT 35 DAT 91 DAT

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/m 2 ), but 120 traps/ha 

were required at higher levels of infestation (over 10 individuals/m 2 ). 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 m 2 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 m 2 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 

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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. 


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.

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. 

79



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. Albert.Ester@wur.nl 

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. 


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 the15 th or 16 th 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.


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 19 th of April until 

the 17 th of May no difference were found between treated and untreated. On the 17 th 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 14 th 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 21 st of June, with the 

exception of 28 May for A. obscurus and 11 June for A. lineatus. On 28 th of June and 2 nd 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 15 th and 16 th 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, 

83

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. 

Numbers of 

Agriotes spp. 

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

Numbers of 

Agriotes spp. 

50 

40 

30 

20 

10 

0 

10-5 

50 

40 

30 

20 

10 

0 

3-5 

10-5 

M = timing treatment 

17-5 

M 

Winter wheat 

Grass seed 

24-5 

31-5 

7-6 

Data of monitoring 

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

17-5 

M = timing treatment 

M 

24-5 

31-5 

14-6 

A. lineatus 

A. obscurus 

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. 

7-6 

Data of monitoring 

14-6 

A. lineatus 

A. obscurus 

21-6 

21-6 

28-6 

28-6

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 Entomology 

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. 

85



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 (

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. 


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). 

89

90 

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. 


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. 


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.



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: lorenzo.furlan@inwind.it 

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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92 


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 9 th 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 

93

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 (Langenbuch, 

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

ait 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 nonfeeding 

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: 

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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 cropwireworm 

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.,

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. 


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. 

97

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 evoluzione 

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: Elateridae): 

Absence of geographic and temporal variation. – Eur. J. Entomol. 92: 437-442.

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 republiky. 

– 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. 


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. 


some notes on that of Athous haemorroidalis F. Part II. – Ann. Appl. Biol. 8: 193-215. 


some notes on that of Athous haemorroidalis F. Part III. – Ann. Appl. Biol. 9: 306-324. 

99

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, Elateridae) 

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 Morphologie 

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, 


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 (Coleoptera: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.



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. 


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. 

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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 

60 

40 

20 

0 

10 

50 

13 

76 

2 

44 

1999 2000 2001 2002 2003 2004 

13 

14 

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. 

32 

24 

6 

42

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. 


100 

80 

60 

40 

20 

0 

36 


early ploughing late ploughing early ploughing late ploughing 

Figure 2. Tillage, Neemcake and potatoes damaged by wireworms 


+ Neemcake 

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. 

4 

103

104 


100 

80 

60 

40 

20 

0 

50 

early harvest late harvest 


8 

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 


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 Drahtwurmbefalls 

(Agriotes spp. L.) im Ökologischen Kartoffelanbau. – In: Beiträge zur 7. Wissenschaftstagung 

zum Ökologischen Landbau, Ökologischer Landbau der Zukunft. B. Freyer 

(ed.), Universität für Bodenkultur, Wien: 133-136. 

77 

72 

77



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. 


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. 


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. 


60 

50 

40 

30 

20 

10 

0 

10 

without grass-clover annual grass-clover two years old grassclover 

13 

23 

39 

triennial grassclover 

N=44 N=44 N=23 N=7 

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


60 

50 

40 

30 

20 

10 

0 

19 

11 

without grass-clover in crop rotation 

with grass-clover in crop rotation 

2 

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

4 

Vicia faba Pisum sativum Lupinus luteus Trifolium pratense 

N=9 

N=5 

N=8 

N=1 

3 

N=7 

9 

N=4 

18 

N=6 

25 

N=3

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. 


60 

50 

40 

30 

20 

10 

0 

15 

21 

spring summer autumn winter 

N=17 

N=16 

N=11 

N=22 

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

5 

18 

107

108 


60 

50 

40 

30 

20 

10 

0 

15 14 

low middle strong 

N=67 

N=42 

N=8 

Figure 4: Perannual weed density and potatoes damaged by wireworms 


The project was financed by the Federal Ministry of Consumer Protection, Food and Agriculture 

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. 


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 Drahtwurmbefalls 

(Agriotes spp. L.) im Ökologischen Kartoffelanbau. – In: Beiträge zur 7. Wissenschaftstagung 

zum Ökologischen Landbau, Ökologischer Landbau der Zukunft. B. Freyer 

(ed.). Universität für Bodenkultur, Wien: 133-136. 

23



pp. 109-115 

Metarhizium anisopliae as a biological control for wireworms 

and a report of some other naturally-occurring parasites 

Todd Kabaluk 1 , Mark Goettel 2 , Martin Erlandson 3 , Jerry Ericsson 1 , Grant Duke 2 , Bob 

Vernon 1 

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 10 6 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, Saskatchewan. 

This article reports findings from a sample of studies carried out from 2000-2004. 

109

110 


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 10 6 /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, 10 6 , 10 7 , or 10 8 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 10 6 /g wet soil. Conidia were acquired from fresh cultures. 

Thermogradient profiling of M. anisopliae isolates 

Experiments were carried out by pipetting 5.0x10 3 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.

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/cm 2 . 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/cm 3 . 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 10 7 

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 10 14 

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 3 rd and 4 th anterior segments with 1.43 x 10 7 

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. 


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). 

% MAM 

100 

90 

80 

18C 

Duration of exposure 

35 

30 

12C 

70 

0 hours 

25 

60 

50 

12 hours 

24 hours 

20 

240 hours (total mortality) 

40 

48 hours 

15 

30 

96 hours 

10 

20 240 hours 

10 

5 

0 

0 

0 20 40 60 80 0 20 40 60 80 

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 18 o C and 12 o C. 

111

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. 

% emigration 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Emigration from M. anisopliae environment Deterence from entering a M. anisopliae environment 

No food in Met environment 

Food in Met environment 

% deterred 

0 

0 10 10 10 

0 10 10 

6 10 7 10 8 10 7 10 8 

0 

0 

80 

70 

60 

50 

40 

30 

20 

10 

Conidia / g soil 

No Travel 

Returnees 

TTl Deterred 

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.

% mortality 

100 

80 

60 

40 

20 

0 

Total 

mortality MAM 

all beetles treated 

two beetles treated 

not treated 

Agriotes obscurus 

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

100 

80 

60 

40 

20 

0 

Agriotes lineatus 

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 

% mortality 

70 

60 

50 

40 

30 

20 

10 

0 

Wireworms in the field Field wireworms incubated in the lab 

70 

After 27 days in the field 

After 48 days in the field 

Granules Conidiasoil 

mix 

Coated 

seed 

Control 

60 

50 

40 

30 

20 

10 

0 

Granules Conidiasoil 

mix 

Coated 

seed 

Control 

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. 

113

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). 

Number of holes per cm 2 on potato tubers 

Tuber 

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 conidiainjections, 

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). 


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 Invertebrate 

Pathology 21: 101-106.

Zacharuk, R.Y. & Tinline, R.D. 1968. Pathogenicity of Metarrhizium anisopliae, and other 

fungi, for five Elaterids (Coleoptera) in Saskatchewan. – Journal of Invertebrate Pathology 

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. 

115

116



pp. 117-122 

Evaluation of different sampling techniques for wireworms 

(Coleoptera, Elateridae) in arable land 

Nina Brunner 1 , Bernhard Kromp 1 , Peter Meindl 1 , Christian Pázmándi 2 , Michael 

Traugott 2 

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. 


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 loamyclayey 

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

subterranean pitfall-traps were hand-sorted for wireworms or extracted by Berlese. Handsorting 

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 headcapsule 

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 biweekly 

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. 


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 handsorting. 

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. 

119

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. 

Field Species Mbo n=200 

Mbi 

n=200 

SPT 

n=200 

PH n=200 

SC 

n=120 

Total 

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 


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 


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 .

25 

20 

15 

10 

5 

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. 


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. 

121

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 Feldkulturen. 

– 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: 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. 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. 


spp.) in the field, and the relationship between bait-trap catches and wireworm damage to 

potato. – Crop Protection 15(6): 521-527. 


spp.) on potato with particular reference to the U.K. – Agricultural and Forest Entomology 

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



pp. 123-126 

Bait and pheromone trapping of Agriotes sp. in Lower Austria 

(first results) 

Marion Landl 1 , Lorenzo Furlan 2 , Johann Glauninger 1 

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. 


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 handsorted 

and afterwards kept in Tullgren-funnels for further extractions. The wireworms were 

counted and will be identified in following investigations. 


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. 


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. 

captured wireworms 

50 

40 

30 

20 

10 

0 

4 4 

2 

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). 

0 

16 

40

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. 


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. 

A. ustulatus 

20,00 

15,00 

10,00 

5,00 

0,00 

24.04.04 

03.05.04 

12.05.04 

21.05.04 

30.05.04 

08.06.04 

17.06.04 

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. 

26.06.04 

05.07.04 

14.07.04 

23.07.04 

01.08.04 

10.08.04 

125

126 


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. 


on potato with particular reference to the U.K. – Agricultural and Forest Entomology 3: 85- 

98.



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 12 C 

and 13 C for carbon or 14 N and 15 N for nitrogen. Their standardized ratio is expressed by the 

delta 13 C and delta 15 N 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. 


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. 


Laboratory experiments 

Table 1 shows that there is no significant trophic shift from the wheat fed to the tissue of 

A. obscurus for delta 13 C. 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 15 N 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

trophic shift for delta 15 N of 2.4 ± SE 0.42 ‰ between vascular plants and animals, which fits 

to the data obtained in our study. 

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

trophic shift between them. 

delta 13 C delta 15 N 

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 13 C and delta 15 N values roughly matched 

among the three fields, but those of the soil showed large differences in the delta 13 C values. 

delta 15 N 

12 

10 

8 

6 

4 

2 

0 

-2 

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

delta 13 C 

Figure 1. Delta 13 C and delta 15 N 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 13 C values slightly more positive 

than, or equal to, the soil samples, and the delta 15 N 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 

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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 15 N to 

connect them to plant roots of matching delta 15 N 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. 

delta 15 N 

14 

12 

10 

8 

6 

4 

2 

0 

-2 

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

delta 13 C 



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 15 N 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 15 N 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 13 C values within the range of C4plants, 

which have delta 13 C values around – 13 ‰ (Larcher, 1994). Maize, for instance, has a 

mean delta 13 C 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 

13 C value. 

One individual of Veronica filiformis had roots with a remarkably high delta 15 N value of 

10.92 ‰. The other four root samples of V. filiformis, from the fields “Au Ost” and “Kappenhofwiese”, 

had delta 15 N 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 15 N value high enough to make it a carnivore. For the other one, the

oots 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). 

delta 15 N 

12 

10 

8 

6 

4 

2 

0 

-2 

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

delta 13 C 



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.). 


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. 

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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 macroinvertebrates. 

– 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. 


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: Coleoptera: 

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 (Coleoptera: 

Elateridae) in strawberry (Rosaceae) plantings with wheat (Gramineae) as a trap 

crop. – Can. Entomol. 132: 231-241.



pp. 133-142 

Pheromone composition of European click beetle pests 

(Coleoptera, Elateridae): common components – selective lures 

Miklós Tóth 1 , Lorenzo Furlan 2 

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. 


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. 


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).

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). 

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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.

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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138 

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.

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. 

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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.

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. 


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 electroantennographic 

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). 

141

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, 


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 femaleproduced 

sex pheromones and their geographical differences in pheromone gland extract 

composition from click beetles (Col., Elateridae). – J. Appl. Ent. 120: 463-466.

Diabrotica



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: peter.cate@ages.at 

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 

km 2 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 10 th 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. 


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 redistributed 

to the provincial offices and the Ministry of Agriculture, Forestry, Conservation 

and Water Resources. 


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 

145

146 

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. 

22 

Bregenz 

Feldkirch 

Bludenz 

Voralberg 

9 

Landeck 

Tirol 

Innsbruck 

Kufstein 

Salzburg 

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 

Tirol 

Lienz 

5 

Salzburg 

Oberösterreich 

Kärnten 

Linz 

Wels 

Steiermark 

Eisenerz 

Niederösterreich 

Results of the 2004 Monitoring Programme in Austria 

province number of traps 

number of traps 

with beetles 

number of 

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 

26 

6 

Villach 

Steyr 

Klagenfurt 

Gmund 

206 

Bruck 

160 

Baden 

Burgenland 

Wien 

Eisenstad 

Wiener Neustadt 

Graz 

Krems 

7 

226



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. 


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-methoxicinnamaldehyde 

(Bedoukian Inc, Danbury, USA) were purchased commercially and were 

147

148 

>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) 


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.

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 

149

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).

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. 

151

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.

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 

Bait type pheromone floral 

Sex caught males 

Sex ratio (vs. 

natural) 

practically 

100% males 

females & 

males 

higher % of 

females 

pheromone & 

floral 

males & 

females 

close to natural 

no chemical 

bait 

females & 

males 

higher % of 

females 

Detection highly sensitive sensitive highly sensitive not sensitive 

Monitoring reliable reliable reliable 

Capacity limited limited 

Non-target 

insects caught 

many very many 

high (>10000 

beetles) 

very few - high 

selectivity 

with very 

high 

populations 

limited 

very many 

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 

Bait type pheromone floral 

Assembling 

and design 

pheromone & 

floral 

no chemical bait 

easy easy complicated simple 

Maintenance dirty (sticky) dirty (sticky) clean dirty (sticky) 

Killing agent sticky material sticky material insecticide sticky material 

Costs (used for 

detection) 

Costs (used for 

season-long 

monitoring) 

cheaper cheaper more expensive not applicable 

more expensive more expensive cheaper more expensive 

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. 

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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. 


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 femaleproduced 

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 Helicoverpa 

(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



pp. 157-161 

The impact of the fungal BCA Metarhizium anisopliae 

on soil fungi and animals 

Martin Kirchmair 1 , Lars Huber 2 , Elke Leither 2 , Hermann Strasser 1 

1 

Institute of Microbiology, Leopold-Franzens University Innsbruck, Technikerstrasse 25, 

6020 Innsbruck, Austria; e-mail: martin.kirchmair@uibk.ac.at 

2 

Institute of Zoology, Johannes Gutenberg-University of Mainz, 55099 Mainz, Germany; 

e-mail: lhuber@mail.uni-mainz.de 

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. 

157

158 


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). 


In the tramline of treated plots a M. anisopliae density of 10 4 -10 5 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

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 10 3 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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160 

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”).


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 

1 st 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. – Pedobiologia 

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. 

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pp. 163-167 

Biocontrol potential of entomopathogenic nematodes against nut and 

orchard pests 

Stefan Kuske 1 , Claudia Daniel 2 , Eric Wyss 2 , Jean-Paul Sarraquigne 3 , Mauro Jermini 4 , 

Marco Conedera, 5 Jürg M. Grunder 1 

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 2.2x10 6 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 (2x10 6 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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164 

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. Semifield 

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. 


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 

bacteriophora 

nema TOP E-nema, Raisdorf (Germany) 

H. indica no commercial 

product 

E-nema, Raisdorf (Germany) 

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 Northwestern 

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.

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. 

Experimental Hazelnut weevil 

Target pests 

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 

period 

August October June 

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, 1m 

depth & 

capacity 

2 miniparcels 

Dosage 2.2 x 10 6 IJ/m 2 2 x 10 6 IJ/m 2 2 x 10 6 IJ/m 2 

Irrigation 

Mean number 

Yes (Micro-sprinkler) No (Rain) No (Rain) 

of pest 

larvae/replicate 

53 

43 

38 

Replicates 3 6 5 


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 

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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 Laboratory 1 Semi- Laboratory 

field 

1 Semi- Laboratory 

field 

1 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.


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. – 9 th 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.. – 9 th 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. 

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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: H.Malinowski@ibles.waw.pl; A.Sierpinska@ibles.waw.pl 

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-yearold 

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 

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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. 


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. 


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.

60 

50 

40 

30 

20 

10 

60 

50 

40 

30 

20 

10 

0 

No. of beetles/ tree 

0 

11. 17. 22. 25. 4. 7. 11. 13. 18. 28. 5. 6. 8. 14. 15. 23. 

24. 

29. 

2000 2001 

before hibernation 

August September October 

No. of beetles/ tree 

3. 

7. 

10. 

11. 

16. 

26. 

27. 

30. 

2. 

7. 

8. 

2000 2001 2002 

after hibernation 

11. 

14. 

17. 

21. 

24. 

31. 

12. 

March April May June 

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. 

171

172 

1999/ 2000, I 

1999/ 2000, C 

2000/ 2001, I 

2000/ 2001, C 

2001/ 2002, I 

2001/ 2002, C 

Maximal no. of 

beetles/ tree: 

50 - 80 

3 - 4 

12 - 49 

3 - 7 

2 - 4 

1 - 2 

100 80 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 

300 

250 

200 

150 

100 

50 

Height of pine trees [cm] 

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

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. 

60 

50 

40 

30 

20 

10 

0 

Annual increment in height [cm] 

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). 

173

174



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. 


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. 

175

176 

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). 


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

Abundance 

Abundance 

Abundance 

7·10 5 

6·10 5 

5·10 5 

4·10 5 

3·10 5 

2·10 5 

1·10 5 

0·10 5 

3,5·10 5 

3,0·10 5 

2,5·10 5 

2,0·10 5 

1,5·10 5 

1,0·10 5 

0,5·10 5 

0,0·10 5 

9·10 5 

8·10 5 

7·10 5 

6·10 5 

5·10 5 

4·10 5 

3·10 5 

2·10 5 

1·10 5 

0·10 5 

1 2 

3 

4 

5 

6 

8 10 12 14 16 18 20 22 

1 

9 

10 

11 

6 

Retention time (min) 

12 13 14 

8 10 12 14 16 18 20 

9 

10 

11 

6 

15 


12 17 

7 

8 

7 16 

8 10 12 14 16 18 20 22 

15 


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. 

a 

b 

c 

7 

18 16 

19 

177 

1 � α-pinene 5 � τ -terpinene 

2 � β-pinene 6 � terpinolene 

3 � o-cymene 7 � caryophyllene 

4 � D-limonene 8 � farnesene 

9 � β-Myrcene 

10 � 2-ethyl-hexan-1-ol 

11 � 1,4-p-menthadien-7-ol 

12 � 1,3,8-p-menthatriene 

13 � borneol 

14 � 3-ethenyl-1,2-dimethyl-cyclohexa-1,4diene 

15 � bornylacetat 

16 � 2,4-bis(1,1-dimethylethyl)-phenol 

17 � bis(1-methylethylidene)-cyclobutene 

18 � α-curcumene 

19 � (E)-γ -bisabolene

178 


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 Intensivobstanlagen. 

– 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, 12 th Symposium on Insect-Plant Relationships in 

Berlin, 7.-12.08.2004: 146.



pp. 179-184 

Persistence of the insect pathogenic fungus Metarhizium anisopliae 

(Metsch.) Sorokin on soil surface and on oilseed rape leaves 

Christina Pilz 1 , Siegfried Keller 2 , Rudolf Wegensteiner 1 

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 10 7 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 10 7 and 2 x 10 6 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. 


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 15 th to 29 th 

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. 

179

180 

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 

2x10 7 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 (2x10 6 spores/ml and 2x10 7 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

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). 

CFU/g fresh mass leaves 

100000 

80000 

60000 

40000 

20000 

0 

0 1 17 24 168 

hours after treatment 

181 

untreated 

treated 

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).

182 

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). 

cfu/g fresh mass leaves 

25000 

20000 

15000 

10000 

5000 

0 

1 16 24 72 168 


2x10*6 sp/ml 

2x10*7 sp/ml 

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

concentrations. 

cfu/g dry mass soil 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

0 1 16 24 72 168 


first treatment 

second treatment 

Figure 3: Persistence of M. anisopliae spores in soil samples after a first and second treatment with 

spore suspension (2x10 7 sp/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).

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). 

infection rate% 

100,00 

80,00 

60,00 

40,00 

20,00 

0,00 

0 1 16 24 72 168 


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- 

183

184 

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. 


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



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 Peveling 2 , Peter Nagel 2 , Siegfried Keller 1 

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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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). 

Frequency of occurence 

100% 

75% 

50% 

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 broadleaved 

forests in Baselland had the lowest frequencies.

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 

Frequency of occurence 

100% 

75% 

50% 

25% 

0% 

a a ab c ab a b c 

BL ZH 

Broad-leaved forest 

Coniferous forest 

Cropland 

Meadow 

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 nearnorthern 

habitats. – Can. J. Bot. 76: 1198-1204. 

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188 

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.



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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190 

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.

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, 23 rd February, 2000, Auer, Italy. (ii) “Bioactive Fungal 

Metabolites – Impact and Exploitation”; held 22 nd –27 th April, 2001 at the University of 

Wales, Swansea; and the Closing Meeting of BIPESCO FAIR6 CT-98-4105" , 24 th 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. 


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).

IOBC/wprs Bulletin Vol. 28(2) 2005

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