Verslag van bezoek aan "Eidg. Forschungsanstalt für Obst-, Wein- eund Gartenbau Wädenswil" te Wädenswil (Zürich) en deelname aan bijeenkomst IOBC/WPRS: working group on insect pathogens and insect parasitic nematodes

PROJECT

4102 (reisverslag Zwitserland)

REIS VERSLAG INHOUD

Verslag van bezoek aan "Eidg. Forschungsanstalt für Obst-, Wein-und Gartenbau Wädenswil" te Wädenswil (Zurich) en deelname aan bijeenkomst IOBC/WPRS: working group on insect pathogens and insect parasitic nematodes.

Zürich 1993. R.W.H.M. van Tol

PB - Boskoop september 1993

Nadruk of vertaling, ook van gedeelten, is alleen geoorloofd na schriftelijke toestemming van de directie van het proefstation en de auteur. Het ministerie van Landbouw, Natuurbeheer en Visserij, de Stichting Proefstation voor de Boomkwekerij, de Stichting

Boomteeltproeftuin voor Noord-Brabant, Limburg en Zeeland (Horst), de Stichting Boomteeltproeftuin "De Boutenburg" (Lienden) en de Stichting Boomteeltproeftuin Noord-Nederland (Noordbroek) stellen zich niet aansprakelijk voor eventuele schadelijke gevolgen, ontstaan door het gebruik van de gegevens die in deze uitgave zijn gepubliceerd.

INLEIDING

Van 5 to 9 september 1993 werd de 4e bijeenkomst van de IOBC/WPRS werkgroep over insektepathogenen en insekteparasitaire aaltjes gehouden te Zürich (Zwitserland). Via contact met het proefstation voor de fruitteelt, wijn- en tuinbouw te Wädenswil was ik in de gelegenheid om een reisbeurs bij de EEG te krijgen. Deze beurs dekte grotendeels de kosten voor deelname aan het symposium en het bezoek van dit proefstation. Het hoofddoel van dit bezoek was de deelname aan het symposium. Voorafgaand aan dit symposium heb ik het proefstation één dag kunnen bezoeken. In dit verslag wordt een samenvatting gegeven van de gesprekken met enkele personen werkzaam op het gebied van

gewasbescherming. In de bijlage staat algemene informatie over het proefstation in Wädenswil, het programma van het IOBC/WPRS- symposium, de publicatie behorend bij mijn gepresenteerde poster (wordt

gepubliceerd in IOBC/WPRS Bulletin 1993), een adreslijst van de

deelnemers aan dit symposium en tot slot de rapportage aan de EEG naar aanleiding van mijn bezoek van het proefstation.

SAMENVATTING BEZOEK WÄDENSWIL

Op 3 september 1993 werd ik ontvangen op de "Eidgenossische

Forschungsanstalt für Obst-, Wein- und Gartenbau Wädenswil" door Dr. J. Grunder. Dr. Grunder is hoofd van de afdeling Entomologie en Nematologie. Daarnaast is er nog een afdeling Fytopathologie en een afdeling Gewasbeschermingsmiddelen en onkruidkunde (zie bijlage). Op elk van deze afdelingen zijn deskundigen voor ziekten behorend bij de drie verschillende gewasgroepen (fruit, wijn en tuinbouw). Dr. Grunder is van oorsprong nematoloog. Zijn eigen onderzoek concentreewrt zich dan ook op de aaltjesproblematiek inclusief de insecteparasitaire aaltj es.

De boomkwekerij wordt in Zwitserland niet tot de landbouw gerekend. Deze keuze is in het verleden door de kwekers zelf genomen. Veel

kleine kwekers die eikaars concurrent zijn voelden destijds niets voor samenwerking. De nadelen van deze historische beslissing zijn

tegenwoordig goed merkbaar in deze sector. Er is geen georganiseerde structuur, met als gevolg dat er weinig mensen en geld beschikbaar zijn voor onderzoek e.d. in deze specifieke sector.

IP (Integrierte Pflanzenbau) komt goed op gang in de tuinbouw en de wijnbouw, echter in de sierteelt (behalve onder glas) voorlopig nog niet vanwege de al eerder genoemde ontbrekende structuren in deze teelt. Dr. Rueegg is verantwoordelijk voor de boomkwekerij-kant in de sierteelt. Hij moet geheel alleen werken en is nogal pessimistisch over het nut van een aparte waardering van deelnemers aan IP (bv. labeling van IP-produkten). Hij gelooft niet in een controle op

IP-teelt. Intelligente kwekers zouden uit zichzelf moeten streven naar geïntegreerde teelt en niet vanwege de label op zijn produkt. In

Zwitserland zijn slechts enkele grote producenten/afnemers van

boomkwekerij- en andere siergewassen (m.n. de supermarktketen Migros). Deze firma importeert ook een groot deel van zijn planten uit Europa en maakt daarbij geen onderscheid tussen planten uit Zwitserland en uit de importlanden. Dit bemoeilijkt een eerlijke vergelijking tussen IP-produkten en buitenlandse produkten (zonder IP-merk). De heer Frey (vnl. werkzaam in de sierteelt onder glas) houdt zich bezig met deze

problematiek. Hij is aanzienlijk positiever ingesteld dan de heer Rueegg. Sinds kort hebben de grote bedrijven (Migros) interesse

getoond om mee te werken aan IP-produktie/waardering. Hij ziet ook in dat de grootste problemen in de nabije toekomst liggen bij de

praktische invulling/begeleiding van IP. Het idee om via

2

-structurele opzet via organisaties als een DLV en PD in Nederland is problematisch aangezien dit soort separate organisaties in Zwitserland niet bestaan. In Zwitserland moeten de onderzoekers naast eigen

onderzoek zowel de voorlichting verzorgen als ook de analyses van binnenkomend ziek plantenmateriaal. In bepaalde periodes van het jaar is men bijna voltijds bezig met analyses en voorlichting/advisering. Dr. Frey ziet wel wat in het opzetten van een separate organisatie die de IP-begeleiding zou moeten gaan verzorgen. Hij is zeker van plan om binnen afzienbare tijd naar Nederland te komen om zich op de hoogte te stellen van het IP-programma bij ons in Boskoop.

Naast dit thema heb ik uiteraard met enkele mensen gesproken over de biologische bestrijding van de gegroefde lapsnuitkever en enkele andere insekteplagen die met aaltjes bestreden worden (Dr. Grunder, Dr. Steiner). In de rapportage aan de EEG (zie bijlage) staat een uitgebreide samenvatting hierover.

In Zwitserland zijn tegenwoordig ook veel problemen met een verwante keversoort (Otiorhynchus craetagL). Deze kever lijkt veel op

0.sulcatus, echter de levenscyclus is nog slecht bekend en de

biologische bestrijding van de larven met aaltjes is nog niet zo succesvol. Het vraatpatroon van deze kever onderscheidt zich van die van de taxuskever door en veel fijner gekarteld patroon aan de rand van de bladeren en tevens loopt deze karteling dieper het blad in. In Nederland is dit, voorzover ik weet, geen kever die veel voorkomt en problemen geeft. Hoewel ik nog niet zo lang geleden in Brabant in een bos met Rhododendrons ditzelfde fijne kartelpatroon massai ben

tegengekomen !

Enkele interessante gesprekken heb ik verder nog gevoerd over met name meeldauw bestrijding. Fons van Kuik heeft de informatie hierover

inmiddels ontvangen.

Ten aanzien van bestrijdingsmiddelen geldt in Zwitserland dat middelen toegelaten worden voor plagen in alle teelten, dus niet per sector of per gewas. Biologische middelen moeten wel een toestemming krijgen voor gebruik in Zwitserland, maar dit houdt feitelijk niet meer in dan dat ze geregistreerd worden en niet dat er een uitgebreide wettelijke keuring is.

3

-BIJLAGE

- informatie algemeen over proefstation in Wädenswil

- programma IOBC/WPRS symposium "insect pathogens and insect parasitic nematodes

- publicatie behorend bij poster op symposium - deelnemerslijst symposium

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Union Internationale des Sciences Biologiques

j* « I h ORGANISATION INTERNATIONALE DE LUTTE BIOLOGIQUE ET INTEGREE jk | f1 11 1^ CONTRE LES ANIMAUX ET LES PLANTES NUISIBLES j f\

M 11 in SECTION REGIONALE OUEST PALEARCTIQUE jt IIOU

. 1 •> ^ International Union of Biological Science £

1//\Y INTERNATIONAL ORGANIZATION FOR BIOLOGICAL AND INTEGRATED 7

CONTROL OF NOXIOUS ANIMALS AND PLANTS *

WEST PALAEARCTÏC REGIONAL SECTION

Sunday, 5 17.00-20.00 20.00-22.00 Monday, 6 08.00-09.30 09.30-09.45 09.45-10.30 10.30-11.00 11.00-11.45 11.45-12.30 12.35-14.00 14.00-14.25 14.25-14.50 14.50-15.15 15.15-15.45 15.45-16.10

4th meeting of the IOBC/WPRS working group on insect pathogens and insect parasitic nematodes—

Zurich, 5-9 September 1993 ' %

Programme

- ".conlnvekerij - 1 -1 Î - + Building1 & Room2 CLS GEP : r K O O

\

CLS Registration in CLS building Welcome party in GEP pavilion

Late registration in CLS building

Plenary Session "Insect pests difficult to control"

HG G3 P. Smits: Opening address

G. Benz: Insect pathology from 1960 to 1993 at the ETH-lnstitute of Entomology: some reminiscences and unpublished data.

(HG GG) Coffee

J. Weiser: Insect pests difficult to control with biopesticides.

A. Vey: Interactions between entomopathogenic fungi, and mecha­ nisms of resistance and immunity.

(MM) Lunch

M. Tomalak: Genetic improvement of Steinernema feltiae for inte- * grated control of the WFT, Frankliniella occidentals (Thripidae). C. Chastel: Tabanid spiroplasmas in France: ecology and taxonomy. M.G. Chukrii, Voloschuk, L. & Popushoi, I.S.: Application of vectors for viral infection among pests.

(HG GG) Coffee

J. Eilenberg: Fungal, bacterial and protozoan pathogens on Delia

radicum and Delia floralis (Dipt., Anthomyiidae).

'See plan: CLS = Entomology, GEP = pavilion, HG = main building, MM = mensa

2B, F, G = floors (exemple F26.3 = room Nr. 26.3 on F floor), GG = gallery on G floor

Building & Room

16.10-16.35 HG G3 S. Henning: Pathogenicity and practical use of two protozoan patho­ gens on Prostephanus truncatus (Horn) (Col., Bostrichidae) in Togo. 16.35-17.00 C. Mendes, L. Lacey, J. Amarel & M. Klein: Biological control of

Popillia japonica (Col., Scarabaeidae) on Terceira Island (Azores,

Portugal): potential of Bacillus popilliae.

17.00-17.25 R. Wegensteiner: Chytridiopsis typographi (Protozoa, Microsporidia)

and other pathogens in Ips typographus (Col., Scolytidae). Night meeting

20.15-21.45 HG G3 Discussion meeting: "Which pathogens for difficult pests?" Tuesday, 7

09.00-10.30 Postersessions

4 rooms each for 7-9 posters; 4 discussion leaders, 5 min presen­ tation by author + 5 min discussion per poster

HG F26.3 1. Viruses + Bacteria

J. Adamek: Non-occluded Baculovirus (Nudibaculovirinae) of

Pyrrho-coris apterus (Heteroptera, Pyrrhocoridae).

X. Li & G. Benz: 2-dimensional gel electrophoresis and immunoblot analysis oiAdoxophyes orana F.v.R. (Lep., Tortricidae) granulosis virus.

X. Li, & Benz, G., Zürich: Synthesis of viral proteins in vivo.

N. Luisier & G. Benz: Improving the efficiency of the granulosis virus of

Adoxophyes orana F.v.R. for microbiological control.

J.-L. Zeddam: Use of nucleic acid probes for epidemiological survey of Phthorimaea operculella (Lep., Gelechiidae) granulosis virus. H.-J. Joeressen, P. Maier & G. Benz: Differential cytotoxic activity of Jwo J-Endotoxins of Bacillus thuringiensis on mammalian cells. -&B. Keller & S. Vriesen: Insects on poplars: Regional distribution in

[Germany and their susceptibility to Bacillus thuringiensis isolates. N. Monteny: Humoral or cellular reaction induced by Plasmodium

yoelii yoelii in Anopheles stephensi (Dipt., Culicidae).

HG F26.5 2. Fungi

—^ jF. Cravanzola, & O. Ozino Marletto: Action of Beauveria brongniartii against Melolontha melolontha (Col., Scarabaeidae) and its persist-• ency in Valle d'Aosta.

B. Ekbom: Tolypocladium cylinchosporum as a biological control agent against the window midge Sylvicola cinctus.

J. Gillespie: Purification and partial characterization of a fungal protease inhibitor in the haemolymph of the tobacco hornworm,

Manduca sexta.

Hirte, H. Triltsch & H. Sermann: Growth and surviability of the I entomopathogenic fungus Verticillium lecanii in the soil.

Building & Room

P. James: The effect of destruxin A on the ultrastructure of the Malpi-ghian tubules of the desert locust Schistocerca gregaria.

S. Keller: Two Melolontha populations with different susceptibility to infections with the fungus Beauveria brongniartii.

R. Kleespies: Electron microscope investigations on the ultrastructure of blastospores and conidia of Metarhizium anisopliae.

M. Singh: Fungal diseases found on Delia radicum and D. floralis (Dip., Anthomyiidae) in Norway during 1992-1993.

T. Steenberg: Tolypocladium sp. (Fungi imperfecta Hyphomycetes) in larvae of Agrotis segetum (Lep., Noctuidae).

HG GG north

3. Nematodes A

L. Gerritsen: Use of antiserum to discriminate between Xenorhabdus strains and form variants.

K. Huneke, & Peters, A. & Ehlers, R.-U.: Movement patterns of dauer juveniles in response to host cues.

K. Jung: The influence of osmotic stress on insect parasitic nematodes.

Z. Mrafcek: Current view on the taxonomy of the family Steinernematidae.

0. Strauch, & R.-U. Ehlers: Morphological determination of pre-dauer versus propagative juveniles of Heterorhabditis spp.

1. Vänninen: Dynamics and effect on infectivity of endogenous lipid

reserves in two aging Steinernema spp. _

"fr. Van Tol: Influence of temperature on the control of the black vine) weevil with strains of some insect-parasitic nematodes. |

J. Wybenga: Lipid content of insect parasitic nematodes. HG GG south

/ H

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A

4. Nematodes B

D.H. Gouge, & N.G.M. Hague: Development of Steinernema feltiae (Nematoda: Steinernematidae) in Sciarid species (Diptera: Sciaridae). R. Gwynn: Investigations of U.K. soils for entomopathogenic

nematodes.

D. Hay: Efficacy of U.K. Steinernema spp. against the mushroom sciarid fly larvae [Lycoriella solani).

j S.M. John: Glasshouse and field control of black vine weevil with

Steinernema and Heterorhabditis spp. nematodes in Belgium.

D. Schneider: Biological control of insects in forest nureseries with , entomopathogenic nematodes.

>v ƒ W. Steiner: Distribution of entomopathogenic nematodes in the Swiss

^ Alps.

D. Sulistyanto, A. Peters, H. Hokkanen & R.-U. Ehlers: Evaluation of entomopathogenic nematode strains for the control of Delia radicum (Dipt., Anthomyiidae) and Tipula spp. (Dipt., Tipulidae).

Building & Room

A. Wulff, A. Peters & R.-U. Ehlers: Pathogenicity of the Steinernema

feltiae-Xenorhabdus bovienii complex to Tipula oleracea

10.30-11.00 (HGGG) Coffee

11.00-12.30 Second round, same posters, 4 different discussion leaders

12.30-14.00 (MM) Lunch

» Paper sessions of subgroups

Subgroup Nematodes, organisation R.-U. Ehlers

14.00-14.25 HG G3 N.G.M. Hague & A. Schirocki: The effect of temperature on the susceptibility of the black vine weevil to different isolates of Steinerma and Heterorhabditis.

14.25-14.50 P. Westerman: Infectivity and pathogenicity of the insect parasitic nematodes Hederorhabditis spp. and Steinernema spp. for

Otiorrhyn-chus sulcatus (Col., Curculionidae) at different temperatures.

14.50-15.15 A. Vainio: Effect of pesticides on long-term survival of Steinernema

feltiae in the field.

15.15-15.45 (HGGG) Coffee

15,45-16.10 S. Pollitt, A. Peters & R.-U. Ehlers: Control potential of a naturally occuring Steinernema feltiae population.

16.10-16.35 J.W.A. Scheepmaker: Control of mushroom flies: dispersal and

survival of nematodes in mushroom compost.

16.35-17.00 N. Simoes & F.S. Rosh: Survival of entomophilic nematodes in soil. 17.00-17.25 P. Westerman: The vertical migration ability of Heterorhabditis spp.

and Steinernema spp. at 9°C, and the relationship to efficacy against

Otiorrhynchus sulcatus (Col., Curculionidae) at 9°C.

Subgroup Fungi, organisation B. Papierok

14.00-14.25 HG F26.5 B. Papierok: Entomopathogenic fungi associated with cocoons of nettle caterpillars (Lepidoptera: Limacodidae) in oil palm plantations in Sumatra.

14.25-14.50 A.K. Charnley: Trehalases produced by the entomopathogenic fungus

Metarhizium anisopliae.

14.50-15.15 N. Jenkins: Development of a new procedure for the mass production

of conidia of Metarhizium fiavoviride.

15.15-15.45 (HGGG) Coffee

15.45-16.10 L.S. Osborne & Z. Landa: The utilisation of the entomogenous fungus

Paecilomyces fumosoroseus against the sweetpotato whitefly, Bemisia tabaci (Sternorrhyncha, Aleyrodidae).

16.10-16.35 I. Ben-Ze'ev: Natural occurrence and artificial inoculation experiments

with Conidiobolus coronatus and C. thromboides in a glasshouse population of Bemisia tabaci (Sternorrh., Aleyrodidae).

16.35-17.00 G. Tropea Qarz\a:Neozygites parvispora a pathogen of western flower

Building & Room

17.00-17.25 HG F26.5 C. Santiago-Alvarez & S.E.M. Sanchez: Entomophthorales fungi in Andalucia.

20.30-22.00 CLS B4 Business meeting scientific committee.

Wednesday, 8 Paper sessions of subgroups (continuation)

HG G3 Subgroup Nematodes 09.00-09.25 09.25-09.50 09.50-10.15 10.15-10.45 10.45-11.00 11.00-12.30 (HG GG)

O. Strauch & R.-U. Ehlers: Sex ratio in Heterorhabd'rtis spp. J. Gründer & P. Lüthy: On the role of the bacterium Xenorhabdus

luminescens, the microsymbiont of the nematode CH-HW 79. (Heterorhabditis sp.).

K. Krasomil-Osterfeld & R.-U. Ehlers: Induction of secondary form variants of Xenorhabdus luminescens XSH 1.

Coffee

R.-U. Ehlers: COST Action 812 "Entomopathogenic nematodes": Scientific cooperation in Europe.

Discussion hot topics and future cooperation and meetings

HG F26.5 Subgroup Fungi 09.00-09.25 09.25-09.50 09.50-10.15 10.15-10.45 10.45-12.30 (HG GG)

R.P. Bateman: Physical properties and atomisation of ULV formul­ ations of myco-insecticides.

W. Ravensberg: Side-effects of pesticides on Verticillium lecanii: in

vivo tests on whitefly and aphids.

S. Keller: Side effects of pesticides on insect pathogenic fungi: general remarks.

Coffee

Discussion hot topics and future cooperation.

HG F26.3 Subgroup Viruses and Bacteria

09.00-09.25

09.25-09.50 09.50-10.15

10.15-10.45 (HG GG)

G. Fediere, A. Tahara, Y.C. Veyrunes, S. Aboi Eia, X. Lery, J.-L. & Zeddam & Y. Giannoti: Genomic characterization of a Picorna like virus isolated from Sesamia cretica (Lep., Noctuidae), the most important corn borer in Egypt. .

X. Li: Restriction endonuclease analysis of the granulosis virus of

Adoxophyes orana F.v.R. (Lep., Tortricidae).

E. Vargas-Osuna, A. Diaz-Duran, H.K. Aldebis & C. Santiago-Alvarez: Interactions between two natural virus pathogens of Ocnogyna baetica (Lep., Arctiidae) larvae.

Building & Room

10.45-11.10 HG F26.3 A. Cherry: Oil formulation of insect viruses.

11.10-11.35 H.-J. Joeressen & G. Benz: Comparison of the action of two Bacillus

thuringiensis isolates in vivo and in vitro.

11.35-12.00 C. Santiago-Alvarez, A. Cabello, E. Vargas-Osuna & H.K. Aldebis: Interactions between Bacillus thuringiensis var. aizawai and a NPV on

Spodoptera littoralis (Lep., Noctuidae) larvae.

12.00-12.25 P. Damgaard: Natural occurrence of Bacillus thuringiensis on grass

and cabbage foliage.

12.30-13.30 (MM) Lunch

14.00-22.00 Lake of Zurich: Excursion and social dinner on MS GLÄRNISCH, departure from Bürkliplatz (tramway nr. 9 from ETH).

Thursday, 9 Plenary Session "Interaction pathogen-defense mechanism insect"

09.00-09.25 HG G3 A. Peters: Interaction between insect defense mechanisms and

entomopathogenic nematodes.

09.25-09.50 L.J.M. Gerritsen: Pathogenicity of new combinations of Heterorhabditis

spp. and Xenorhabdus luminescens against Galleria mellonella (Lep.) and Tipula oleracea (Dip.).

09.50-10.15 A. Navon: Quantifying interactions among herbivore larvae, Bacillus

thuringiensis Ô-endotoxin, and plant alleiochemicals.

10.15-10.45 (HGGG) Coffee

10.45-11.10 J.J. Lipa: NPV and BT interactions in Agrotis spp. (Lep., Noctuidae)

and methods of their evaluation.

11.10-11.35 A. Wiesner: Induction and regulation of immune reactions in Galleria

mellonella (Lepidoptera).

11.35-12.00 L. Thomsen: Chitin synthetases in the protoplastic Entomophthorales.

12.00-12.30

12.30-14.00 (MM)

Closing session, evaluation of meeting, issues and place of next meeting.

Lunch

Afternoon Free, visits to other labs, private discussions, meetings?

Workshop on Entomophtorales by S. Keller

(special programme)

14.00 Field Collection tour in vicinity of Zurich.

Friday

INFLUENCE OF TEMPERATURE ON THE CONTROL OF THE BLACK VINE WEEVIL WITH STRAINS OF SOME INSECT-PARASITIC NEMATODES

R.W.H.M. Van Toi

Research Station for Nursery Stock

P.O. Box 118, 2770 A.C. Boskoop, The Netherlands Summary

In a climate room experiment two of the three strains of Heterorhabdltls

sp. (NWE) tested reduced the number of vine weevil larvae by 80 to 100% after six hours of 12°C soil temperature followed by 9°C until the end of the experi­ ment. These two strains gave no control at continuous 9°C but achieved total control at continuous 12°C. A laboratory experiment with larvae in small tubes confirm the results of the experiments in the climate rooms. One strain infected in this experiment also at 9°C which suggests that the temperature limit for penetration is lower than the limit for successful migration to the larvae with this strain. A strain of Steinernema tested was not even effective at continuous 12°C.

1. Introduction

The black vine weevil (Otlorhynchus sulcatus) is a major pest in nursery stock. The larvae damage the roots of many plants and are difficult to control. Hatched larvae overwinter in soil and continue to feed on plant roots. In winter large larvae start to feed on the main roots and the root collar of the plants, causing economically important damage. Outdoor application of insect-parasitizing nematodes in autumn, as late as possible, would be most effective to reduce the larval population. Weevils have then stopped laying eggs, most larvae are big enough to be successfully infected by the nematodes and the plants have not yet suffered economic damage. The problem with late applica­ tions of nematodes outdoors is the low soil temperature, which reduces the efficacy of many strains. In earlier experiments in 1991 it was demonstrated that several strains of Heterorhabdltls sp. are effective in pots outdoors. A few of these strains were also effective in the open ground. Climate room experiments showed that there is a relation between nematode activity at low temperatures and the efficacy against the larvae of the black vine weevil (Van Tol, 1993a; Van Tol, 1993b) . A soil temperature of 9°C was not sufficient to obtain any control but a continuous temperature of 12°C was enough to achieve control with the better strain.

The study reported in this paper aimes to determine the critical period at 12°C required to achieve successful infection of the larvae.

2. Material and methods

Four nematode strains of different origins were tested (Table 1). The trials were carried out in climate rooms and in climate chambers. The substrate used in pots consisted of 55% peat pellets, 40% sphagnum-moss peat and 5% aeolian sand. The test plant was Thuja occldentalls 'Brabant'. The data were statisti­ cally processed using ANOVA.

Table 1 - Biological agents used in the experiments

nematode origin code experiment*

Heterorhabdltls sp.^ United Kingdom UK-H-211 M M M i

Heterorhabdltls sp.^ Germany D-H-SH I,il

Heterorhabdltls sp.+ Netherlands N1-H-F85 I

Steinernema carpocapsae U.S.A. US-S-25 il

* I = climate room experiment; II = laboratory experiment + nematodes belonging to the North-West European group (NWE)

I - Climate room experiment

Four treatments (Table 1), including "untreated", at five different tempera­ ture schedules were carried out in fourfold, using four test plants per replicate. Plants in one-litre containers were inoculated with 30 weevil eggs per plant and incubated at 20°C in a climate room for two months. On 30 October 1992 the plants were divided into three series and transferred to climate rooms at 9 and 12°C. At 2 November 1992 each pot was inoculated with IS 000 nematodes and placed on a grid to prevent the nematodes from migrating to other plant3. Five temperature treatments were applied. In two treatments the plants, after inoculation with nematodes, were kept at either 9°C or 12°C until the end of the experiment. In the other treatments plants were kept at 12°C for 6, 18 or 96 hours after inoculation and then placed at 9°C until the end of the experiment. The nematode suspension applied was cooled to 9°C or 12°C before inoculation. On 14 December 1992 all plants were checked for the presence of living larvae. II - Laboratory experiment

Three treatments (Table 1) at five different temperature schedules were carried out in tenfold. Tubes of 50 ml were filled with a mixture of peat and roots from Thuja-plants. In each tube one larva was placed. Moisture of the soil was kept constant in all treatments. The tubes were cooled down to 9 or 12°C. At the start of the experiment each tube was inoculated with a cooled down suspension of 500 nematodes. In two treatments the tubes were kept at 9°C or 12°C until the end of the experiment. In the other treatments tubes were kept at 12°C for 6, 12 (only strain D-H-SH and US-S-25) or 13 hours after inoculation and then placed at 9°C until the end of the experiment. Svery 3 to 4 days the larvae were checked. Dead larvae were removed and checked for infection.

3. Results

The results of the two experiments are presented in table 2 and figure 1. The experiment in the climate rooms (I) shows that temperature affects the efficacy of the nematode strains. It also appears that the efficacy of the three 3trains of the NWE group differs at 12°C. At that temperature the strains UK-H-211 and N1-H-F85 performed best. Strain D-H-SH gave no statistically significant control at 12°C. At 9CC there was no control with any of the three strains

tested. One temperature shock of 12°C for 6 hours was already enough for almost complete control of the larvae with the strains UK-H-211 and N1-H-F85.

The lab experiments (II) confirm the results of the climate room experiments. In the lab experiment, however, the nematodes of UK-H-211 were also able to infect and kill larvae at 9°C. The strains D-H-SH and US-S-25 were not effective at 9°C and also not after a temperature shock of 12°C. Only strain D-H-SH gave some infection at 12°C continuous.

Table 2 - Percentage reduction of O. sulcatus larvae compared with "untrea­ ted" (untr. ) , achieved by three strains of Heterorha.bd2.Z1s sp. under different temperature conditions in climate rooms (exp.I), 42 days after inoculation with nematodes.

Percentage reduction treatment® O.S.* untr.UK-H-211 D-H-SH N1-H-F85 9°C _{1.9 0a" 16a Oa Oa} 6h.l2°C 2.7 0a 100b 44a 82b 18h. 12°C 2.4 0a 79c 38ab 63bc 96h.12°C _1.3 0a 100c 39ab 85bc 12°C _0.5 0a 100b 40 ab 100b

* Figures in the same row followed by the same letter are not statistically significantly different, with a 95% confidence limit.

# average number of 0. sulcatus larvae in the untreated plants, ê soil temperature during the experiment. After 6 to 96 hours

at 12°C the temperature was maintained at 9°C until the end of the experiment.

days after application nematodes

- O - 9 ° C - + - 6 n . - A - 1 8 h . 1 2 ° C 12°C 12°C

days after application nematodes

- o - 9 ° C - + - 1 2 h . 1 2 ° C - a- 12°C

both 12°C S25 HSH

both

Figure 1 - Percentage infected dead larvae in time (exp.II) after application of UK-H-211 (top figure) and D-H-SH and US-S-25 (bottom figure).

4. Discussion

The results from the climate room experiment confirm that temperature influ­ ences the efficacy of the tested strains of the NWE group. At 9°C no strains were effective and at 12°C two strains (UK-H-211 and N1-H-F8S) gave 100% con­ trol. As found earlier in 1991 (Van Tol, 1993a), the D-H-SH strain was not

effective, even at 12°C. It is remarkable that only one temperature shock of 12°C for six hours after application of the nematode strains UK-H-211 and Nl-H-F85 was enough to achieve 80 to 100% control. It seems probable that the temperature shock of 12°C for a few hours enables the nematodes to find the larvae in the soil. Penetration and successive infection and death of the lar­ vae can then occur at 9°C soil temperature. In the laboratory experiment the nematodes of UK-H-211 were also able to infect and kill Tarvae at 9°C. In the lab experiment larvae were placed in a small soil volume (50 ml) so that the nematodes did not seed to search for their potential hosts, but only had to penetrate the larvae. It seems therefore probable that the temperature limit for penetration is lower than the limit for successful migration to the larvae. Many researchers also found that different strains of Heterorhabditis sp. and

Steinernema sp. differ in their efficacy to migrate and locate hosts and in their chances of penetrating the host (Barbercheck & Kaya, 1991; Gerritsen & Smits, 1993; Westerman & Godthelp, 1990; Westerman, 1991). Gaugler et al.(1990) stated that the probability of successful penetration and establishment of a single insect-parasitic nematode is low, even in very susceptible hosts. This means that there is a minimum number of nematodes needed to get a successful infection. Low soil temperatures probably limit the number of nematodes that can find the larvae in soil. In large volumes of soil (open ground) there is less chance of finding the hosts, and consequently also less chance of a suc­ cessful infection.

The experiments discussed here indicate that every subprocess of the infec­ tion of the larvae by nematodes has its optimal temperature. Locating and

finding the larvae in the soil is influenced by temperature, 3oil volume and probably by antagonism. Successful penetration and infection of the larvae probably needs a minimum number of nematodes per larva. Knowing more about the subprocesses of infection would help optimize the strategy and timing of nema­ tode applications to control the larvae of the black vine weevil and makes it

possible to select more specific for better nematode strains. The results cf

the different experiments done suggest that the searching ability of the nema­ todes is one of the most limiting processes for infection at low temperatures. Future research will focus on these different subprocesses.

References

BARBERCHECK, M.E. & H.K. KAYA, 1991 - Effect of Host Condition and Soil Textura on Host Finding by the Entomogenous Nematodes Heterorhabdizis bacteriophora

(Rhabditida: Heterorhabditidae) and Steinernema carpocapsae (Rhabditida: Stei-nernematidae). Environ. Entomol. 20(2), 582-589.

GAUGLER, R., J.F. CAMPBELL S T.R. MCGUIRE, 1990 - Fitness of a genetically improved entomopathogenic nematode. J. Invertebr. Pathol. 56, 106-116.

GERRITSEN, L.J.M. & P.H. SMITS, 1993 - Variation in pathogenicity of recombi­ nants of Heterorhaditis and Xenorhabdus luminescens strains. Fundamental and Appl. Nematology. In press.

VAN TOL, R.W.H.M., 1993a - Control of the Black Vine Weevil (Otiorhynchus sul-catus) with different isolates of Heterorhabditis sp. and Metarhizium aniso-pllae in Nursery Stock. Proc. Exper. & Appl. Entomol., N.S.V. Amsterdam, Vol. 4, 181-186.

VAN TOL, R.W.H.M., 1993b - Efficacy of control of the Black Vine Weevil (Otio­ rhynchus sulcatus) with strains of Heterorhabditis sp., Steinernema sp. and the

fungus Metarhizium anisopliae in Nursery Stock. Med. Fac. Landbouww. Gent. In press.

WESTERMAN, P.R. & J.M. GODTHELP, 1990 - The host-searching ability of the in­ sect parasitic nematode Heterorhabditis sp. in sand columns. Med. Fac. Land­ bouww. Gent 55/2br 691-698.

WESTERMAN, P.R., 1991 - Comparison of migration and host searching ability of various Heterorhabditid species and isolates in sand columns. IOBC/WPRS 3ull. 14, 32-35.