Ralf-Udo Ehlers

Nematodes as biocontrol agents

PART I. NEMATODE MORPHOLOGY AND TAXONOMY Morphology and Taxonomy of Nematodes Used in Biocontrol - S P Stock, University of Arizona, USA, and D J Hunt, CABI Bioscience, Surrey, UK PART II. ENTOMOPATHOGENIC NEMATODES Biology and Behavior - C Griffin, National University of Ireland, UK, N Boemare, Universite Montpellier II, France, and E E Lewis, Virginia Technology Institute, USA Mass Production - R-U Ehlers and D I Shapiro-Ilan Formulation and Quality - P S Grewal, and A Peters, e-nema GmbH, Germany Application Technology - D J Wright, Imperial College London, UK, A Peters, S Schroer, Christian-Albrechts University Kiel, Germany, and J Patterson Fife, Battelle Memorial Institute, USA Forum on Safety and Regulation - R-U Ehlers Lawn, Turfgrass and Pasture Applications, P S Grewal, A M Koppenhofer, Rutgers University, USA, and H Y Choo, Gyeongsang National University, Republic of Korea Glasshouse Applications, M Tomalak, Institute of Plant Protection, Poland - S Piggot, Littlehampton, UK and G B Jagdale, Ohio State University, USA Nursery and Tree Application - R W H M van Tol, Wageningen-UR, Wageningen, The Netherlands and M J Raupp, University of Maryland, USA Mushroom Applications - S Jess, Department of Agriculture and Rural Development for Northern Ireland, H Schweizer, Queens University of Belfast, and M Kirkpatrick, NIHPBS Loughgall, County Armagh, UK Orchard Applications - D I Shapiro-Ilan, L W Duncan, University of Florida, Lake Alfred, USA, L A Lacey, USDA-ARS, Washington, USA and R Han, Guangdong Entomological Institute, Guangzhou, China Soft Fruit Applications - R S Cowles, Connecticut Agricultural Experiment Station, USA, S Polavarapu, (Deceased), R N Williams, Ohio State University, USA, A Thies, e-nema, France, and R-U Ehlers Vegetable and Tuber Crop Applications - G Belair, Agriculture and Agrifood Canada, Canada, D J Wright, and G Curto, Servizio Fitosanitario Regione emilia-Romagna, Italy Cereal, Fiber, Medicinal, and Oilseed Crop Applications - H E Cabanillas, USDA ARS, USA, R J Wright, University of Nebraska-Lincoln, USA and R V Vyas, Gujarat Agricultural University, India Forestry Applications - P Torr and M J Wilson, University of Aberdeen, UK and S Heritage, Forestry Research, Northern Research Station, Roslin, UK Applications for the Control of Pests of Humans and Animals - I Glazer, Volcani Center, Israel, M Samish, Kimron Veterinary Institute, Bet-Dagan, Israel, and F G del Pino, Universitat Autonoma de Barcelona, Spain Application for Social Insect Control - D H Gouge, University of Arizona, USA A Systems Approach to Conservation of Entomopathogenic Nematodes, M Barbercheck, Pennsylvania State University, USA, and C W Hoy, Ohio State University, USA Interactions with Plant-parasitic Nematodes - E E Lewis and P S Grewal Compatibility and Interactions with Agrochemicals and Other Biocontrol Agents - A M Koppenhofer and P S Grewal PART 3. ENTOMOPHILIC NEMATODES Application of Beddingia siricidicola for Sirex Wood Wasp Control - R A Bedding, CSIRO, Australia and E T Iede, EMBRAPA Florestas, Brazil The Entomophilic Thripinema - J E Funderburk and K Sims Latsha, University of Florida, USA Mermithid Nematodes - E G Platzer, B A Mullens, University of California, Riverside, USA and M M Shamseldean, Cairo University, Egypt PART 4. SLUG-PARASITIC NEMATODES Biology, Production, and Formulation of Slug-parasitic Nematodes - M J Wilson and P S Grewal Field Application of Slug-parasitic Nematodes - A Ester, Applied Plant Research Ltd, The Netherlands and M J Wilson PART 5. PREDATORY NEMATODES Potential of Predatory Nematodes to Control Plant-parasitic Nematodes - A L Bilgrami and C Brey, Rutgers University, USA PART 6. FUNGAL FEEDING NEMATODES Potential of Fungal Feeding Nematodes for the Control of Soilborne Plant Pathogens - N Ishibashi, Saga University, Japan PART 7. CONCLUSIONS AND FUTURE DIRECTIONS Critical Issues and Research Needs for Expanding the Use of Nematodes in Biocontrol - P S Grewal, R-U Ehlers and D I Shapiro-Ilan.

Mass production of entomopathogenic nematodes for plant protection.

Abstract. Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are commercially used to control pest insects. They are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the major food source for the nematodes. The biology of the nematode–bacterium complex is described, a historical review of the development of in vitro cultivation techniques is given and the current use in agriculture is summarised. Cultures of the complex are pre-incubated with the symbiotic bacteria before the nematodes are inoculated. Whereas the inoculum preparation and preservation of bacterial stocks follow standard rules, nematodes need special treatment. Media development is mainly directed towards cost reduction, as the bacteria are able to metabolise a variety of protein sources to provide optimal conditions for nematode reproduction. The process technology is described, discussing the influence of bioreactor design and process parameters required to obtain high nematode yields. As two organisms are grown in one vessel and one of them is a multicellular organism, the population dynamics and symbiotic interactions need to be understood in order to improve process management. Major problems can originate from the delayed or slow development of the nematode inoculum and from phase variants of the symbiotic bacteria that have negative effects on nematode development and reproduction. Recent scientific progress has helped to understand the biological and technical parameters that influence the process, thus enabling transfer to an industrial scale. As a consequence, costs for nematode-based products could be significantly reduced.

Liquid culture of the entomopathogenic nematode-bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens

A process technology for production of the entomopathogenic biocontrol nematode-bacterium complex Heterorhabditis megidis/Photorhabdus luminescens in monoxenic liquid culture in laboratory scale bioreactors is described. Dauer juvenile yields varied between 21 and 68 million dauer juveniles/l medium. The maximum density was reached at 13 to 25 days after inoculation of P. luminescens. The reason for the high variability in yield was identified. After the 24-h bacterial preculture the bioreactor is inoculated with nematode dauer juveniles which develop to self-fertilizing hermaphrodites. The exit from enduring dauer stages (recovery) was between 18 and 90% of the inoculum density. Low dauer juvenile recovery resulted in the development of two-generations within 20 to 25 days. In contrast, high dauer juvenile recovery led to a one-generation process terminated within 15 days. Factors influencing dauer recovery are still unknown.

Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture

Photorhabdus luminescens are bacterial symbionts of entomopathogenic nematodes of the genus Heterorhabditis. The bacto-helminthic complexes are used in biocontrol of insect pests in cryptic environments. For in vitro production, liquid media are incubated with P. luminescens for 24 h prior to the inoculation of nematode dauer juveniles. The nematodes develop to self-fertilizing hermaphrodites and produce offspring. The exit from the developmentally arrested dauer stage (recovery) is a response to a yet undescribed food signal. Major process instability is caused by low and unsynchronized recovery of the dauers. In living insects, dauer recovery is approximately 95% within 1 day. In liquid cultures of P. luminescens the recovery is spread over several days and varies between 0 and 81%. In complex culture media no food signal was detected. A food signal is produced by P. luminescens and excreted into the culture medium. The maximum food signal production was recorded during the late exponential growth phase. Compared to the food signal found in insects, the efficacy of the bacterial signal is much lower. The reasons for the variable activity of the bacterial food signal and its function during the nematode life cycle are discussed.

Endotokia matricida in hermaphrodites of Heterorhabditis spp. and the effect of the food supply

Intra-uterine birth causing maternal death ( endotokia matricida ) is relatively common in rhabditid nematodes and typical for entomopathogenic nematodes of the genus Heterorhabditis . A detailed description of this phenomenon is given, including a summary of embryo development, egg-laying, juvenile hatching and development. It is demonstrated that low food supply significantly promotes the beginning of endotokia matricida but has no influence on the time scale of the process. The biological function of endotokia matricida and the intra-uterine induction of the dauer formation is discussed. Endotokia matricida in Heterorhabditis spp. is a well synchronised process of the destruction of the maternal tissues correlated with the juvenile development including the uptake of the symbiotic bacteria by the late pre-dauer stage. It secures the development of dauer juveniles at a moment when the external food supply is reducing and provides offspring which are well equipped with energy reserves and symbiotic bacteria for long term survival and subsequent infection of insects in the soil. Endotokia matricida bei Hermaphroditen von Heterorhabditis spp. und der Einfluss der Nahrungsversorgung . - Der Schlupf juveniler Nematoden im mutterlichen Uterus mit anschliessender Totung des Muttertieres ( endotokia matricida ) ist relativ weit verbreitet bei rhabditiden Nematoden und typisch fur entomopathogene Nematoden der Gattung Heterorhabditis . Die Endotokia matricida wird beschrieben sowie die Embryonalentwicklung, das Eilegeverhalten, der Schlupf und die Entwicklung der Juvenilen. Der Beginn der endotokia matricida wird durch eine niedrige Nahrungsverfugbarkeit in der Kultur signifikant gefordert, diese hat jedoch keinen Einfluss auf den zeitlichen Ablauf der endotokia matricida . Die biologische Funktion der endotokia matricida und die intra-uterine Induktion der Dauerlarvenbildung werden diskutiert. Die endotokia matricida bei Heterorhabditis spp. ist ein gut synchronisierter Prozess, bei dem die Zerstorung des Muttertiers korreliert mit der Entwicklung der Juvenilstadien, einschliesslich der Aufnahme der symbiotischen Bakterien durch das Pra-Dauer Stadium. Sie sichert die Entwicklung von Dauerlarven zu einem Zeitpunkt, wenn die externe Nahrungsversorgung sich verschlechtert und bringt so Nachkommen hervor, die gut mit Energiereserven und symbiotischen Bakterien ausgestattet sind, um im Boden lange zu uberdauern und Insekten zu befallen.

Insect Biocontrol with Non-endemic Entomopathogenic Nematodes (Steinernema and Heterorhabditis spp.): Conclusions and Recommendations of a Combined OECD and COST Workshop on Scientific and Regulatory Policy Issues

Fifteen invited experts from 10 Organisation for Economic Cooperation and Development (OECD) and European countries participating in the European Commissions Cooperation in the Field of Science and Technical Research (COST) Action 819, along with 12 other participants, met to review and debate the potential problems associated with the introduction and commercial use of non-indigenous nematodes for insect biological control. The consensus view of the participants was that entomopathogenic nematodes (EPNs) possess specific biological and ecological features, which make their use in biological control exceptionally safe. All the scientific evidence available supports the conclusion that EPNs are safe to the environment, as well as to production and application personnel, the general public and the consumers of agricultural products treated with them. Only a few potential, but very remote, risks could be identified. Therefore, it was recommended that EPNs should not be subject to any kind of registration. T...

Storage and formulation of the entomopathogenicnematodes Heterorhabditis indica and H. bacteriophora

Successful control of insect pests through theapplication of entomopathogenic nematode dauerjuveniles of H. bacteriophora and H.indica can only be achieved when the nematodematerial reaches the end user in good condition.Storage and formulation techniques must provideoptimum conditions to guarantee a maximum survival andinfectivity of the nematodes. Nematode survival wastested at temperatures ranging between 5–25 °C.A maximum survival of H. indica was achieved at15 °C and the highest mortality at 5 °C.H. bacteriophora survived best at 7.5 °Cand least at 25 °C. An increase of the saltconcentration had positive effects on dauer juvenilesurvival in aqueous suspensions. Low pH between 6 and4 reduced the bacterial growth and prolonged survivalof stored dauer juveniles. Of the organic acidsascorbic, benzoic, citric and sorbic acid, onlyascorbic acid had a positive effect on H. indicasurvival. Extracts of the dried spice plants cinnamon,cloves, rosemary and oregano were tested. Enhancementof H. indica survival was recorded for cinnamonand cloves. Survival and infectivity of nematodesstored in attapulgite and bentonite clays and spongewere recorded over several weeks at different storagetemperatures. Infectivity was not influenced by thedifferent formulation materials. When stored insponge at 25 °C nematodes survived less than 1week and the formulation in clay could only prolongthis period for another week. At 5 °C thesurvival of H. bacteriophora in sponge wassuperior to that in clay, whereas H. indicasurvived less well in sponge than in clay at15 °C. Storage in aerated water at 5 °Cfor H. bacteriophora and at 15 °C for H. indica resulted in the lowest mortality. Forstorage at controlled conditions (temperature, pH andosmolarity), aerated water is superior to all othermethods tested and the addition of preservatives willincrease survival.

Combined releases of entomopathogenic nematodes and the predatory mite Hypoaspis aculeifer to control soil-dwelling stages of western flower thrips Frankliniella occidentalis

The effect of the predatory miteHypoaspis aculeifer Canestrini (Acarina:Laelapidae) on soil-dwelling stages of thewestern flower thrips (WFT) Frankliniellaoccidentalis Pergande (Thysanoptera: Thripidae)and the influence of combined releases of H.aculeifer and two entomopathogenic nematodes(EPNs) Heterorhabditis bacteriophora Poinar(Rhabditida: Heterorhabditidae) (strain HK3,HK3) and Steinernema feltiae Filipjev(Rhabditida: Steinernematidae) (Nemaplus®,SFN) were investigated in pot trials usingseedlings of green beans (Phaseolus vulgarisL.). Ten H. aculeifer adults per pot and 400infective juveniles (IJs) cm−2 soil, of the twoEPN strains were used. In comparison withuntreated control, H. aculeifer reduced theproportion of adult F. occidentalis emergenceby 46%, while SFN and HK3 led to a reductionin adult thrips emergence by 46% and 61%,respectively. Significant differences in adultWFT emergence were found between combinedtreatments of EPNs and H. aculeifer, andindividual applications of EPNs and/or H.aculeifer, with significantly lower adultthrips emergence in the combined treatments.These findings highlight the potential for acombined use of EPNs with H. aculeifer for thecontrol of soil-dwelling stages of thrips.

Regulation of biological control agents

General Aspects of Regulation 1 Regulation of biological control agents and the EU Policy Support Action REBECA 1.1 Biological control and regulation of biological control agents 1.2 Regulation of biological control agents in Europe - the REBECA Policy Support Action 1.3 History of biocontrol registration 1.4 The precautionary principle in risk assessment 1.5 Stakeholders 1.6 References 2 Regulation according to EU Directive 91/414: data requirements and procedure compared with regulation practice in other OECD countries 2.1. Introduction 2.2. Methodology and Terminology 2.3. Legal framework and regulatory procedures 2.4. Analysis of formal data requirements 2.5. Practical experience with the regulatory process 2.6. Initiatives taken to facilitate the registration of BCAs in the EU 2.7. Major changes due to the New Regulation 1107/2009 2.8. Overall conclusions 3 An international comparison of invertebrate biological control agent regulation: what can Europe learn? 3.1 Introduction 3.2 Comparative analyses of international regulation 3.3 Conclusions and recommendations 3.4 References 4 Regulation of plant protection in organic farming 4.1 What is organic farming? 4.2 Regulation of organic farming 4.3 Plant protection in organic farming 4.4 Authorization of new pesticides for organic farming 4.5 Organic plant protection in practice: regulation and other determinants 4.6 References 5 Policy Aspects of Regulation 5.1 The Regulatory State 5.2 Dysfunctional aspects of regulation 5.3 Governance 5.4 Solution oriented stakeholder engagement 5.5 Regulatory innovation 5.6 References 6 Cost-benefit, risk and trade-off analysis of regulation 6.1 The risk society and regulation of biological control 6.2 Methods for assessing the cost-benefit relation and economic efficacy of regulation 6.3 Costs of regulation 6.4 Benefits of regulation and cost-benefit ratio 6.5 Trade-off analysis 6.6 Agriculture policy and biological control 6.7 References Risks and Risk Assessment 7 Risks of microbial biocontrol agents and regulation: are they in balance? 7.1 Introduction 7.2 Regulation as it exists today 7.3 Requirements of Annex II B of Directive 91/414 7.4 Conclusion 7.5 References 8 Ecology and human pathogenicity of plant-associated bacteria 8.1 Molecular ecology of plant-associated bacteria 8.2 Plants as reservoir for opportunistic human pathogenic bacteria? 8.3 Caenorhabditis elegans: a model to assess pathogenicity factors 8.4 Influence of antagonistic bacteria on indigenous microbial communities 8.5 Conclusions 8.6 References 9 Metabolite toxicology of fungal biocontrol agents 1. Introduction 2. Mycotoxins of fungal biological control agents 3. Standard procedure for toxic metabolite assessment 4. Conclusion 5. References 10 Risks of biocontrol agents containing compounds of botanical origin or semiochemicals 10.1 Introduction 10.1 Botanicals and semiochemicals: definitions for a concept in evolution 10.2 Risk assessment 10.3 Pheromones 10.4 Botanicals and plant allelochemicals 10.5 Conclusion 10.6 References 11 Risks of invertebrate biological control agents - Harmonia axyridis as a case study 11.1 Benefits and risks of biological control 11.2 Harmonia axyridis as a model high risk biological control agent 11.3 Concluding remarks 11.4 References Proposals for Balanced Regulation Procedures 12 Facilitations in the regulation of plant protection products containing baculoviruses 12.1 Introduction 12.2 The OECD consensus document 12.3 Genetic composition of baculovirus isolates 12.4 Potential risks from plant protection products containing baculoviruses 12.5 Current regulatory situation in the EU 12.6 Proposal for facilitated regulation of baculoviruses as active ingredients in plant protection products 12.7 Data Protection 12.8 Remark on genetically modified baculoviruses 12.9 Regulatory situation 12.10 Proposal on threshold levels for microbial contaminations in baculovirus products 13 Proposals for bacterial and fungal biocontrol agents 13.1 Introduction 13.2 Pre-Submission Meeting 13.3 Identification of low risk products 13.4 Risk assessment methodology 13.5 Proposed waivers 13.6 References 14 Proposals for regulation of botanicals 14.1 Introduction 14.2 Widely used botanicals 14.3 Environmental impact and human health risks of botanicals 14.4 Overview of regulation and regulatory efforts for botanicals 14.5 Bottlenecks under the current system 14.6 Proposals of the REBECA project 14.7 Evaluation of the REBECA proposals 14.8 Acknowledgements 14.9 References 15 Proposals for regulation of semiochemicals 15.1 Introduction 15.2 Semiochemicals in plant protection 15.3 Environmental impact and human health risks of semiochemicals 15.4 Fourth stage review of semiochemicals in the EU 15.5 Bottlenecks under the current system 15.6 Proposals of the REBECA project 15.7 Evaluation of the REBECA proposals 15.8 Acknowledgements 15.9 References 16 Regulation of invertebrate biological control agents in Europe: recommendations for a harmonised approach 16.1 Introduction 16.2 Principles of a balanced regulatory system for IBCAs 16.3 Standardised Licence (Permit) Application Form and Guidance Document 16.4 Role of ERA in a regulatory system 16.5 Validation of ERA and Licence (Permit) Application process 16.6 Implementation of a pan-European Regulatory System 16.7 Wider issues concerning implementation 16.8 Conclusions 16.9 References 17 Proposals on how to accelerate registration of biological control agents 17.1. Abstract 17.2. Introduction 17.3. Fees and financial support 17.4. Improve communication between regulators and applicants 17.5. Improve communication among regulators of biological control agents 17.6. QPS approach in risk assessment 17.7. Define low risk biological control agents/substances for fast track authorization 17.8. Guidance documents based on experience from the 4th stage evaluation 17.9. Timelines 17.10. Centralized registration authority 17.11. Optimal legislative framework 17.12. Efficacy evaluation 17.13. Perspectives 17.14. References Index

Identification and ecological characterisation of three entomopathogenic nematode-bacterium complexes from Turkey

Two heterorhabditid nematode strains (TUR-H1 and TUR-H2), and their bacterial symbionts isolated from soil samples taken at the campus of the Agriculture Faculty of the University of Ankara, Turkey, were identified by molecular methods and by cross-breeding with Heterorhabditis bacteriophora . The bacterial symbionts shared >99% similarity in the 16S rDNA sequence with Photorhabdus luminescens subsp. laumondii . Results of the restriction fragment length analysis of the ITS region assigned both nematode strains to the species H. bacteriophora . Cross-breeding confirmed the species designation for strain TUR-H2. Crosses of TUR-H1 with a hybrid strain of H. bacteriophora or with TUR-H2 resulted in infertile offspring. Both strains reproduced in monoxenic cultures of the symbionts P.luminescens isolated from H. bacteriophora and H. megidis and the resulting dauer juveniles retained cells of the bacteria. Reproduction on the symbionts isolated from H. indica failed. Infectivity at variable soil moisture and heat tolerance of the two heterorhabditid strains was compared with a Turkish isolate of Steinernema feltiae . Significantly more nematodes invaded the insect Galleria mellonella in a sandy soil assay at 10% water content than at lower values. A higher water content significantly reduced the invasion rate. Steinernema feltiae was better adapted to a temperature of 32°C than the heterorhabditid strains. Hardly any nematodes of all strains survived for longer than 4 h at 36°C.