David I. Shapiro-Ilan

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.

Insect pathogens as biological control agents: Back to the future.

The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and MCAs: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control.

Production technology for entomopathogenic nematodes and their bacterial symbionts.

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode–bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 137–146 DOI: 10.1038/sj/jim/7000230

Superior efficacy observed in entomopathogenic nematodes applied in infected-host cadavers compared with application in aqueous suspension.

Two greenhouse experiments were conducted to compare the efficacy of entomopathogenic nematodes applied in aqueous suspensions with application in infected cadavers. One experiment targeted the diaprepes root weevil Diaprepes abbreviatus with Heterorhabditis indica and the other targeted the black vine weevil Otiorhynchus sulcatus with H. bacteriophora. Entomopathogenic nematode application in infected cadavers were more effective than nematode application in aqueous suspensions in all cases. The increased efficacy observed in the cadaver applications may have been due to the additional physiological stress in the aqueous application (during temporary storage in water or upon application). Superior efficacy in the cadaver application might also have been due to compounds in the infected host cadaver that can enhance nematode infectivity.

Survey of entomopathogenic nematodes and fungi endemic to pecan orchards of the Southeastern United States and their virulence to the pecan weevil (Coleoptera: Curculionidae).

Abstract The pecan weevil, Curculio caryae (Horn), is a major pest of pecans in the Southeastern United States. Entomopathogenic nematodes and fungi are potential alternatives to chemical insecticides for C. caryae control. Our objective was to survey pecan orchards in the southeastern United States for entomopathogenic nematodes and fungi and determine the virulence of the new isolates to C. caryae larvae. Soil was collected from 105 sites in 21 orchards in Arkansas, Georgia, Louisiana, and Mississippi. Entomopathogens were isolated by exposing soil to C. caryae and greater wax moth larvae, Galleria mellonella, (L.). We isolated entomopathogenic fungi and nematodes from 16 and 6 of the 21 orchards surveyed, respectively. The entomopathogenic fungi included Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin, and nematodes included Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema glaseri (Steiner), and Steinernema rarum (Doucet). This is the first report of Steinernema rarum in the United States. Soil characteristics in orchards were analyzed for pH, organic matter, and nutrients; we detected a negative relationship between fungal occurrence and manganese levels in soil and a positive relationship between M. anisopliae occurrence and calcium or magnesium levels. In laboratory assays, virulence of 15 nematode and 22 fungal isolates to C. caryae larvae was tested in small plastic cups containing soil. Results indicated poor susceptibility of the C. caryae larvae to entomopathogenic nematodes. Several fungal isolates that caused significantly higher mortality in C. caryae larvae than other strains (including a commercial strain of B. bassiana) should be investigated further as potential control agents of C. caryae.

Virulence of Entomopathogenic Nematodes to Pecan Weevil Larvae, Curculio caryae (Coleoptera: Curculionidae), in the Laboratory

Abstract The pecan weevil, Curculio caryae (Horn), is a key pest of pecans in the Southeast. Entomopathogenic nematodes have been shown to be pathogenic toward the larval stage of this pest. Before this research, only three species of nematodes had been tested against pecan weevil larvae. In this study, the virulence of the following nine species and 15 strains of nematodes toward fourth-instar pecan weevil was tested: Heterorhabditis bacteriophora Poinar (Baine, HP88, Oswego, NJ1, and Tf strains), H. indica Poinar, Karunakar & David (original and Hom1 strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK211 strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN strain), and S. glaseri (Steiner) (NJ43 strain). No significant difference in virulence was detected among nematode species or strains. Nematode-induced mortality was not significantly greater than control mortality (in any of the experiments conducted) for the following nematodes: H. bacteriophora (Baine), H. zealandica (NZH3), S. carpocapsae (All), S. feltiae (SN), S. glaseri (NJ43), and S. riobrave (355). All other nematodes caused greater mortality than the control in at least one experiment. Heterorhabditis megidis (UK211) but not H. indica (original) displayed a positive linear relationship between nematode concentration and larval mortality. Results suggested that, as pecan weevil larvae age, they may have become more resistant to infection with entomopathogenic nematodes.

Source of trait deterioration in entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae during in vivo culture

The stability of traits important for biological control was studied in the entomopathogenic nematode-bacteria complexes Heterorhabditis bacteriophora and Steinernema carpocapsae . Five experimental lines of each species were subcultured for 20 serial passages in Galleria mellonella larvae to assess trait stability. Subculturing impaired performance of both H. bacteriophora and S. carpocapsae . Virulence, heat tolerance and fecundity deteriorated in all H. bacteriophora experimental lines, and four out of five experimental lines deteriorated in host-finding ability. All S. carpocapsae experimental lines deteriorated in heat tolerance and nictation, and four out of five experimental lines declined for reproductive capacity, whereas virulence declined in two experimental lines. Determination of whether trait deterioration was due to changes in nematode, bacteria, or both symbiotic partners was tested by exchanging nematodes or bacteria from control populations with nematodes or bacteria from the most deteriorated experimental lines and assessing trait recovery. The source of deterioration varied according to trait, but only the bacterial partner played a role in trait reductions for every trait and species, whereas the nematode was the main source only for S. carpocapsae nictation. These results emphasise the important role each symbiotic partner plays in the stability and expression of beneficial traits.

Impact of the host cadaver on survival and infectivity of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) under desiccating conditions.

Entomopathogenic nematode species of Steinernema carpocapsae, Steinernema riobrave, or Heterorhabditis bacteriophora were used to compare survival and infectivity among infective juveniles (IJs) emerging in water from hosts in White traps (treatment a), emerging in sand from hosts placed in sand (treatment c), and emerging from hosts placed on a mesh suspended over sand (treatment m). Nematode survival and infectivity was recorded in sand at three-day intervals during 21 days of storage in desiccators at 75% relative humidity and 25 degrees C. Infectivity was measured by exposing 5 Galleria mellonella for 16 h to IJs. Treatment did not affect percent survival of H. bacteriophora IJs. Percent survival of S. riobrave and S. carpocapsae IJs was lowest in treatment a. Across all treatments, by 10 days after the beginning of the experiments, IJ survival declined to 93, 43, and 28% of levels on day 1 for H. bacteriophora, S. riobrave, and S. carpocapsae, respectively. For the three treatments, infection rate over time was described by a negative exponential function for S. riobrave and S. carpocapsae and by a sigmoid function for H. bacteriophora.

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.

Principles of Epizootiology and Microbial Control

An epizootic is defined as an outbreak of disease with an unusually large number of cases. A central question in insect pathology is: what are the factors that cause an epizootic? The question is addressed through epizootiology, the study of animal disease dynamics on a population level. The major factors influencing an epizootic can be divided into four basic components: the pathogen population, the host population, transmission, and the environment. The question pertaining to the causes of an epizootic is of great interest to all aspects of insect pathology, and particularly to microbial control efforts. Microbial control can be defined as the use of entomopathogens (viruses, fungi, bacteria, protists, or nematodes) for pest suppression. This chapter presents a summary and analysis of epizootiological principles and concepts of microbial control. The goal is to promote expanded studies in epizootiology, and foster research and implementation toward improved microbial control programs.