Parwinder S. Grewal

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.

INFECTION BEHAVIOR OF THE RHABDITID NEMATODE PHASMARHABDITIS HERMAPHRODITA TO THE GREY GARDEN SLUG DEROCERAS RETICULATUM

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum was studied. The dauer (enduring or nonaging) juveniles of P. hermaphrodita invade D. reticulatum within 8–16 hr following external exposure, with the posterior mantle region containing the shell cavity serving as the main portal of entry. The dauer juveniles can recover, multiply, and produce new dauer juveniles in the slug and slug feces homogenates, but not in the soil extract. These results demonstrate that P. hermaphrodita is a facultative parasite of the slug and can complete its life cycle under nonparasitic conditions associated with the host. Although the juvenile and adult nematodes can kill the slug if injected into the shell cavity of the host, only the dauer juvenile can serve as an infective stage in the natural environment.

Host recognition behavior by entomopathogenic nematodes during contact with insect gut contents

Host recognition is an important step in the life cycle of most parasites. We tested whether host recognition by entomopathogenic nematodes occurs through contact with insect gut contents and whether this recognition is important to successful infection. Behavioral responses of 4 species of entomopathogenic nematodes, Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steirnema scapterisci, ere studied during contact with the gut contents of insects of 4 species: Acheta domestica. Blatella germanica, Popillia japonica, and Spodoptera exigua

BOTH HEAT-SHOCK AND COLD-SHOCK INFLUENCE TREHALOSE METABOLISM IN AN ENTOMOPATHOGENIC NEMATODE

Heat-shock response is highly conserved in animals and microorganisms, and it results in the synthesis of heat-shock proteins. In yeast, heat-shock response has also been reported to induce trehalose accumulation. We explored the relationship between heat- (35 C) or cold-shock (1 and 10 C) and trehalose metabolism in the entomopathogenic nematode, Heterorhabditis bacteriophora. Because both heat- and cold-shocks may precede desiccation stress in natural soil environments, we hypothesized that nematodes may accumulate a general desiccation protectant, trehalose, under both situations. Indeed, both heat- and cold-shocks influenced trehalose accumulation and activities of enzymes of trehalose metabolism in H. bacteriophora. Trehalose increased by 5- and 6-fold in heat- and cold-shocked infective juveniles, respectively, within 3 hr of exposure, compared with the nematodes maintained at 25 C (culture temperature). The activity of trehalose-6-phosphate synthase (T6PS), an enzyme involved in the synthesis of trehalose, also significantly increased in both heat- and cold-shocked nematodes during the first 3 hr of exposure. Generally, the trehalose levels and activities of T6PS declined to their original levels within 3 hr when nematodes were transferred back to 25 C. In both heat- and cold-shocked nematodes, trehalase activity decreased significantly within the first 3 hr and generally returned to the original levels within 3 hr when these nematodes were transferred back to 25 C. The results demonstrate that the trehalose concentrations in H. bacteriophora are influenced by both heat- and cold-shocks and are regulated by the action of 2 trehalose-metabolizing enzymes, T6PS and trehalase. The accumulated trehalose may enhance survival of nematodes under both cold and warm conditions, but it may also provide simultaneous protection against desiccation that may result from subsequent evaporation or freezing. This is the first report of the relationship between trehalose metabolism and heat-shock for the Nematoda.

Top‐down and bottom‐up regulation of herbivores: Spodoptera frugiperda turns tables on endophyte‐mediated plant defence and virulence of an entomopathogenic nematode

Abstract.  1. The fungus Neotyphodium lolii forms a symbiotic relationship with its grass host Lolium perenne (perennial ryegrass). The fungus benefits from access to plant nutrients and photosynthate, whereas the plant benefits from acquired chemical defence against herbivory.

Tank-Mix compatibility of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, with selected chemical pesticides used in turfgrass

We evaluated the compatibility of two entomopathogenic nematodes, Heterorhabditis bacteriophora HP88 and Steinernema carpocapsae All strain with selected pesticide formulations used in turfgrass in tank-mixes under laboratory conditions. The nematodes were exposed to the recommended rates of pesticides applied in either 100, 300, or 500 L/ha tank-mix volumes in 24-well plates at room temperature for 3 h and infective juveniles (IJ) viability determined, and then tested against Galleria mellonella larvae at 22–26°C for 96 h to assess IJ pathogenicity. We found that S. carpocapsae viability was not affected by any of the pesticides, while aluminum tris and trichlorfon significantly reduced S. carpocapsae pathogenicity at all concentrations. Thiamethoxam and trichlorfon significantly reduced H. bacteriophora viability, while halofenozide, aluminum tris, trichlorfon, and carbaryl significantly reduced H. bacteriophora pathogenicity. Imidacloprid, at the recommended rate 330–440 g AI/ha, significantly increased H. bacteriophora pathogenicity at 500 and 300 L/ha application volume. The integration of these nematode pesticide combinations in turf pest management programs is discussed.

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 w g/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 w g/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 w g/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.

Water Content and Fatty Acid Composition of Infective Juvenile Entomopathogenic Nematodes during Storage

Limited shelf life of infective juveniles is a major obstacle in realizing the full biological control potential of entomopathogenic nematodes. We studied some of the physiological parameters affecting storage stability. Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri were cultured in the wax moth larva, Galleria mellonella, and the freshly emerged infective juveniles were stored in water at 25 C. Shelflife, estimated by a minimum of 20% survival, varied among species: infective juvenile S. glaseri survived for 36 wk, whereas S. carpocapsae and H. bacteriophora survived only 16 and 7 wk, respectively

Dissolved nutrients and atrazine removal by column-scale monophasic and biphasic rain garden model systems.

Rain gardens are bioretention systems that have the potential to reduce peak runoff flow and improve water quality in a natural and aesthetically pleasing manner. We compared hydraulic performance and removal efficiencies of nutrients and atrazine in a monophasic rain garden design versus a biphasic design at a column-scale using simulated runoff. The biphasic rain garden was designed to increase retention time and removal efficiency of runoff pollutants by creating a sequence of water saturated to unsaturated conditions. We also evaluated the effect of C substrate availability on pollutant removal efficiency in the biphasic rain garden. Five simulated runoff events with various concentrations of runoff pollutants (i.e. nitrate, phosphate, and atrazine) were applied to the monophasic and biphasic rain gardens once every 5d. Hydraulic performance was consistent over the five simulated runoff events. Peak flow was reduced by approximately 56% for the monophasic design and 80% for the biphasic design. Both rain garden systems showed excellent removal efficiency of phosphate (89-100%) and atrazine (84-100%). However, significantly (p<0.001) higher removal of nitrate was observed in the biphasic (42-63%) compared to the monophasic rain garden (29-39%). Addition of C substrate in the form of glucose increased removal efficiency of nitrate significantly (p<0.001), achieving up to 87% removal at a treatment C/N ratio of 2.0. This study demonstrates the importance of retention time, environmental conditions (i.e. saturated/unsaturated conditions), and availability of C substrate for bioremediation of pollutants, especially nitrates, in rain gardens.

Genetic variation and relationships between isolates and species of the entomopathogenic nematode genus Heterorhabditis deciphered through isozyme profiles.

We studied variation in isozyme patterns of 8 metabolic enzymes in 5 species of Heterorhabditis (H. bacteriophora, H. indica, H. marelata, H. megidis, and H. zealandica) comprising 18 isolates. Isozyme banding patterns of all the 8 enzymes were species specific; however, 3 enzymes, i.e., arginine kinase, fumarate hydratase, and malate dehydrogenase, displayed distinct patterns among all the 18 isolates. Phylogenetic analysis of the isozyme patterns produced dendrograms depicting a high degree of genetic variation among Heterorhabditis species, with the average pairwise distance of 0.2000. Trees constructed using different phylogenetic methods showed a relatively close genetic relationship between H. megidis and H. zealandica and between H. bacteriophora and H. indica. Also, H. bacteriophora HP88 was the most distant species from H. megidis (UK isolate), H. marelatus (Oregon isolate), and H. zealandica (X1 isolate) with pairwise distance of 0.1957, 0.2228, and 0.2120, respectively. Phylogenetic analysis also revealed genetic variation among H. bacteriophora isolates with the average pairwise distance of 0.1507. GPS2 and GPS3 were the most closely related isolates with the average distance of only 0.0870, followed by GPS1 and GPS2 with average distance of 0.1087. In contrast, KMD19 and HP88, OH25, and HP88, and OH25 and Acows isolates were the most divergent populations with a pairwise distance of 0.2011 and 37 character differences. Pairwise distance analysis also revealed that genetic divergence among populations of H. bacteriophora is relatively independent of geographic distance. Overall, these results demonstrate strong subspecies structuring in H. bacteriophora.