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Entomopathogenic nematodes (EPNs) are widely used as biological control agents against soil-dwelling insect pests. Three commercially available species are Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) (Hb), Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) (Sc), and Steinernema feltiae Filipjev (Sf). This study evaluated the effects of high temperature exposure on both survival and infectivity of these EPNs. Survival was assessed after diluting Hb, Sc, and Sf in water and treating for 1 to 10 h in glass vials in incubators at 30 to 45°C. Overall, Sc was the most heat tolerant, then Hb, then Sf. Treatments at 30°C had no impact on survival of any species, there was varying survival at 35 and 40°C, and treatments of 1 or 2 h at 45°C resulted in complete mortality. Product formulation was not found to have a consistent, significant effect on survival. Both Sc and Sf were treated at 30, 35, and 40°C, then two surviving infective juveniles were applied to individual Galleria mellonella Linn. (Lepidoptera: Pyralidae) larvae in well plates filled with moist sand. Larval mortality was assessed after 3 days. Doses of 5000 infective juveniles of Sc and Sf were also applied to moist growing mix in plastic cups, treated at 35 and 40°C, then 15 fungus gnat (Bradysia impatiens Johannsen; Diptera: Sciaridae; FG) larvae were added to each cup. Emerging adult FG were caught on sticky card traps and counted 2 weeks after larvae were added. Treatment at 30°C had no impact on Sc or Sf infectivity. Treatment at 35°C did not reduce infectivity of Sc, but infectivity of Sf against both hosts was reduced after 4 and 8 h exposures. After just 1 h exposure at 40°C Sf did not cause any infection, while 2 h treatment slightly reduced Sc infectivity against G. mellonella, and 4 h significantly reduced Sc infectivity against FG. Without high temperature exposure Sf caused higher infection of FG than Sc did, so growers may want to rely on Sf unless soil temperatures surpass 4 h at 35°C, or if they reach 40°C for any duration, in which case Hb or Sc may be more effective.

Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae),the western corn rootworm (WCR), is a major pest of corn (Zea mays L.) in the United States and Europe. WCR management options comprise mainly transgenic hybrids, insecticide applications and crop rotation. WCR is highly adaptable to management practices and field-evolved resistance to transgenic corn, insecticides and crop rotation in the United States Corn Belt has been reported. Therefore, the motivation for this project was to look into alternative options for WCR management. The goal of this dissertation is to characterize the natural enemies from irrigated commercial cornfields in Nebraska and examine their potential as biological control agents of the WCR. We surveyed five cornfields to document populations of arthropod predators, entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPN). Yellow sticky cards and dry pitfalls captured a diverse community of above-ground natural enemies but their impact on WCR population dynamics is unlikely. In the laboratory, we isolated EPF and EPN species from soil samples using a baiting technique with Galleria mellonella L. andTenebrio molitor L. Entomogenous fungi with a variety of ecological roles were detected in every cornfield. Entomopathogenic fungi made up the majority of isolates, primarily represented by Metarhizium, but other genera of known and potential EPF include Beauveria, Penicillium, Pseudogymnoascus, and Purpureocillium. In the laboratory, forty-eight strains were screenedagainst WCR larvae. Results showed thatMetarhizium anisopliae, M. robertsii, Pseudogymnoascussp. and BotaniGard (Beauveria bassiana) caused mortality higher than the control and should be explored further in field studies. Six strains that were tested against the WCR can also infect prepupae of western bean cutworm (Striacosta albicostaSmith), another damaging pest of corn in Nebraska. We also determined that EPN strains of Heterorhabditis bacteriophoraPoinar andSteinernema spp. are present in Western Nebraska cornfields. An inoculation project with commercial and New York strains of EPN did not cause significant mortality in WCR populations, potentially due to native Steinernemaspp. being present in the control plots. Describing the natural enemy community from WCR-infested fields is a necessary first step in the exploration of biological control as a management tool against this devastating pest. Advisors: Julie A. Peterson and Lance J. Meinke

Entomopathogenic nematodes (EPNs), microscopic roundworms that infect and kill insects, have gained importance as biological control agents for the safe, environmentally friendly control of insect pests. However, their adoption by managers is limited by variable field performance, to which variability in abiotic and biotic soil properties is a likely contributor. Abiotic soil properties such as texture, moisture, and porosity can affect the ability of EPNs to survive, move, and locate hosts. Also, many soil organisms can interact with EPNs in helpful or harmful ways, though their effects on EPN infectivity remain poorly quantified. Increased knowledge of the extent and relevance of abiotic and biotic soil properties’ effects on EPN performance will help managers better utilize EPNs against pests, both through increased ability to predict whether or not EPNs will be effective in a given soil and through soil management practices to increase their suitability for EPNs. My research is focused on examining the effects of biotic interactions on the ability of EPNs to infect hosts, and also to relate biotic and abiotic soil properties to EPN control of a pest insect in field soils. In laboratory trials, I evaluated the effect of soil microarthropod communities on EPN infectivity against the model host Galleria mellonella. Individuals of the EPN Heterorhabditis bacteriophora were applied into loam soil mesocosms containing or lacking a microarthropod community. Five G. mellonella larvae were introduced to each mesocosm sixteen days later and removed after an additional five days. The cadavers were dissected and average number of invading EPNs determined for each replicate, as well as the overall percentage of infection. Percent EPN infection of G. mellonella and average EPN establishment was significantly lower in soils with fauna than in soils without fauna. In addition, linear regression analysis suggested a significant negative correlation between the abundance of predatory mesostigmatid mites and EPN establishment within hosts. These results indicate negative effects of soil animals on EPN infectivity, likely due to predation. In the field, the relationship between EPN efficacy and biotic as well as abiotic soil properties was tested in turfgrass athletic fields. A series of field bioassays with the EPNs H. bacteriophora and Steinernema feltiae were conducted against the root-feeding turfgrass pest Popillia japonica. Sentinel P. japonica grubs were buried in replicated plots in two school soccer fields, in both the high-traffic areas in front of the goals and the low-traffic areas in the corners of the fields. EPNs were applied to the plots and the EPN infection of the grubs recorded. Additional measurements of abiotic soil properties and the abundances of four microarthropod groups were recorded. EPNs achieved modest control of P. japonica grubs at one of the two sites. Efficacy of S. feltiae was much higher in low-traffic soils than in high-traffic soils at one of the two tested fields in 2016 but not 2017. Sand content was the only soil property tracking EPN efficacy along a non-metric multidimensional scaling matrix, indicating abiotic properties take precedence over biotic properties in determining EPN efficacy in turfgrass field soils. While biotic interactions may reduce EPN infectivity under laboratory conditions, in the field setting of turfgrass soccer fields, abiotic soil properties are more important influences of EPN efficacy.

The purpose of the following work was to explore the potential use of these nematodes and fungi as control agents of adult Japanese beetles. Twenty strains of Steinernema and Heterorhabditis nematodes were evaluated for their ability to cause mortality to the adult beetles. All strains caused significant mortality, compared to the control treatment. The ability of four nematode strains to infect and reproduce in adult beetles was also examined. While both genera were able to infect and kill the beetles, only Steinernema strains able to produce offspring. Steinernema scarabaei and Beauveria bassiana were placed in outdoor pheromone traps, in order to test them in the field. Beetles caught in the trap were observed for subsequent mortality and evidence of infection. S. scarabaei was unable to cause infection in the trap, and B. bassiana provided greater than 80% control, with over 70% of cadavers in that treatment developing mycelial growth.

The conservation of naturally-occurring biological control agents, such as entomopathogenic nematodes (EPN), of soil-dwelling pests in agriculture may decrease the need for chemical pesticides, result in economic savings, promote a safer environment for both farm workers and native wildlife, and protect groundwater and surface water run-off. Thorough evaluation of abiotic and biotic interactions between EPN and the soil community and environment are important to assess both impacts to EPN populations and potential impacts on soil fauna resulting from the introduction of EPN. Two experiments were conducted to evaluate the response of soil fauna to the application of EPN to the soil in no-till and conventional-till corn. Each experiment used a different application method (Bait experiment = EPN delivered via infected insect cadaver, Inundation experiment = EPN delivered in aqueous solution). Both experiments were designed as a stripped split split plot over four blocks. Each experiment was repeated on 6 different dates. Variables included: 4 blocks x 2 tillage regimes x 2 sampling times x 5 treatments. The treatments included: three nematode species treatments Heterorhabditis bacteriophora (CEFS strain), Steinernema riobrave (commercially available but does not naturally occur in NC), and Steinernema carpocapsae (CEFS strain) and two control treatments, water and soil for inundation, and water and a dead insect control for bait. Response of soil fauna was measured at the levels of abundance (large traditional taxonomic affiliations of soil invertebrates), diversity, and community composition based on the finest level of taxonomic identification of invertebrates collected. Responses of soil fauna differed between the two application methods. Experimental factors, including sampling date and time, tillage regime, and blocks significantly affected abundance, diversity and community composition of soil invertebrates in both experiments. Significant changes in abundance of individual soil invertebrate taxa due to the effect of nematode treatment were found in both experiments. Both positive responses and negative responses, were detected in various taxonomic categories. And, large taxonomic groupings of invertebrates exhibited responses that differed significantly from the responses of individual taxa within those large groupings.

The position taken is that populations of natural enemies occupying both soil and foliar habitats are regulated by lower trophic levels. A series of hypotheses were tested on the influence of lower trophic levels on population persistence of natural enemies with divergent life history traits; the entomopathogenic nematode, Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) and the larval parasitoid, Diadegma insulare Cresson (Hymenoptera: Ichneumonidae). Initial studies on the entomopathogenic nematode, were aimed at understanding their ecology in vegetable landscapes and thereafter investigations were conducted to determine the influences of insect host availability and plant diversity on population densities and the mechanisms underlying their effects. Investigations for D. insulare, focused on its responses to densities of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) and flowering coriander. Several strains of entomopathogenic nematodes that were able to infect and reproduce in common vegetable insect pests were recovered along grassy banks adjacent to cultivated areas of a vegetable production system. Soil moisture was the principal factor associated with the presence of these endemic nematode populations. Persistence of H. bacteriophora populations differed in plots with different insect host availability, but persistence differed among strains and was related, in part, to the abilities of the nematodes to survive under test site conditions. For those strains with high survival rates, populations increased with increasing host availability, whereas no such effects were observed for populations with low survival rates. Higher nematode population densities were observed in plots with more diverse plants and insect herbivores. A correlation was found between nematode population density and chrysomelid beetle abundance, suggesting that persistence was at least partially as a result of nematodes recycling within these insects. D. insulare aggregated twice as much in areas with high compared with low populations of diamondback moth. Parasitism was greater when flowering coriander was present, but this effect was only observed at low population densities.In summary, bottom-up effects of plant and herbivore communities on natural enemies above and below-ground were similar. Population persistence for the two natural enemies was higher with increasing insect hosts and plant diversity; but the effects occurred at different spatial and temporal scales.

Biological pesticides (i.e., biopesticides) are living systems, which introduce additional challenges with respect to formulation and delivery not previously encountered with conventional chemical pesticides. Understanding the effects of the different physical phenomena within a spray system is important to begin identifying the equipment characteristics and operating conditions that are least detrimental to the biological agents. Specifically, this work considered the effects of pressure differentials and hydrodynamic stress on damage to a benchmark biological pest control agent, entomopathogenic nematodes (EPNs). Four EPN species were evaluated in this work: Heterorhabditis bacteriophora, H. megidis, Steinernema carpocapsae, and S. glaseri. Additionally, temperature influences due to pump recirculation were investigated. Results from this work indicate that S. carpocapsae nematodes were able to withstand greater pressure differentials and more intensive hydrodynamic conditions than the other EPN species. Consequently, EPN species is an important factor to consider when defining spray operating conditions. Operating pressures within a spray system should not exceed 2000 kPa (290 psi) for H. bacteriophora and S. carpocasae, and 1380 kPa (200 psi) for H. megidis. Other EPN species may require lower pressure. Experimental results of EPN damage after passage through an abrupt contraction and two common types of hydraulic nozzles (flat fan and cone) were compared to flow parameters from numerical simulations of the experimental flow fields using FLUENT, a commercial computational fluid dynamics (CFD) program. Based on the flow field characteristics, the rotational flow regime within a cone type nozzle produces hydrodynamic conditions that are less damaging to EPNs compared to the extensional flow developed within the narrow, elliptic exit orifice of the flat fan nozzle. It was found that the tensile stress loading that occurs during flow into a constricted region, which characterizes an extensional flow, is damaging to the biological material. An empirical model comparing average energy dissipation rates computed in FLUENT to observed EPN damage was developed. Overall, the model was able to predict the EPN damage after treatment with the hydraulic nozzles well, in many cases within 5%. These results show that CFD is a feasible method to evaluate the flow conditions within an equipment component to assess its compatibility with a biological agent. Finally, extensive recirculation of the tank mix can cause considerable increases in the liquid temperature. It was found that either a diaphragm or roller pump is better suited for use with biopesticides, compared to a high-capacity centrifugal pump, which contributes significant heat to the spray system.

Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.) contain symbiotic fungi (Neotyphodium spp.) that provide several ecological advantages to host plants: enhanced resistance to drought, disease, and insect herbivory. The resistance to insect herbivory is the result of fungus-produced alkaloids that are toxic to most herbivores. However, black cutworm, Agrotis ipsilon, is a generalist herbivore that is able to feed and develop on endophytic perennial ryegrass. As some insects can use plant secondary compounds to defend themselves against predators, I hypothesized that the cutworms fed on endophytic grasses would exhibit greater defense against a lethal endoparasitic nematode, Steinernema carpocapsae. To test this hypothesis, I developed a method to remove the fungal endophytes from the grass seed through a heat treatment. Laboratory experiments involving 4-5th instars support the hypothesis that A. ipsilon feeding on plants with high (>90%) incidence of endophyte are less susceptible to entomopathogenic nematodes than those feeding on plants with no or low incidence of endophyte. Field studies show decreased susceptibility to S. carpocapsae when larvae were confined to areas of endophytic grass (>75% infected). Early (2-3rd) instars were equally susceptible to nematode attack regardless of host plant. Endophytic grass fed to cutworm larvae did not influence nematode attachment behavior, or their ability to penetrate and successfully develop into adults. I examined the effects of ergot alkaloids that are produced by N. lolii such as, ergotamine, ergonovine, ergocryptine, ergocristine, and seed extract on nematode viability and infectivity. Ergonovine malate increased and ergocristine decreased the rates of nematode infectivity, whereas other treatments had no significant effect. I investigated the effects of ergocristine on Xenorhabdus nematophila, the symbiotic bacterium of S. carpocapsae. Bacterial growth and pathogenicity were significantly reduced when bacteria were grown in nutrient broth containing 200 ìg/ml concentration of ergocristine. Further research demonstrated that several fungal alkaloids, including ergocristine, persist in or are sequestered by black cutworm and another noctuid, the fall armyworm. I conclude that A. ipsilon developing on endophytic grasses may acquire some level of resistance against entomopathogenic nematodes. This resistance is mediated through the effects of alkaloids; thus our results underscore the ability of N. lolii to affect trophic interactions through the production of alkaloids.