Microbe–mediated Tritrophic Interactionsbetween Helicoverpa Zea And Tomato (Solanum Lycopersicum)

Helicoverpa zea(Lepidoptera: Noctuidae)is crucial polyphagous pest in America,most of its host plants are economic important.In this research,we employed the study system of tomato-H.zea to evaluate the role of H.zea associated microbes which may improve the research area of tritrophic interactions of plant-insect-microbe.Insects and plants live in a microbial milieu including bacteria,viruses,fungi and nematodes,and their interaction associated with microbes turn to be sophisticated and complex.Insects can possess specific immune responses to protect themselves from different types of pathogens.Activation of immune cascades can inflict significant developmental costs to the surviving host.A previous study has demonstrated differential fitness costs in a host infected by one of two viruses from the same family,Baculoviridae.The timing of mounting immune responses is crucial for a surviving host that experienced baculovirus challenge to characterize the infection kinetics.In the second chapter,we investigated the time–dependent immune responses elicited by inoculations from one of two wild–type baculovirus species,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)and Helicoverpa zea single nucleopolyhedrovirus(HzSNPV),in their common host H.zea.As H.zea is a semi–permissive host of AcMNPV and fully permissive to HzSNPV,we hypothesized that there are differential immune responses and fitness costs associated with fighting off each virus species.Newly molted fourth–instar larvae were inoculated with an LD15 of one of the two viruses.We then assessed the innate immune responses of total haemocyte numbers,haemolymph phenoloxidase(PO)activity,FAD–glucose dehydrogenase(GLD)activity and haemolymph protein concentration at multiple time points post virus inoculation.We also assessed larval weight gain,development time and pupal weight of the survivors.We found that H.zea larvae inoculated by either virus showed significantly higher GLD activities compared to their corresponding control larvae.PO activity,total haemocyte numbers and protein concentration were not induced to higher levels,but instead were lower than control larvae at some time points.H.zea larvae that survived challenge from either virus exhibited reduced pupal weight,but survivors inoculated with AcMNPV grew slower than the control larvae while survivors of HzSNPV inoculation grew faster.The results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.Although the tritrophic interactions of plants,insect herbivores and their natural enemies have been intensely studied for several decades,the roles of entomopathogens in their indirect modulation of plant–insect relationships is still unclear.In the third chapter,we employed a sublethal dose of a baculovirus with a relatively broad host range(AcMNPV)to explore if feeding by baculovirus–challenged H.zea caterpillars induces direct defenses in tomato plant.We examined induction of plant defenses following feeding by H.zea,including tomato plants fed on by healthy caterpillars,AcMNPV–challenged caterpillars,or undamaged controls,and subsequently compared the transcript levels of defense related proteins(i.e.,trypsin proteinase inhibitors,peroxidase and polyphenol oxidase)and other defense genes(i.e.,proteinase inhibitor II and cysteine proteinase inhibitor)from these plants,in addition to comparing caterpillars' relative growth rates.As a result,AcMNPV–challenged caterpillars induced the highest plant anti–herbivore defenses.We examined several elicitors and effectors in the secretions of these caterpillars(i.e.,glucose oxidase,phospholipase C,and ATPase hydrolysis),which surprisingly did not differ between treatments.Hence,we suggest that the greater induction of plant defenses by the virus–challenged caterpillars may be due to differences in the amount of these secretions deposited during feeding or to some other unknown factor(s).Insect herbivores possess a diverse and abundant gut microbiota that may influence plant growth in nature.The application of plant beneficial bacteria to improve agricultural production and soil quality has long been of interest.Thus,these insect–associated microbiota have the potential to be developed into effective bio–fertilizers.The bacterium,Enterobacter ludwigii,was isolated from the regurgitant of field–collected H.zea.The bacterium can be secreted by the insect onto tomato seeds during fruit feeding and is also commonly found in the soil.In the fourth chapter,we applied E.ludwigii to germinated tomato seeds and measured tomato plant growth and productivity under controlled greenhouse conditions.Since there are often trade–offs between plant growth and plant defenses,we examined whether the E.ludwigii–mediated faster growth corresponds with weaker anti–herbivore defenses.When E.ludwigii was applied to germinated tomato seeds,the plants exhibited faster root,shoot and hypocotyl growth,and produced more fruits and seeds than untreated control plants.The plants treated with bacteria exhibited the same activity levels of two key enzymes involved in anti–herbivore defenses,polyphenol oxidase and peroxidase,and induced the same levels of mortality and growth inhibition in H.zea larvae as untreated plants.Thus,our results demonstrate that the application of E.ludwigii to seeds can promote tomato plant growth and yield without compromising anti–herbivore defenses.