Effects Of Entomopathogenic Fungus On Microbial Community In Plant Phyllosphere And Host Gut

Microbial pesticide is one of critical components in integrated pest management?IPM?.Entomopathogenic fungi may infect arthropod pests through the external cuticle and trigger a series of complex interactions and pathological phenotypes.Apart from cuticle adhesion to insect,entomopathogenic fungi can also disperse on plant phyllosphere during application.Little,however,is known in terms of the impact of these organisms on the indigenous microbial community in plant phyllosphere and insect intestinal tract.The broad-spectrum insecticide,Metarhizium anisopliae CQMa421wasahighlyeffectivestrainincontrollingtherice leafroller,Cnaphalocrocis medinalis Guenee and Locusta migratoria manilensis.In this project,the distribution of M.anisopliae,its efficacy in controlling the rice leaf roller,and its effects on rice phyllosphere microbial diversity after application in field trials were investigated.Our results indicated that M.anisopliae CQMa421 was highly effective in controlling the rice leaf roller.Phylloplane specific distribution of the fungus was observed over time by colony forming units?CFUs?,displaying a more rapid decline of M.anisopliae CFUs seen in the top leaf layer compared to lower layers.Application of the fungus resulted in transient changes in the endogenous microbial diversity with variations seen in the bacterial observed species and Shannon index.Notable increases in both parameters were seen at 6 day post application of M.anisopliae.Application of M.anisopliae increased the relative distribution of bacterial species implicated in plant growth-promotion and organic pollutant degradation,e.g.Methylobacterium,Sphingobium and Deinococcus.No significant difference was seen at 12 day.These data showed that entomopathogenic fungi insecticides were not only highly effective in controlling the rice pest but also minimal impact of M.anisopliae on endogenous microbial diversity with transient changes in bacterial abundance/diversity that may result in no negative influence on crops microbiology ecology.Insect immune responses can be cascaded when entomopathogenic fungi infecting.However,there is lacked systematic research on microbial community of host intestinal tract after changed insect immune responses with entomopathogenic fungi infecting.In order to explore how to change for the host survival and its gut microbe when fungi infecting,and uncover the underlying pathogenic mechanism,following bioassays were performed:L.migratoria inoculated topically with M.anisopliae CQMa421 and M.acridum CQMa102,and Prodenia litura inoculated with M.anisopliae CQMa421 and Beauveria bassiana Bb0725 respectively.The results showed that all of those entomopathogenic fungi strains could kill corresponding pest and resulted in bacteria lethal phenomena.The bacteria lethal rate in lower M.anisopliae spores-concentration with 1×10⁵ spores/mL?LMC?groups during bioassay for L.migratoria was higher than that in higher M.anisopliae spores-concentration with 1×10⁹ spores/mL?HMC?groups.Those results suggested that some opportunistic pathogenic bacteria entered the host body and resulted in host death during the M.anisopliae infecting.In order to research impacts of fungi infecting on microbial community of insect intestinal tract,L.migratoria and P.litura midgut bacterial flora were investigated during M.anisopliae CQMa421 infecting.The results suggested that the entomopathogenic fungus altered L.migratoria midgut bacterial flora with reduced species richness and diversity.Midgut microbial community structure with?Un?Weighted Uni Frac distance decreased after the M.anisopliae infected.Escherichia-Shigella,which could cause gastrointestinal diarrhea complications,was the highest distributing Biomarker in L.migratoria midgut of HMC group owing to external cuticle infecting by M.anisopliae CQMa421.However,potential entomopathogenic bacteria Pseudomonas was the Biomarker in LMC group.Moreover,community structure of L.migratoria and P.litura midgut bacterial flora changed significantly after M.anisopliae CQMa421 infecting,and Pseudomonas and pathogenic Providencia significantly wide distributing in LMC.Furthermore,the experiment implemented to detect the expression level of immune related genes?peptidoglycan recognition proteins:PGRP1,PGRP3 and Toll signaling pathway:Spatzle?in hemocyte and midgut by real-time fluorescence quantitative PCR,suggesting that the innate host immune system was disrupted after fungal infection.In addition,phenol oxidase enzymatic activity in hemolymph maintained a higher level during M.anisopliae infecting,but lower level in midgut.Disordered innate immunity in hemolymph and lower immunity levels?Attacin?in midgut might be a primary reasons that generating bacteria lethal phenomena,reduced species richness,diversity and community structure of L.migratoria midgut bacterial flora after the M.anisopliae infected.And opportunistic pathogenic bacteria entered the body and resulted in host death.All above results revealed an new pathogenic mechanism of entomopathogenic fungus:after entomopathogenic fungus'cuticle infecting,the host resist fungus by increased humoral and cellular immunity,but decreased intestinal immunity.And then,insect gut microbial community changed and partial infected insects die due to opportunistic pathogenic bacteria.These research findings also provide theoretical foundation for development and large scaled application of fungi insecticides and are helpful to clarify the pathogenic mechanism of entomopathogenic fungus.