The Virulence And Synergistic Effect Of Beauveria Bassiana And Trichoderma Asperellum To Induce Plant Defense Against The Asian Corn Borer In Maize

Asian corn borer,Ostrinia furnacalisn（Guenée）（Lepidoptera:Crambidae）is a major devastating insect pest in maize,which can cause serious yield loss of corn in China.By studying the entomopathogenic fungi induces resistance in corn against O.furnacalis will help to provide a new idea in IPM strategies for ACB control.So far,many approaches have been used to increase the virulence（pathogenicity）of entomopathogenic fungal isolates to herbivorous insects.The current study is an attempt to estimate synergistic effect of Beauveria bassiana OFDH1-5 and Trichoderma asperellum GDFS1009 on O.furnacalis in order to explore larval immune response through RNA sequencing and differentially expression analysis.Entomopathogenic fungi as biocontrol agents are the best possible and eco-friendly solution to develop resistance against O.furnacalis and improve maize yield.However,knowledge about underlying molecular mechanisms,metabolic shifts and hormonal signaling is limited.In this study,the virulence of B.bassiana,T.asperellum and the binary combination of their LC₅₀in seven proportions（B.bassiana:T.asperellum=1:1,1:2,1:3,1:4,2:1,3:1,4:1）against O.furnacalis was estimated by in vitro pathogenicity bioassay.Then the selected proportion with strong synergistic potential was used in vivo pathogenicity analysis by pot and field experiment.Here in,we used axenic and consortium of entomopathogenic B.bassiana and pathogen-antagonistic T.asperellum in maize through seed coating and soil drenching inoculation methods and observed that consortium results in higher chlorophyll and antioxidants level with a decrease in O.furnacalis survival.We performed a comprehensive transcriptome and untargeted metabolome profiling for the first time at vegetative and silking stage in fungal inoculated maize leaves at 0-,12-,24-,48-,and 72-h post insect infestation.Weighted Gene Coexpressed Network Analysis is a useful method to identify modules/networks of coexpressed genes.Further,correlation of these modules with phenotypic traits were useful to detect key genes within the networks.However,no such large-scale study regarding the synergistic interaction of B.bassiana OFDH1-5 and T.asperellum GDFS1009 in triggering omics based regulatory mechanisms of defense genes,biochemical enzymes and phytohormones induces by O.furnacalis herbivory,are available.Results revealed significant decrease in plant damage and increase in larval mortality due to fungal treatments.We found that B.bassiana OFDH1-5 and T.asperellum GDFS1009 treated corn plants mediated the plant defense response by increasing proline,superoxide dismutase（SOD）,peroxidase（POD）,polyphenol oxidase（PPO）and protease activities in a pot trial.The results showed that seed coating method was the most effective in case of maize endophytic colonization.Transcriptom sequencing to analyze gene expression differences in the midgut tissues of O.furnacalis larvae revealed a total of 59immune-related differentially expressed genes DEGs were identified in O.furnacalis larvae including,cytochrome P450,heat shock protein,ABC transporter,cadherin,peptidoglycan recognition protein（PGRP）,cuticular protein,etc.Further,transcriptomic response was confirmed by q RT-PCR.In Field grown plants,total of 130 Negative and 491 Positive metabolomic features were detected using UHPLC-QTOF-MS.We identified 13 candidate genes highly correlated with JA-ET signaling pathway and antioxidants viz protein kinase gene（GRMZM2G025459）,Acyl-Co A dehydrogenase（GRMZM5G864319）,thioredoxin gene（GRMZM2G091481）,Glutathione S-transferase（GRMZM2G116273）,glutathione peroxidase（GRMZM2G144153）,GDSL-like lipase（AC212068.4＿FG005）,defensin（GRMZM2G392863）,protease inhibitor（GRMZM2G004466）,Patatin-like phospholipase gene（GRMZM2G154523）,Chitinase gene（GRMZM2G453805）,Beta-glucosidase18（GRMZM2G031660）and cytochrome p450（GRMZM2G139874）,in vegetative plants and 10 SOD,POD,PPO,Protease and Proline encoding genes viz superoxide dismutase Cu,Zn and Mn,（GRMZM2G124455,GRMZM2G169890,GRMZM2G025992,GRMZM2G106928）,peroxidase genes（GRMZM2G047968,GRMZM2G054300 and GRMZM2G144153）,polyphenol oxidase gene（GRMZM5G851266）,protease associated gene（GRMZM2G002756）,proline-rich extension gene（GRMZM2G065214）and 4 genes viz GRMZM2G154523（PLP）,GRMZM2G074401（omega-3 fatty acid desaturase）,GRMZM2G453805（insect cell wall degrading chitinase gene）,GRMZM2G156632（Protease inhibitor）participating in JA signaling pathway at silking stage not reported previously via Weighted Gene Co-expression Network analysis（WGCNA）.Intramodular significance and RT-q PCR expressions showed that these genes are the true candidate genes and are important in the maize defense against O.furnacalis.We observed that the level of OPDA was modulated at 24-and 72-h of insect feeding of the consortium-based seed priming.Our results concluded that,coculture of B.bassiana and T.asperellum has the synergistic potential to suppress the immune response of O.furnacalis and can be used as sustainable approach to boost the induced plant resistance through activation of defense-related enzymes.Fungal consortium can boost up plant defense responses at molecular level by increasing the expression of defense related compounds and genes more.We assumed that plant defense against ACB attack is under the influence of multiple genes at the same time.Our results provide insights into the genetics,biochemicals and metabolic diversity and are useful for future biocontrol strategies against ACB attack.Seed priming technology with entomopathogenic fungi or biocontrol agents had positive significance on plant defense and growth and it can be widely used to enhance the plant growth and improve plant resistance to herbivorous insects.B.bassiana OFDH1-5 and T.asperellum GDFS1009 have a strong synergistic potential and it can be sustainably used to increase antioxidants and jasmonate（JA and OPDA）related defense responses against O.furnacalis herbivory in maize.Besides,the present study not only showed the mechanism of plant response against unfavorable biotic stress condition by seed priming with B.bassiana OFDH1-5 and T.asperellum GDFS1009,but it also laid a foundation for application of seed priming in maize production under insect herbivory in field conditions.