A Study On The Control Effects Of Beauveria Bassiana Combined With RNAi Against A Leaf Beetle Plagiodera Versicolora

The leaf beetle Plagiodera versicolora(Coleoptera: Chrysomelidae),which mainly feeds on the leaves of willow and poplar,is one of the most notorious herbivorous pest insects of Salicaceae plants.The entomopathogenic fungus is recognized as an ideal alternative to chemical pesticides,nonetheless,its efficacy is often limited by insect’s innate immune system.The suppression of the host immunity may overcome the obstacle and promote the toxicity of the fungi.Combining appropriate reference genes and employing RT-q PCR in the aforementioned study will assist in correctly evaluating whether the target genes are down-regulated or not.Therefore,in this study,after screening and specifying suitable reference genes,we explored the feasibility of synergizing the virulence of B.bassiana by RNAi technology using bacterial ds RNA expression system.The main research methods and results are listed as follows:1.Identifying suitable reference genes for RT-q PCR analysis of leaf beetle under different biotic or abiotic conditions.The stability of seven housekeeping genes(including Actin,EF1 A,α-tubulin,RPL13 a,RPS18,RPL8 and UBC)was investigated under both biotic(developmental stages,tissues,sex and pathogen treatment)and abiotic(RNA interference treatment,temperature treatment)conditions.The ge Norm,Norm Finder,Best Keeper,and ΔCt programs were used to analyze gene expression data.The Ref Finder synthesis analysis was applied to suggest a handful of appropriate reference genes for each experimental condition.RPS18 and EF1 A were the most reliable reference genes in different development stages;RPS18 and RPL8 were most stable in female and male adults,different tissues,different temperatures,and pathogen treatment;α-tubulin and RPL13 a were most stable after dietary RNAi treatment.2.Feeding on ds RNA-expressing bacteria resulted in the silencing of immunity genes.Five immune-related genes(PGRP1,Toll1,Domeless,SPN1,and Lysozyme)were selected from the leaf beetle.Then a HT115 strain with an immune gene-L4440 expression vector was constructed,which can synthesize immune gene ds RNA when induced by IPTG.Feeding larvae a concentrated bacterial solution continuously suppressed the expression of immune genes significantly,and the relative expression of targeted genes decreased by 86.3%,67.3%,86.6%,54.6%,and 82.4%,respectively.And the silencing of selected immune genes alone could not lead to significant mortality of leaf beetle.3.RNAi enhances the effectiveness of B.bassiana in killing the willow blue leaf beetle.By infecting the larvae with B.bassiana spores,the mortality rate of B.bassiana-infected larvae was 27%.To increase the virulence of B.bassiana against the leaf beetle,ds RNA-expressing bacteria were fed to suppress the leaf beetle immunity genes.We found that once a combination of B.bassiana and immunosuppressive ds RNA was applied,the mortality of the larvae was significantly increased,and the bacteria expressing ds PGRP1 produced the strongest synergistic effect with B.bassiana,with a lethality rate of 88.3%.In addition,after B.bassiana infection,the silencing efficiency of five immune genes(PGRP1,Toll1,Domeless,SPN1,and Lysozyme)was determined again after feeding ds RNA,resulting a 71.4%,39.0%,72.0%,49.0%,and 68.7% gene silencing,respectively.Taken together,this study provides a comprehensive evaluation of the reference genes of the leaf beetle.And the ds RNA bacterial expression system targeting its immune genes was successfully constructed.Based on this,RNAi technology can enhance the effectiveness of B.bassiana in killing willow blue leaf beetle through synergistical effects.However,the molecular mechanism behind the synergistic efficacy of B.bassiana against the leaf beetle requires further exploration.