Study On Control Technique Against Aphid By Combined Spraying Beauveria Bassiana And RNA Interference

Aphids harm crops by directly sap-feeding and transmitting plant viruses.The destructive power of aphids and insecticide resistance are increasing in production now.One of the solution is to search for green and sustainable aphid control strategy.Beauveria bassiana with wide host range is a widely used biocontrol microorganism.RNAi shows merits including target specificity,no contamination,massive potential targets and so on,being promising in developing’the new generation insecticides’.In this study,Acyrthosiphon pisum was used to determine B.bassiana and Verticillum Lecanii virulence,which ascertain the strain Bb07 of B.bassiana for further study.Subsequently,the transcriptome of the pea aphids with Bb07 infection was analyzed and screened target genes.Finally,RNAi technology was combined withB.bassiana Bb07 to evaluate the control effect of the pea aphids.The aim of this study is to provide a theoretical basis for pest control technology combining insect pathogenic fungi with RNAi.The major results were as follows.1.Virulence determination of B.bassiana to the pea aphidThe fungal conidia were inoculated by spraying and the survival rate was calculated every 12 h.The cumulative survival rates of the pea aphids were 52.4%,52.3%and 43.6,respectively,in 7 days,under the treatment of B.bassiana(Bb06&Bb07)and V.lecanii(Vl03)with conidia concentration of 1×10~9 spores/m L.This result showed that three strains used in this study had low virulence to the pea aphids.The Bb07 strain of B.bassiana,isolating from the peach aphids,was chosen as the subsequent test strain.According to the results of virulence measurement and the literature,it is speculated that 36 h is the initial stage of B.bassiana infection of aphids,which causes the defense response.Post 72 h is a time point before the survival rate sharply decreased(96 h).2.Response gene mining of the pea aphids by infection of B.bassiana Bb07Compare transcriptome were performed at 0 h,36 h and 72 h to explore the differentially expressed genes upon the infection of Bd07.A total of 567 differentially expressed genes were obtained.In group of 36 h vs 0 h,there were 295 differentially expressed genes,of which 256 were up-regulated and 39 were down-regulated.In group of 72 h vs 0 h,there were 99 differentially expressed genes,of which 48 were up-regulated and 51 were down-regulated.In group of 72 h vs 36 h,there were 173differentially expressed genes,of which 20 were up-regulated and 153 were down-regulated.Based on the NR annotation,most of the differentially expressed genes related to immunity and metabolism were up-regulated in the group 36 h vs 0 h,whereas most of the differentially expressed genes related to immunity and metabolism were down-regulated in the group 72 h vs 36 h.We speculated that B.bassiana entered the hemolymph around 36 h,B.bassiana propagated in hemolymph of aphids and produced a large number of toxins such as beauvericin at 72 h.According to the NR annotation of differentially expressed genes and the GO and KEGG pathway analysis,a total of 7 target genes were selected as target candidates:Apgene4714,Apgene14740,Apgene14741,Apgene16968,Apgene15105,Apgene20844 and Apgene15782.Among them,Apgene4714 were unknown genes and significantly up-regulated at 36 h and 72 h.Apgene14740 and Apgene14741 annotated as cathepsin of lysosome pathway.Apgene16968 annotated as cuticle protein possibly involved in the first barrier for the defense of B bassiana infection.Apgene15105annotated as cytochrome P450 family gene which could detoxify toxins produced by B bassiana.Apgene20844 annotated as zinc finger protein gene and was related to the osmotic stress of insects.Apgene15782 was Trypsin serine proteases and was related to the insect immunity.3.Assessment of aphids control by combining B bassiana Bb07 with RNAi technologyBy spray tower based sequence-specific ds RNA application,four out of the seven candidate gens showed significant silencing efficiency with the concentrations of either334 ng/μL or 1002 ng/μL ds RNA.The results indicated that the spraying based RNAi could induce gene silencing while unsuccessful in some genes.It was found that the germination rate of conidia was not affected by the mixture of ds RNA with the spore suspension of B.bassiana Bb07.Aphid control capacity was then evaluated by combining B.bassiana Bb07 with RNAi.With the ds RNA concentration 1002 ng/μL,co-silencing of Apgene4714 and Apgene15782 with B bassiana Bb07 reduced the survival rate to 28.3%at 7 days in the pea aphids(p=0.0026).Furthermore,the combination of“ds4714+ds15782+Bb07”were also tested in Myzus persicae and Aphis citricidus.The survival rate of 7 days was significantly decreased to 42.5%(p=0.0004)and 52.1%(p=0.0006)in M.persicae and A.citricidus.It was proved thatB.bassiana in combination with RNAi technology could increase the virulence against aphids.In conclusion,transcriptome based gene mining can be used to serve as the candidates for RNAi-based aphids control in combination with B.bassiana.Here,we showed that the combination of gene4714-ds RNA,gene15782-ds RNA with Bb07 were potential different aphids,which highlight that the combination of RNAi and biocontrol microorganisms is an effective and potential pest control technology.