Screening Of Beauveria Bassiana With High Pathogenicity Based On ARTP And FACS

Beauveria bassiana is an entomopathogenic fungus.Due to its high efficiency,specific target and no residue,it is often used as a biological control agent.However,because B.bassiana is highly sensitive to abiotic stress and shows low toxicity under natural conditions,it has been greatly hindered in practical applications.Atmospheric room temperature plasma(ARTP)technology has the advantages of low mutagenesis temperature,abundant types of mismatched sites,and rapid mutation.As a high-efficiency mutagenesis tool for microbial mutant libraries,it plays a significant role in the process of microbial breeding.In order to efficiently obtain B.bassiana strains with high biochemical potential,this study used the fluorescence activated cell sorting(FACS)to induce mutations in B.bassiana by ARTP.The mutagenesis library was screened and the highly pathogenic B.bassiana strain was obtained.The main research contents and results are as follows:(1)Compare the mutagenic efficiency of ARTP mutagenesis technology and ultraviolet mutagenesis technology against B.bassiana.The time required to induce conidia of B.bassiana was 90 s and 30 min respectively.(2)ARTP mutagenesis of B.bassiana was performed,and a mutation library containing 8000 mutants was obtained.Then,based on the forward scatter(FSC)signal of the conidia of the mutant,6 candidate mutants with significantly larger spores than wild type were selected.(3)Evaluation of characteristics of spore yield,germination rate,heat resistance and virulence of candidate mutants.The results showed that among the six candidate mutants,the FSC readings of candidate mutants B2,B3 and B6 were significantly higher than wild-type strains by 25.1%,12.2% and 37.4%,respectively.Compared with the wild type,B1 has a 29.8% lower tolerance to heat stress at 40 ° C.B2 and B3 have 25.3% and 25.9% lower spore production.The germination capacity and sporeproduction of B4 are 23.3 and 30.5 lower.The spore yield of B5 was 39.3% lower.The mutant B6 was screened as B.bassiana strain with high biocontrol potential.Compared with the wild-type strain,the FSC reading of the mutant strain B6 was37.4% higher than that of the wild-type strain,and the half-lethal time was reduced by32.6%.The infection toxicity was significantly higher than that of the wild-type strain.Under the same culture conditions,B6 also showed a 13.5% higher germination rate and 12.1% spore production than the wild type.In addition,the heat resistance and conidial production of B6 were not significantly different from those of the wild type.(4)Finally,this study also compared the transcriptome of B6 and wild-type infected Galleria mellonella larvae.The analysis showed that 1491 co-expressed genes showed significant expression changes(| FC | ? 2,Q ? 0.001)in the Galleria mellonella larvae 3 days after B6 infection,including 622 up-regulated genes and 869down-regulate genes.Of the 35 GO entries,25 have more down-regulated genes than up-regulated genes,indicating that most of the transcriptional activities of the differential genes are inhibited by infection with highly virulent strains,such as fatty acid synthesis and other metabolic processes.However,the immune response of the giant wax moth larvae and the process of protein digestion and absorption are highly activated.This paper confirms that it is feasible to use the combined application of ARTP and FACS to screen B.bassiana strains with high toxicity.The analysis of the transcriptome infecting highly virulent strains has promoted the understanding of insect-pathogen interactions,and a new strategy to improve the control efficiency of insect pathogenic fungi from the transcriptome perspective.