| The fall armyworm（FAW）,Spodoptera frugiperda,is a major migratory invasive pest belonging to Lepidoptera,Noctuidae.It has invaded China since 2018 and can harm more than 300 plant species,causing huge economic losses.The greater wax moth,Galleria mellonella,belongs to Lepidoptera,Pyralidae.G.mellonella is a common agricultural pest that endangers apiculture.As an entomopathogenic fungus（EPF）,Beauveria bassiana is one of the most widely used microbial pesticides in the biological control of pests.However,Lepidopteran pests have strong resistance to B.bassiana,resulting in unsatisfactory insecticidal efficiency.The EPF strains with higher toxicity can be screened and obtained by ultraviolet mutagenesis.However,the ultraviolet mutagenesis and transcriptome of B.bassiana are rarely studied at present.In this study,the B.bassiana strain ARSEF2860 was subjected to ultraviolet mutagenesis,and the mutants with higher growth rate,conidial yield and chitinase activity were screened,and finally two mutants,named as 6M and 8M,were obtained.The following comparative studies were conducted with wild-type and mutant strains:biological characteristics（growth rate,sporulation,spore germination rate）;stress resistance（anti-osmotic pressure,anti-oxidation and anti-ultraviolet）;Compatibility with common chemical insecticides（matrine,spinetoram,chlorantraniliprole,and emamectin benzoate）;Protease,lipase,cellulose and chitinase activity;Determination of insecticidal toxicity against G.mellonella and S.frugiperda in the laboratory;Transcriptome sequencing and analysis of wild-type and mutant strains.In this study,two mutagenic strains with significantly enhanced insecticidal activity compared with wild-type B.bassiana were screened by UV mutagenesis,and transcriptome sequencing and comparative analysis were conducted.The results can provided new ideas for identifying novel virulence genes and carrying out genetic engineering transformation of EPF.The main results are as follows:1.UV mutagenesis of B.bassiana:By UV mutagenesis and mutant screening of B.bassiana ARSEF 2860,two mutants with higher growth rate,higher spore production and higher germination rate were obtained,which were named 6M and 8M,respectively.2.Observation of colony morphology,determination of growth rate,germination rate and spore yield:Compared with the wild type strain,the shape and edge of mutants on the PDA medium are irregular,but there is no obvious difference in colony color and conidia morphology.The colony diameter of mutants exceeded that of wild type from the 8th day.The germination rate of two mutants at the 24,27 and 30 h were significantly higher than that of the wild type.From the 11th day,the conidia yield of 8M mutant exceeded that of wild-type and 6M mutant,and from the 15th day,the conidia yield of 6M mutant exceeded that of wild-type.Therefore,the growth rate,germination rate and spore production of mutants were higher than those of wild type.3.Analysis of stress resistance（osmotic pressure resistance,antioxidant,and UV resistance）:Wild type and mutants were inoculated on PDA medium containing H2O2,sorbitol,sodium chloride or Congo red,and their morphology and growth rate were compared.The results showed that there were some differences in colony morphology.The colony diameter of 6M mutant on the 14th and 16th day on H2O2 medium was larger than that of wild type and 8M strain.The diameter of the two mutants on sorbitol medium was larger than that of the wild type from the sixth day.Similarly,on Na Cl and Congo red medium,the colony diameter of mutants was larger than that of wild type.The germination rates of wild type,6M and 8M mutants decreased by 26%,6%and 4%respectively after 2min of ultraviolet irradiation.After irradiation for 3 min,they decreased by 49%,19%and23%respectively.After irradiation for 5 min,they decreased by 68%,25%and 31%respectively.Therefore,the stress resistance of mutants is better than that of wild type.4.Insecticide compatibility analysis:We compared the germination rate and growth rate of wild-type and mutant strains on liquid and solid media containing four commonly used chemical insecticides.The results showed that only emamectin benzoate had a significant negative effect on the germination rate and growth rate of wild-type and mutants,while matrine,spinetoram,and chlorantraniliprole had little effect.Therefore,the mutant strain is similar to the wild-type in terms of insecticide compatibility,and they are not suitable to be used in combination with emamectin benzoate.5.Analysis of chitinase,protease,cellulase and lipase activities:Compared with the wild type strain,6M and 8M mutants showed higher protease,lipase and cellulose activities,and the diameter of halo formed by hydrolysis is obviously larger.The chitinase activity of6M strain was higher than that of wild type on the 5th,9th and 10th day.The chitinase activity of 8M strain was higher than that of wild type on the 5th and 6th day.6.Determination of insecticidal activities against G.mellonella and S.frugiperda in the laboratory:Insecticidal activities of 6M and 8M mutant strains against G.mellonella larvae were significantly higher than those of the wild type.Compared with the wild type,two mutants can significantly decrease the hatching rate of S.frugiperda eggs and increase the cumulative mortality rate of larvae..7.Transcriptome sequencing and comparative analysis:10173 unigene were identified by transcriptome sequencing of the conidia of wild-type and mutants,which were annotated in NR,Swiss-Prot,Pfam,Egg NOG,GO and KEGG databases.In the 6M vs.WT gene set,367 genes were up-regulated and 325 genes were down-regulated.In 8M vs.WT gene set,157 genes were up-regulated and 132 genes were down-regulated.Then GO and KEGG enrichment analysis,gene set enrichment analysis（GSEA）and protein interaction network analysis（PPI）were carried out.To sum up,this study proved that ultraviolet mutagenesis is a very effective and economical method to obtain EPF strains with higher virulence and higher stress resistance.Comparative analysis of transcriptomes can identify more potential virulence genes,thus providing new ideas for the genetic engineering of EPF and the improvement of biocontrol strategies. |
