| Metarhizium anisopliae is a broad-spectrum insect pathogenic fungus.It has the advantages of no pollution to the environment,no residue,no harm to humans and animals,and is considered as an eco-friendly biological insecticide.Adverse environmental conditions such as ultraviolet light and high temperature will affect the efficacy of microbial preparations.In the field test,ultraviolet light and high temperature reduced the survival rate and virulence of conidia,thus hindering the effect of conidia of Metarhizium anisopliae.If it can improve the sporulation and UV resistance of Metarhizium anisopliae,it will help to maintain the vitality,germination ability and toxicity of conidia and improve the application ability of Metarhizium anisopliae.To improve the application ability of Metarhizium anisopliae,this study used Metarhizium anisopliae 421 as the starting strain,and used Atmospheric and room temperature plasma(ARTP)and ultraviolet(UV)compound mutagenesis to breed excellent Metarhizium anisopliae strains with ultraviolet resistance,heat resistance and strong toxicity.The biological characteristics of the mutant strains were explored,and the solid-state fermentation of the mutant strains was optimized by single factor and response surface conditions.The research results are as follows:(1)ARTP and UV compound mutagenesis treatment of Metarhizium anisopliae,preliminary screening by comparing the speed of sporulation and growth diameter,and selection of strains using ultraviolet resistance and virulence as rescreening indicators.And the genetic stability test was carried out on the mutant strains obtained by breeding.Finally,an excellent strain AU34 with high sporulation,UV resistance,strong toxicity and genetic stability was selected at ARTP 40 s and ultraviolet 120 s.The sporulation time of this strain is faster than that of the original strain,and the sporulation is 1.63±0.22(108 spores/cm2),which is 163%higher than the original strain.After 5 minutes of UV irradiation,the survival rate of the mutant strain AU34 was 3.18%,which was more resistant to UV than the original strain.In addition,the mutant strain AU34 was highly toxic to Plutella xylostella,with a corrected mortality rate of 85.71%and a half-lethal time(LT50)of 6.12 d.After subculture for 6 generations,the sporulation amount of the mutagenic strain AU34 had no obvious change,and had good genetic stability.(2)The biological characteristics of the mutagenic strain AU34,mainly heat resistance,UV tolerance,and osmotic stress resistance were investigated.It was found that the mutagenic strain AU34 has stronger tolerance to temperature than the original strain.The germination rate of mutagenesis strain AU34 was higher than that of the original strain at 3 5?-50?.Both strains grew slowly at 55? and could not grow at 60?.and the morphology of the mutagenic strain AU34 is fuller than that of the original strain after different time treatments.The UV tolerance and virulence of the mutagenic strain AU34 and the original strain were compared at different concentrations,and the results showed that AU34 had strong UV resistance and pathogenicity.The growth diameters of the two strains under different salt concentrations were measured.The results showed that with the increase of salt concentration,the growth and sporulation ability of the two strains gradually decreased,but the permeability resistance of AU34 was relatively good.(3)The effects of different culture media,vitamins,additives and water content on sporulation of Metarhizium anisopliae were explored,and the solid fermentation conditions of the mutagenic strain AU34 were optimized through single factor and response surface test methods.The best medium formula is corn flour:rice hull mass ratio is 1.79:1,vitamin B1 addition amount is 0.04%,CaCO3 addition amount is 0.09%,water content is 60.85%,pH 6.5,inoculation amount is 6%.Under the optimized conditions,theoretical sporulation was 12.44×108 spores/g,the actual test showed that the sporulation was 11.96× 108 spores/g. |
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