Study On The Biological Characteristics And Wettable Powder Preparation Of Metarhizium Rileyi SZCY200812 Isolated From Spodoptera Frugiperda

Metarhizium rileyi is an important entomopathogenic fungus with good potential for biological control.However,there is no biocontrol agent or product for M.rileyi.How to efficiently prepare aerial conidia of insect fungal preparations as raw materials for biological pesticide preparations has become an urgent problem to be solved.In this study,the biological characteristics of M.rileyi SZCY200812 strain isolated and purified from Spodoptera frugiperda in corn field,liquid phase fermentation and solid phase fermentation conditions based on Plackett-Burman and response surface methodology were screened and optimized,which provided guidance to produce biocontrol agents of M.rileyi.The main results are as follows:1.The Biological Characteristics of the M.rileyi SZCY200812 Causing the Epidemic of S.frugiperda:The pathogenicity,biological characteristics,and environmental stress resistance of M.rileyi SZCY200812 isolated from S.frugiperda larvae in corn field were studied under indoor conditions.It was found that the strain was suitable for growth on SMAY medium at 25℃ and 24L:0D.The conidia produced by the strain had the strongest temperature tolerance and ultraviolet tolerance,showing good environmental resistance.2.Effects of different nutrients on the growth of M.rileyi SZCY200812 strain:Five carbon sources,five nitrogen sources were used in M.rileyi SZCY200812 culture to study effects on growth rate,colony diameter,spore yield per unit area and germination rate.The result showed that there were different nutrients had significant differences in colony growth,sporulation per unit area and conidial germination rate.Oleic acid and gelatin were the best for colony growth of M.rileyi SZCY200812.3.Effects of different medium sSubculture on biological characteristics and chitinase activity of M.rileyi SZCY200812:The colony diameter,sporulation per unit area,germination rate per unit time,virulence to S.frugiperda and chitinase activity of the strain were determined by subculture in different media.The results showed that the basic biological parameters,pathogenicity to 3rd instar larvae and chitinase activity of M.rileyi SZCY200812 strain in F₁,F₃ and F₅ generations of SAMY medium supplemented with S.frugiperda pupal shell were significantly higher than those of basic SMAY medium and SMAY medium supplemented with oleic acid,and showed an increasing trend from generation to generation.After the same culture time,the strain was cultured in the SAMY medium supplemented with the pupal shell of S.frugiperda to the F₅ generation.Compared with the F₁ generation,the colony diameter increased by 5.1%,the sporulation increased by33.3%,the spore germination rate per unit time increased by 6.8%,and the chitinase activity increased by 17%.The LT₅₀ decreased from 4.99 d of the F₁ generation to 4.58 d of the F₅generation,and the cumulative corrected mortality of the 3rd instar larvae of S.frugiperda increased from 86.04%of the F₁ to 89.63%.Based on the above results,the addition of pupal shell of S.frugiperda can increase the sporulation,enhance the pathogenicity of the strain,and enhance the chitinase activity of the strain.4.Optimization of Liquid-state Fermentation Conditions of M.rileyi SZCY200812by Response Surface Methodology:The single factor and Box-Behnken experimental design were used to optimize the culture medium type,liquid volume,p H,shaking speed and culture temperature in the liquid fermentation process.The results showed that SDY culture medium,liquid volume 100/250 m L,p H 7,shaking speed 180 r/min and temperature25℃ were preliminarily determined by single factor test,and the liquid fermentation yield per 10 m L was 0.8433 g at 120 h.The regression model of mycelial biomass and independent variables of M.rileyi SZCY200812 liquid fermentation culture was established by response surface method.According to the results of the model,the liquid volume was 100.82/250m L,the shaking speed was 178.85 r/min,the temperature was 24.80℃,and the p H was6.73.5.Optimization of solid fermentation culture conditions and determination of virulence of M.rileyi SZCY200812 by Response Surface Methodology:By optimizing the solid-phase fermentation culture conditions of M.rileyi SZCY200812,the toxicity of the conidia to the larvae of S.frugiperda was determined,which laid a foundation for improving the large-scale production of the conidia.It was found that rice was an ideal carrier for the solid-phase sporulation of M.rileyi SZCY200812 strain.Culture temperature,photoperiod and yeast extract content were the main factors affecting the sporulation of M.rileyi SZCY200812.The optimum solid-phase fermentation parameters of M.rileyi SZCY200812were as follows:temperature 22.83℃,photoperiod 18.68L:5.32D,yeast extract 4.98 g/100g.Under these conditions,the spore production of M.rileyi was 5.65×10¹⁰ spores/g on the solid-phase medium of glumeless rice.The spore suspension with a concentration of 10⁷CFU/m L was prepared,and the LT₅₀ of the 3rd instar larvae of S.frugiperda was 3.88 d.6.Formulation preparation of wettable powder for M.rileyi SZCY200812:The conidia of M.rileyi SZCY200812 strain were used as materials.The compatibility of conidia of M.rileyi SZCY200812 strain with carrier,dispersant and wetting agent was determined by control variable method,and the biological index of each treatment was calculated.The toxicity of the wettable powder to S.frugiperda was determined by impregnation method.The suitable composition and proportion of M.rileyi SZCY200812 wettable powder were conidia powder 20%,wetting agent sodium benzene sulfonate 1%,dispersant NNO 3%,kaolin to 100%.The viable bacteria content of the wettable powder was 5.12×10⁸ spores/g,the wetting time was 76 s,the suspension rate was 72.31%,the p H was 6.62,and the fineness was 99%.The LT₅₀ of this preparation against the 3rd instar larvae of S.frugiperda was 5.37d.Under these conditions,the indicators of M.rileyi SZCY200812 wettable powder were in line with national standards,and the 10⁸ spores/g wettable powder had a good infection and pathogenic effect on the larvae of S.frugiperda.