| Beauveria bassiana is an important biological control agent for various insect pests.The application of B.bassiana to control agricultural and forestry pests has multiple advantages such as environmentally friendly and unlikely to cause insecticide resistance.In the mass production of B.bassiana,the liquid-solid dual-phase fermentation technology has been commonly used and the parameters of solid phase fermentation affect the yield and quality of spores.Previously,a B.bassiana strain,Bb06,was obtained in our laboratory and showed high virulence to the underground pest Gryllotalpa orientalis.However,the solid phase fermentation parameters of Bb06 are not clear,which limits its large-scale production and application in pest control.Therefore,this study aims to optimize the solid phase fermentation parameters of Bb06,thus providing a basis for the large-scale production of Bb06 spores.The results of this study are as follows:1.Optimization of ratio of substrate component for Bb06 fermentationThe solid phase fermentation medium uses rice as the basic component and rice husk,wheat bran and wheat straw as the supplementary materials.The best ratio of solid fermentation substrate was determined by using sporulation quantity and spore germination rate as indicators.The results showed that the maximum number of spores was(23.80±0.57)×10~9/cm~2when rice:wheat bran:rice husk:wheat straw was 50%:40%:5%:5%.In addition,different ratios of rice,wheat bran,rice husk,and wheat straw had no significant effect on the spore germination rate.In all the treatments the germination rate was more than 96%.2.Optimization of water content of solid substrateBased on the results of the above mentioned experiments,the ratio of rice,wheat bran,rice husk and wheat straw was set as 50%:40%:5%:5%,and distilled water was added in the substrate to 20%,30%,40%,50%and 60%.After fermentation,the sporulation yield of Bb06strain was determined.The results showed that the sporulation quantity was the highest when the water content was 40%.3.Optimization of fermentation temperatureAccording to the results of above mentioned experiments,the fermentation substrate consisted of rice,wheat bran,rice husk and wheat straw at a ratio of 50%,40%,5%and 5%was used,and the water content was set as 40%.Different temperatures were set as 21℃,23℃,25℃,27℃and 29℃using a incubator,and sporulation was measured after fermentation.The results showed that the maximum sporulation was achieved when the temperature was set at 25℃.4.Optimization of fermentation time,inoculation quantity and p HThe optimum fermentation time,inoculation quantity and pH of Bb06 were studied by setting different parameters under the conditions of 50%∶40%∶5%∶5%ratio of rice,wheat bran,rice husk and wheat straw,and water content of substrate at 25℃.The results showed that when the inoculation quantity of fermentation liquid was 15%,the p H was 6,and fermentation time was 8 days,the number of spore yield was the highest.5.Evaluaiton of virulence and control efficacy of fermented Bb06 productsThe spores of Bb06 was obtained by using the above optimized fermentation parameters,and the virulence to G.orientalis was determined.The spores growed on SDAY medium were used as controls.The results showed that there was no significant difference in the insect death rate caused by fermented spores and SDAY-produced spores.Furthermore,simulated field efficacy of fermented spores and spores produced from SDAY medium was determined.The results showed that the correction control efficacies of fermented spores were 47.78%and 75.56%,respectively,at concentrations of 1.0×10~7and 1.0×10~8spore/m L.These efficacies showed no significant difference to those of control group(SDAY-produced spores).Taken together,the findings indicated that the parameters optimized in this study coulb be used in the mass production of Bb06 strain. |
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