Modified Techniques For Mass Production, Formulation And Storage Of Beauveria Bassiana (Deuteromycotina: Hyphomycetes) Conidia And Features Of Its Amylases

The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, is a classic fungal agent highly potential for use in microbial control of insect pests and has been developed into a variety of commercial formulations called mycoinsecticides in the world. However, technical problems involved in mass production, formulation, and normal storage of B. bassiana conidia remain obstacles to intensive development and practical use of B. bassiana-based mycoinsecticides for pest control. Based on previous work on an aphid-derived isolate (SG8702) of B. bassiana in our laboratory, the present study was attempted to search for technical approaches to improving mass production of aerial conidia, optimizing components of emulsifiable formulation, and prolonging shelf life under normal storage conditions. Aspects on production of extracellular amylases by the fungal isolate were first assayed as well.A modified technology for production of high-quality conidia powder on low-quality rice substrate. Low-quality rice used as solid substrate was tested to produce aerial conidia of B. bassiana SG8702 in 15-cm dishes or large trays. The thickness of rice layer piled in dishes, and the source and concentration of supplemented nitrogen were found affecting conidial yield, viability, and water content. The thinner the rice layer the lower the water content in the conidia powder produced. Adding a very small amount of peptone, KNO3 or NH4NO3 to the rice substrate significantly increased conidial yield and viability, but there was little difference among the nitrogen sources tested. A supplement of 0.1% KNO3 (w/w) was found most favorable to conidial production, resulting in a conidial yield of 57.3-67.9 mg per gram of rice in repeated solid fermentation. In an expanded experiment including the use of 6.5 kg rice, each gram of rice yielded a maximal conidial powder of 55.5 mg, equivalent to 5.83×109 conidia. The powdery product was in high quality with pureness of 1.48 ×1011 conidia/g, viability of 98.72%, and water content of 5.25%. The results indicate that the low-quality rice can be utilized for efficient production of high-quality B. bassiana conidia for microbial control of insect pests.Production and activity of extracellular amylases in liquid culture. Cuticle-degrading enzymes of B. bassiana such as proteases and chitinases have been well investigated but little attention has been paid to production of its amylases. A liquid medium including 0.3% soluble starch, 0.5% glucose, 0.5% peptone, and 0.5% yeast extract (w/v) was optimal for production of an amylase in liquid culture of B. bassiana SG8702 among 13 media generated from central composite design. Shaking the liquid culture containing 106 conidia/mL for 3.5 d at the regime of 25 ?1@ and 120 r/min resulted in mycelial biomass of 16.7 mg/mL and amylase yield of 527.1 units/mg. An initial range of pH 4.0-6.0 in the liquid culture was found being most favorable to amylase production with the yield of 1315.8-1439.2 units/mg. The activity of B. bassiana amylase was highest at 40@ and pH 4.0. Its stability to pH apparently varied with temperature and time length for reaction. The amylase was considerably stable at pH 3.0-8.0 for 20 min at 20@ or at pH 4.0-6.0 for 1 h at 37@. The activity of the amylase remained >90% after 20-min maintenance at 40@, but lost 52% and 91% after the same period of maintenance at 50@ and 60@, respectively. Moreover, the presence of Ca2+ had little effect on the activity of the amylase whereas other cations including Cu2+, Mn2+, Na+, Hg2+, Fe2+ and Mg2+ could be significantly inhibitory to its activity at different levels. This is the first report on the production and features of amylase produced by B. bassiana.Optimization of components for emulsifiable formulatons. Primary components of an emulsifiable formulation of B. bassiana SG8702 conidia developed previously were further optimized. As a formulation carrier, light liquid paraffin was found affecting little the viability of suspended conidia during long-term storage at ambient te...