球孢白僵菌耐热突变菌株筛选及发酵条件优化

Screening for thermotolerant mutant strains of Beauveria bassiana and optimization of fermentation conditions

摘要:

背景 球孢白僵菌(Beauveria bassiana)是一种安全、有效的昆虫病原真菌，在人工养殖生产白僵蚕过程中具有重要作用。高温环境会显著降低球孢白僵菌活性，导致白僵蚕产量下降。目的 筛选获得球孢白僵菌高耐热性突变菌株并优化其发酵条件，为高温环境下农户养殖及工厂化生产白僵蚕提供优质高耐热性球孢白僵菌菌株。方法 活化球孢白僵菌菌株并制备液体培养基及孢子悬浮液，利用PCR仪梯度高温热激处理孢悬液，确定最佳热激温度；最佳热激温度下处理孢悬液并培养获得单菌落，将其扩大培养制备孢悬液并再次进行最佳热激温度处理与培养，筛选获得高耐热性球孢白僵菌菌株；对菌株耐热性进行验证，采用Box-Behnken响应面分析法优化球孢白僵菌液体发酵培养条件，并开展大田试验。结果 最佳热激温度为55.2 ℃，获得的高耐热性突变菌株经鉴定为球孢白僵菌(Beauveria bassiana)，命名为Beauveria bassiana B1-3。经耐热性验证，球孢白僵菌B1-3可在33 ℃侵染家蚕，僵化率高达92.93%，最佳发酵条件为温度33.56 ℃、pH 6.74、装液量23.17%，发酵生物量12.75 g/L，产孢量为原始菌株的1.52倍。结论 成功筛选获得高耐热性球孢白僵菌菌株并对其发酵条件进行优化，为高温环境下农户养殖和工厂化生产白僵蚕提供试验基础。

Abstract:

Background Beauveria bassiana functions as a safe and effective entomopathogenic fungus, playing a pivotal role in the artificial production of Bombyx batryticatus. However, elevated temperatures markedly suppress the fungal activity, leading to a substantial decline in the yield of B. batryticatus.Objective To isolate thermotolerant mutant strains of B. bassiana and optimize their fermentation conditions, thereby developing high-quality thermotolerant strains for small-scale and industrial production of B. batryticatus under high-temperature conditions.Methods We activated the wild-type strain of B. bassiana to prepare a conidial suspension and used a PCR-based gradient heat shock system to treat the suspension for determining the critical thermal tolerance threshold (55.2 ℃). Successive cycles of heat shock and cultivation were conducted to isolate stable mutants. The thermal tolerance and pathogenicity of the selected strain were validated. Subsequently, the fermentation conditions were optimized via the Box-Behnken response surface methodology for field application.Results A highly thermotolerant mutant strain, designated B. bassiana B1-3, was successfully isolated. It demonstrated exceptional thermal adaptability, infecting silkworms at 33 ℃ and causing a mortality rate of 92.93%. The fermentation under optimized conditions (33.56 ℃, pH 6.74, and liquid load of 23.17%) resulted in the biomass of 12.75 g/L and a sporulation capacity 1.52 times that of the wild-type strain.Conclusion We isolate a thermotolerant B. bassiana strain and optimize its fermentation conditions, providing an effective solution for small-scale and industrial production of B. batryticatus under high-temperature stress.