具有杀线虫活性的郭霍氏芽孢杆菌发酵条件优化及稳定性评价

Optimization of fermentation conditions and evaluation of stability of Bacillus kochii

摘要:

【背景】芽孢杆菌是一类常见的生防菌，在植物病害防治中展现出巨大潜力。【目的】对一株具有杀线虫活性的郭霍氏芽孢杆菌(Bacillus kochii) DDWB进行发酵条件优化和稳定性评价，为菌株的开发提供理论支持。【方法】以发酵液上清杀线虫率及细菌发酵液OD₆₀₀值为指标，通过单因素法与正交试验设计，对菌株的培养基与发酵参数进行优化；同时对菌株发酵液酸碱、温度、紫外、遗传及储存稳定性进行评价。【结果】DDWB菌株培养基优化后为：蔗糖2%，酵母提取物1%，氯化钾2%；优化后初始pH值为8.0，装液量为150 mL/250 mL锥形瓶，发酵时间为48 h，接种量为8%，转速为160 r/min，发酵温度为31℃；稳定性测定结果显示发酵液对酸碱敏感，紫外光照射4 h后活性物质发生降解，但发酵液对温度不敏感且杀线虫活性相关基因可稳定遗传。【结论】本研究优化了DDWB菌株的发酵条件，并对发酵液中活性物质的稳定性进行了多因素评价，使得菌株可以快速扩繁并长期稳定保持对根结线虫的抑制活性，为进一步评价菌株田间防效和研究生防机制奠定了基础。

Abstract:

[Background] As common biocontrol bacteria, Bacillus exhibit great potential in the prevention and control of plant diseases. [Objective] In this study, we optimized the fermentation conditions and evaluated the stability of Bacillus kochii DDWB with nematicidal activity, aiming to provide theoretical support for the development and application of the strain. [Methods] With the nematicidal activity of supernatant and the OD₆₀₀ of fermentation broth as indexes, we optimized the culture medium and fermentation parameters of DDWB strain via single factor tests and orthogonal design. Furthermore, we analyzed nematicidal activity about pH, temperature, UV irradiation, heredity, and storage stability of the fermentation broth. [Results] The optimized culture medium for DDWB strain contained 2% sucrose, 1% yeast extract, and 2% potassium chloride. The optimized fermentation conditions were initial pH 8.0, the filling volume of 150 mL/250 mL conical flask), the fermentation time of 48 h, the inoculum volume of 8%, the rotation speed of 160 r/min, and the fermentation temperature of 31 ℃. The fermentation broth was sensitive to acid and alkali, and its nematicidal activity degraded after 4 h of UV irradiation. However, the fermentation broth was not sensitive to temperature, and the genes related to nematicidal activity could be stably inherited. [Conclusion] We optimized the fermentation conditions and evaluated the stability of active substances in the fermentation broth of DDWB strain, aiming to make the strain rapidly propagate and maintain its long-term and stable nematicidal activity against root-node nematodes. The findings laid a foundation for evaluating the field efficacy and biocontrol mechanism of DDWB strain.