响应面法优化爪哇虫草菌的培养条件及其对斜纹夜蛾的毒力和保护酶活性影响

Response surface methodology-based optimization of culture conditions of Cordyceps javanica with effects on virulence and protective enzyme activity of Spodoptera litura

摘要:

【目的】优化爪哇虫草菌Bd01的固态发酵培养条件，测定分生孢子对斜纹夜蛾3龄幼虫的毒力，研究被爪哇虫草菌侵染后寄主体内的保护酶活性变化。【方法】采用单因素试验确定爪哇虫草菌Bd01最佳的固态培养基及培养条件，利用Box-Behnken响应面法优化发酵参数，采用浸渍法测定分生孢子对斜纹夜蛾3龄幼虫的毒力，同时利用分光光度计法测定斜纹夜蛾3龄幼虫体内酶活性变化。【结果】以产孢量为指标，通过响应曲面法优化的爪哇虫草菌Bd01最佳产孢条件为：培养基营养成分含量为30.24 g/L，pH值为7.55，光照时长为12.06 h，在该条件下，培养基的产孢量为2.78×10⁸孢子/mL。浓度为1×10⁸孢子/mL的爪哇虫草菌孢子液对斜纹夜蛾3龄幼虫具有一定毒力，处理7 d时致死中浓度(LT₅₀)为3.11 d，致死中时(LC₅₀)为4.68×10⁵孢子/mL，校正死亡率为88.68%。处理后未死亡的斜纹夜蛾幼虫体内保护酶活性与对照组相比发生显著变化。【结论】优化后的培养基能够显著增加爪哇虫草菌的产孢量；爪哇虫草菌对斜纹夜蛾幼虫的致死率和致死效率与浓度呈正相关；斜纹夜蛾幼虫体内的保护酶可能在抵抗爪哇虫草菌侵染的过程中起关键作用。

关键词:爪哇虫草菌;斜纹夜蛾幼虫;培养条件;毒力;保护酶活性

Abstract:

[Objective] To optimize the soliD-state fermentation conditions of Cordyceps javanica Bd01, determine the virulence of the conidia to the 3rd-instar larvae of Spodoptera litura, and study the changes of protective enzyme activities in S. litura after infection by C. javanica. [Methods] The optimum soliD-state medium and culture conditions of C. javanica Bd01 were first determined by single factor experiments and then optimized by Box-Behnken response surface method. The virulence of the conidia to the 3rd-instar larvae of S. litura was determined by the immersion method, and the changes of enzyme activities in the larvae were determined by a spectrophotometer. [Results] With the spore yield as an indicator, the sporulation conditions of C. javanica were optimized as medium nutrient content of 30.24 g/L, pH 7.55, and light duration of 12.06 h. Under these conditions, the spore yield reached 2.78×10⁸ spores/mL. The conidial suspension of C. javanica at the concentration of 1×10⁸ spores/mL was virulent to the 3rd-instar larvae of S. litura. After seven days of treatment, the LT₅₀, LC₅₀, and corrected mortality rate were 3.11 d, 4.68×10⁵ spores/mL, and 88.68%, respectively. The activities of protective enzymes in the survived larvae of S. litura after treatment were significantly different from those in the control group. [Conclusion] The optimized culture medium can significantly increase the spore production of C. javanica. The lethal rate and lethal efficiency of C. javanica to S. litura larvae were dependent on the concentration. The protective enzymes in S. litura larvae may play a key role in the defense against the infection of C. javanica.

Key words:Cordyceps javanica;Spodoptera litura larvae;culture conditions;virulence;protective enzyme activity