生防细菌ML-3抗菌蛋白发酵条件的优化及其防治应用  

Optimization of Fermentation Conditions of Biocontrol Bacterium ML-3 Antifungal Protein and Its Application

作　　者：李静舒[1] 赵佳[2] LI Jing-shu;ZHAO Jia(Shanxi Radio and TV University,Taiyuan 030027;Biotechnology Research Center,Shanxi Academy of Agriculture Science,Taiyuan 030031)

机构地区：[1]山西广播电视大学,太原030027 [2]山西省农业科学院生物技术研究中心,太原030031

出　　处：《生物技术通报》2020年第6期83-92,共10页Biotechnology Bulletin

基　　金：山西省面上青年基金项目(201701D221169)。

摘　　要：明确菌株ML-3抗菌蛋白对马铃薯早疫病的防效效果,为马铃薯早疫病生物防治提供参考依据。本研究采用硫酸铵分级沉淀、琼脂扩散法、正交试验、田间试验以及16S rDNA系统进化分析相结合的方法对菌株ML-3产抗菌蛋白的较优碳源、氮源、发酵条件、最佳硫酸铵沉淀浓度、抑菌稳定性、马铃薯早疫病田间防效和分类地位进行了研究。结果表明:该菌株产生的抗菌蛋白最佳发酵液配方为2%马铃薯、1%蛋白胨和0.5%NaCl,最佳发酵条件为:初始pH值7.0、温度30℃、装液量400 mL/500 mL、接种量5 mL、发酵48 h;抗菌蛋白在温度≤60℃、pH值5-8和紫外照射36 h内具有良好稳定性,而且对K+、Fe^2+、Zn^2+、Na^+稳定性较好;马铃薯早疫病防治的田间用药浓度为≥10 mg/L;使用16S rDNA序列可将该菌株鉴定为枯草芽孢杆菌沙漠亚种(Bacillus subtilis subsp.inaquosorum)。说明菌株ML-3抗菌蛋白在防治马铃薯早疫病和生物农药开发中应用潜力明显。In order to clarify the control effect of antifungal protein of strain ML-3 on potato early blight and to provide reference for biological control of potato early blight,the methods of ammonium sulfate fractionation,agar diffusion,orthogonal test,field test and 16S rDNA sequence analysis were used to study the optimal carbon source,nitrogen source,fermentation conditions,optimal ammonium sulfate precipitation concentration,bacteriostatic stability,field control effect and taxonomic status of strain ML-3.The results showed that the optimal composition of fermentation broth was 2%potato,1%peptone and 0.5%NaCl.The optimal fermentation conditions were initial pH 7.0,temperature 30℃,and fermentation liquid 400 mL in 500 mL Erlenmeyer flasks,inoculation volume 5 mL,and fermentation 48 h.In addition,the antifungal protein presented fine stability,when temperature≤60℃,pH value from 5 to 8,and UV irradiation 36 h.The antifungal protein also had favorable stability to K^+,Fe^2+,Zn^2+,and Na+.Besides,the concentration of antifungal protein should be≥10 mg/L when it was used to control potato early blight in field.Strain ML-3 was identified as Bacillus subtilis subsp.inaquosorum based on 16S rRNA sequence analysis.All results indicate that the antifungal protein of strain ML-3 demonstrates obvious application potential in controlling potato early blight and the development bio-pesticide.

关 键 词：马铃薯早疫病 抗菌蛋白 发酵优化 生防细菌ML-3 田间防效 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治] S476[农业科学—植物保护]