烟草炭疽病拮抗菌分离及其发酵条件优化

Isolation of Antagonistic Bacteria against Tobacco Anthracnose and Optimization of Fermentation Conditions

为获得烟草炭疽病拮抗菌及其最适发酵条件,对烟草根际土壤和根茎叶中细菌进行分离,结合平板对峙法及液体发酵法筛选烟草炭疽病拮抗菌并鉴定,采用液体摇瓶发酵单因素试验和基于Box-Behnken设计的响应面法优化菌株发酵条件。结果表明:烟株根际土壤和根茎叶中共分离出31株对烟草炭疽病有拮抗作用的菌株,其中抑菌率>30%的菌株10株,抑菌率最高的菌株为C-4,抑菌率达72.14%,经16S rRNA序列分析初步鉴定其来自芽孢杆菌属。响应面法优化的Bacillus C-4最适发酵条件为接种量4%,初始pH 7.1,培养温度25.5℃,装液量84.5 mL,摇床转速180 r/min,培养时间48 h;经验证,在最适条件下,发酵液OD600值为1.859,为优化前的1.62倍,抑菌率提高了8.4%。该研究为防治烟草炭疽病微生物菌剂的研制提供了备选菌种资源,同时也证明了响应面法用于优化烟草病害拮抗菌发酵条件的可行性。

In order to obtain the antagonistic bacteria and the optimum fermentation conditions of tobacco anthracnose,the bacteria in the rhizospheric soil,roots,stems,and leaves of tobacco were isolated.Accordingly,the antagonistic bacteria of tobacco anthracnose were screened by the method of solid and liquid medium and their antagonistic activity was determined,as well as identified by 16 S rRNA analysis.The single factor experiment of shake flask fermentation and the response surface method based on Box-Behnken design were used to optimize the fermentation conditions of the best antagonistic bacteria.The results showed that there were 31 strains with antagonistic effects against tobacco anthracnose,and 10 strains with inhibition rate greater than 30%.The strain with the highest inhibition rate was C-4,and the inhibition rate was 72.14%.Sequence analysis initially identified it belonged to the genus Bacillus.The optimal fermentation conditions for Bacillus C-4 were as follows:inoculum volume 4%,initial pH 7.1,culture temperature 25.5℃,liquid volume 84.5 mL,shaker speed 180 r/min,and culture time 48 h.It was verified that under the optimal conditions,the OD600 value of the fermentation broth was 1.859,which was 1.62 times of that before optimization,and the inhibition rate was increased by 8.4%.This study provides an available strain resource for the development of microbial inoculant for the control of tobacco anthracnose.It is effective to optimize fermentation conditions of antagonistic bacteria of tobacco by using the response surface method.