一株生姜青枯病拮抗菌的筛选、鉴定及发酵条件优化

Screening, identification and fermentation condition optimization of biocontrol bacteria YA-1 against ginger bacterial wilt

【目的】筛选对生姜青枯病菌有较强拮抗活性的细菌菌株,鉴定并优化拮抗菌株的发酵条件,为拮抗菌株的田间应用和活性产物开发提供必要的前期基础。【方法】从生姜青枯病发病较重的田块采集17份健康生姜根际根围土样,采用稀释平板分离和室内活性测定,筛选对生姜青枯病菌有较强拮抗活性的细菌菌株;对拮抗菌株进行形态学、生理生化特性和分子生物学鉴定,并通过改变碳源、氮源、发酵温度、初始p H和发酵时间的长短等外界条件对拮抗菌株进行发酵条件优化。【结果】筛选出1株对生姜青枯病菌有较强拮抗活性的细菌菌株YA-1。结合形态学、生理生化特性和16S r DNA鉴定,确定YA-1菌株为枯草芽孢杆菌(Bacillus subtilis)。结合发酵条件的改变和室内活性测定,确定YA-1菌株的最佳发酵条件为:发酵温度32℃、初始p H 7.5、发酵时间48 h,最佳培养基配方为Na Cl 1.0%、Ca CO30.2%、酵母粉0.5%、NH4H2PO40.05%、(NH4)2HPO40.05%和蛋白胨1.0%。发酵条件优化后,处理48 h,抑菌圈平均直径由优化前的23.0 mm提高到29.0 mm。【结论】YA-1菌株对生姜青枯病菌具有较高的拮抗活性,具有开发成生物农药的潜力。

[Objective]Biocontrol bacteria YA-1 with strong antagonism against ginger bacterial wilt was screened and the fermentation conditions were optimized to provide necessary preliminary basis for strain application in field and product development. 【Method】Seventeen healthy ginger rhizosphere soil samples were collected from fields, which were severely hit by ginger bacterial wilt. Dilute plate separation and indoor bioassay were used to screen active strains. Morphological, physiological and biochemical characteristics and bacterial 16 s r RNA gene characteristics were analyzed. At the same time, for the improvement of inhibitory effect, modification of C sources, N sources, fermentation temperature, p H value and fermentation time were made. 【Result】In the bioassay, twelve strains proved to possess antagonistic effect against Ralstonia solanacearum, among which strain YA-1 had the best inhibitory effects. Strain YA-1 was identified as Bacillus subtilis according to morphological, physiological and biochemical characteristics and bacterial 16 s r RNA gene characteristic analysis. The results showed that the best conditions were obtained at fermentation temperature of 32 ℃, p H of 7.5 and fermentation time of 48 hours. And the optimal culture medium was attained when sodium chloride, calcium carbonate, yeast,diammonium phosphate, ammonium dihydrogen phosphate and peptone was 1.0%,0.2%, 0.5%, 0.05%, 0.05% and 1.0%, respectively. When the fermentation conditions were optimized, the average inhibitory zone rose from 23.0 to 29.0 mm. 【Conclusion 】Strain YA-1 displays relatively high inhibition activity to Ralstonia solanacearum and has the potential on biocontrol of ginger bacterial wilt.