1株广谱拮抗植物病原真菌的芽孢杆菌HNA3的鉴定及其活性成分分析

Identification of Bacillus strain HNA3 and its bioactive compounds with a broad spectrum of antifungal activity

采用形态观察、16S rDNA序列和部分特异基因排列分析鉴定从植物根际土壤中分离的1株根际细菌HNA3;采用平板对峙试验和抑菌率计算方法测定其对供试植物病原真菌的拮抗谱和拮抗能力;通过摇瓶发酵和酸沉淀方法研究相关抗菌活性物质的产生条件及其分离方法;采用表面活性检测和有关功能编码基因的序列分析初步鉴定抗菌活性物质成分的性质。结果表明:HNA3菌株为1种解淀粉芽孢杆菌Bacillus amyloliquefa-ciens;BPY(牛肉膏蛋白胨酵母粉)培养基是适合HNA3生长和产生相关抗菌活性物质的适宜培养基;活性物质存在于发酵除菌液中,可通过酸沉淀方法分离获得。活性物质具有较高的稳定性和抗逆性,属于脂肽类物质。本研究结果表明HNA3具有较广谱的拮抗植物病原真菌的能力,具备研发成为一种表面喷施型微生物杀菌剂的特性和应用前景。

This study aims to identify a Bacillus strain HNA3 isolated from the rhizosphere soil and characterize its broad antagonistic capacity towards various plant pathogenic fungi, and to optimize the conditions for the fermentation and the bioactive component separation, therefore, to establish a solid ba- sis for developing the HNA3 strain into a promising bioactive agent with a broad spectrum of antifungal activity. In the present work, the classification status of the HNA3 was identified through morphological observation, 16S rDNA sequence and the order of species-unique genes arrangement analysis. The spec- trum and capacity of the HNA3＇s antifungal activity was identified by the plate dual-culture antagonistic experiment and the antimicrobial inhibition rate, respectively. The flask shaking fermentation and acid precipitation method were used to determine the optimal conditions for the HNA3＇s antimicrobial compound production and separation. By using the methods of surface activity examination and the analysis on the arrangement of candidate function genes and sequences,the basic properties of the bioactive anti- microbial compound were determined. The results showed that the HNA3 was identified as Bacillus arnyloliquefaciens, and the BPY was the appropriate medium for HNA3＇s culture growth, as well as for the production of bioactive antifungal compound. It was also found that the bioactive antifungal com- pound existed in the HNA3＇s culture filtrates and it could be separated through acid precipitation. Fur- ther more,the bioactive compound could be ascribed to the category of lipopeptide and possessed the properties of high stability and stress resistance. In conclusion, the strain HNA3 demonstrates a broad spectrum of antifungal activity,and indicates a promising application prospective by developing it into a type of spraying,agricultural microbial fungicides in the future.