解淀粉芽胞杆菌MH71抗菌物质理化特性及对番茄灰霉病菌的抑菌活性

Physical and Chemical Properties of Antimicrobial from Bacillus amyloliquefaciens Strain MH71 and Its Inhibition Activities against Botrytis cinerea

解淀粉芽胞杆菌Bacillus amyloliquefaciens MH71是一株高效广谱拮抗活性菌株,明确该菌株发酵液中活性物质的理化特性和抑菌活性,为解淀粉芽胞杆菌生物农药开发应用奠定坚实的基础。本研究在对菌株MH71抑菌活性物质理化特性研究的基础上,以番茄灰霉病菌为供试致病菌,探究了抗菌活性物质对番茄灰霉病菌的抑菌作用;采用酸沉淀和硫酸铵沉淀分别获得脂肽类和蛋白类活性物质,SDS–PAGE电泳和HPLC分别对活性物质进行检测。该菌活性物质具有较强的耐热性和p H适应性,100℃处理60 min和121℃处理30 min后抑菌活性分别为原有活性的88.98%和79.60%,在pH 3~11时均有抑菌活性,且对紫外线、蛋白酶K、胃蛋白酶和胰蛋白酶不敏感。该抗菌活性物质对番茄灰霉病菌菌丝的毒力测定结果表明,不同浓度发酵液处理的番茄灰霉病菌菌丝呈现出较多局部膨大或溢缩等异常菌丝。其发酵液经酸沉淀法得到抗菌脂肽类活性物质,经硫酸铵沉淀法得到抗菌蛋白,脂肽类的抑菌活性高于抗菌蛋白类物质,SDS–PAGE电泳检测,得到约40 k D的蛋白条带,HPLC检测表明该抗菌物质中有surfactin存在。

MH71 is a strain with a broad-spectrum of antagonistic activities, and this research was conducted to characterize physical and chemical properties and antimicrobial activity of the active substances produced by the strain. The stability of the antimicrobial substances were studied, and their activities against Botrytis cinerea were tested. By acid precipitation and ammonium sulfate precipitation, lipopeptide and protein active substances were obtained, and these two active substances were then detected by HPLC and SDS-PAGE. Incubated at 100 ℃ for 60 min and 121 ℃ 30 rain, the relative antimicrobial activity of the substances is 88.98% and 79.60%, respectively, and incubated at pH 3-11, the relative antimicrobial activity still remained very high, and moreover, the acitvties were insensitive to UV, protease K, pepsin and trypsin. These results suggested that the antimicrobial substances produced by MH71 had a strong heat resistance and pH adaptability. When the mycelium ofB. cinerea was treated by different concentrations of the MH71 fermentation broth, lots of abnormal hyphae were observed such as swelling or overflow. Compared with the antimicrobial peptides, the lipopeptides showed stronger activities to inhibit B. cinerea. One protein band with a molecular of 40 kD was detected by SDS-PAGE, and HPLC test showed that surfactin existed in the antimicrobial substances produced by MH71.