淡色生赤壳菌Bo-1菌株拮抗物质的分离纯化、解析及活性分析

Isolation,identification and activity analysis of antimicrobial compound from Bionectria ochroleuca strain Bo-1

从浙江省金华市郊水稻内生菌中筛选到1株拮抗水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)P6生理小种的淡色生赤壳菌Bionectria ochroleuca Bo-1菌株。该菌株的菌体和发酵液对Xoo P6生理小种均表现出较强的拮抗能力。以P6生理小种作为指示菌进行活性跟踪,通过分步萃取、硅胶柱层析、高效液相色谱等,分离纯化获得拮抗活性物质结晶;对所获得的结晶物进行核磁共振氢谱分析和质谱分析,通过解析与比对,该物质分子量为344.0835,分子式为C_(18)H_(16)O_7,鉴定为松萝酸(usnic acil)。将纯化的松萝酸以20%叶枯唑(bismerthiazol)和3%中生菌素(zhongshengmycin)为对照,采用96孔板法测定对Xoo P6生理小种的抑菌活性,最低抑制浓度为200μg/mL,表明松萝酸具有较强的抑菌活性,但较中生菌素弱,与20%叶枯唑效果相近。

A strain Bo-1 of Bionectria ochroleuca with antimicrobial activity to Xanthomonas oryzae pv. oryzae （Xoo） race P6 was screened from rice in the suburb of Jinhua. Strain Bo-1 and its ferment fluid had strong antimicrobial activity to Xoo race P6. Using race P6 as the indicator bacterium, an active monomer compound was isolated and purified by means of fractional extraction, silica gel column chromatography separation, and high performance liquid chromatography purification. Through structural analysis and comparison, the target compound was identified as usnic acid with molecular weight of 344.0835 and formula of C18H16O7 on the basis of mass spectrum and nuclear magnetic resonance analyses. Using 3% zhongshengmycin and 20% bismerthiazol as controls, the antimicrobial activity of usnic acid to Xoo race P6 was tested by 96-well plate method. The results illustrated that usnic acid showed better antibacterial activity to Xoo race P6 with MIC value of 200 μg/mL, but lower than 3% zhongshengmycin and equal to 20% bismerthiazol.