枯草芽孢杆菌B006对黄瓜枯萎病菌和辣椒疫霉病菌的抑制作用及其抗菌组分分析

Antifungal Activities and Principal Component Analysis of Bacillus subtilis B006 against Fusarium oxysporum f. sp. cucumerinum and Phytophthora capsici

枯草芽孢杆菌菌株B006发酵液的甲醇提取物对黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum菌丝的抑制作用大于对辣椒疫霉病菌Phytophthora capsici菌丝的抑制作用;但对P. capsici游动孢子萌发的抑制作用明显大于对黄瓜枯萎病菌孢子萌发的抑制作用,提取物原液在5 min内可使辣椒疫霉的游动孢子全部崩解。提取物经60、90和120℃加热处理10 min后仍保持抑菌活性,其盐酸水解液在TLC层析板显现橙红色斑点,说明提取物具有环肽结构。HPLC-ESI-MS分析可得到m/z为995、1009、1023、1037、1051和1436、1450、1478和1492的质谱峰,与抗菌物质surfactin和fengycin的分子量大小一致。以终浓度为106 cfu·g-1的接种量将B006菌剂施入无菌育苗基质中,在黄瓜和辣椒4～6片叶期时用固相萃取法提取根际的抗菌物质,并进行HPLC-ESI-MS分析,可从根际检测到surfactin和fengycin,但未从对照处理根上检测到脂肽类抗生素。本研究对理解枯草芽孢杆菌在田间状况下的防病机理有一定意义。

Methanol extract from cell-free supernatants of Bacillus subtilis B006 suppressed hyphal growth of Fusarium oxysporunm f.sp.cucumerinum more greatly than that of Phytophthora capsici,while suppressed spore germination of F.oxysporunm f.sp.cucumerimerinum less than that of zoospores of P.capsici.The inhibition was not changed when the extract was heated at 60,90,or 120 ℃ for 10 minutes.TLC analysis of the HCL hydrolysis solution of the extract gave rise to orange spots,indicating that the extract belongs to cyclopeptide.HPLC-ESI-MS analysis showed that the extract contained surfactin and fengycin with m/z at 995,1009,1023,1037,1051 and 1436,1450,1478,1492.When cucumber and pepper were planted in sterile nursery substrate inoculated with B006 at 10^6 cfu· g^-1,surfactin and fengycin could also be detected with HPLC-ESI-MS from SPE-extracted lipopeptides of roots of the plants at 4 to 6 leaf stage,while not from that of uninoculated plant roots.This research indicated that B.subtilis B006 produced lipopeptides,an important group of antibiotics,in plant rhizosphere,suggesting its possible role in biological control of soilborne plant disease in vivo.