拮抗灰霉菌的海洋细菌甲基营养型芽胞杆菌的筛选、鉴定及其抑菌活性物质的研究

采用Zo Bell’s 2216E培养基从65份渤海海水和底泥样品中分离到海洋细菌464株,通过平板对峙法获得了对灰霉菌具有较强拮抗作用的海洋细菌47株,其中菌株BH21的抑菌作用最强,抑菌带宽度为(11.8±0.3)mm;采用形态学、生理生化和分子生物学相结合的方法,将菌株BH21鉴定为甲基营养型芽胞杆菌Bacillus methylotrophicus,该菌具有较强耐盐、耐甲醇能力;通过排油圈法确定菌株BH21能够产生具有表面活性的代谢产物,随后以脂肽类物质相关基因的特异序列为引物进行菌株BH21抑菌活性成分相关基因的扩增,确定该菌株中含有itu A、itu B、itu C、itu D、fen D、srf AB和ynd J等7种脂肽物质的合成基因;利用盐酸沉淀和甲醇抽提法获得了BH21发酵液中脂肽粗提物,通过牛津杯法确定该脂肽粗提物具有明显抑制灰霉菌菌丝生长的能力,抑制率达到73.9%。

Screening,Identification of Antagonistic Ocean Bacteria Bacillus methylotrophicus and Its Antimicrobial Substances to Botrytis cinerea pres.

464 strains of ocean bacteria were isolated from 65 samples of sea water and sediment from Bohai using ZoBell's 2216E medium.Through plate confrontation method,47 strains of marine bacteria with strong antagonism against Botrytis cinerea were obtained.Strain BH21 with high salt and methanol tolerance had the strongest antifungal effect and the inhibition zone diameter was 11.8 mm±0.3 mm.Strain BH21 was identified as Bacillus methylotrophicus by morphological,physiological,biochemical and molecular methods.Using the specific primers of lipopeptide gene,the related genes in strain BH21 were amplified and 7 lipopeptide antibiotics synthetic genes including ItuA,ituB,ituC,ituD,fend,srfAB andyndJgenes were identified in the strain.Crude extract of lipopeptide was obtained by the hydrochloric acid precipitation and methanol extraction.Oxford cup analysis demonstrated that the crude extract of lipopeptide could inhibit the mycelial growth ofB.cinerea,with the inhibition rate of 73.9％.