内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析

BEB17是本实验室从健康香蕉植株根部中分离得到的一株具有较强抑制真菌活性的内生解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。在本研究中,我们通过PCR检测多个脂肽类和聚酮类化合物的合成相关基因,并结合基质协助激光解吸附离子化-飞行时间质谱(MALDI-TOF-MS)和液相色谱-质谱联用(LC-MS)分析,发现该菌株能产生表面活性素Surfactin、泛革素Fengycin和伊枯草菌素Iturin三类脂肽类化合物以及Bacillibactin、Difficidin和Bacillaene三类聚酮类化合物。通过比较两种发酵培养基(M9和Landy培养基)和两种提取方法(酸沉淀法和大孔树脂抽提法),结果发现酸沉淀法提取较为高效,Landy培养基更易于BEB17脂肽类和聚酮类化合物的发酵。活性检测表明,两种提取方法提取的粗提物对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense race 4,Foc4)都具有较好的抑制作用,其中大孔树脂抽提法提取的物质抑制效果最好,当其浓度为300μg·m L-1时抑制率可达80.75%,说明该方法制备的粗提物的活性物质含量相对较高。进一步研究表明,该提取物能引起Foc4菌丝膨大和畸形,并抑制其孢子萌发。分析表明,该提取物对Foc4细胞膜具有破坏作用。

Antibacterial activity analysis of lipopeptide and polyketide compounds produced by endophytic bacteria Bacillus amyloliquefaciens BEB17

The strain BEB17 is an endophytic Bacillus amyloliquefaciens isolated from the roots of healthy banana plants in our laboratory, which can produce volatile gases with inhibitory activity against many pathogenic fungi. In this study, we found this strain contained the genes encoding the synthesis of multiple lipopeptide and polyketide compounds via PCR amplification. In agreement, this strain did produce several known bioactive compounds, including Surfactin, Sufengycin, Iturin, Bacillibactin, Difficidin and Bacillaene, identified by using MALDI-TOF-MS and LC-MS. By comparing the two fermentation medium (M9 and Landy medium) and two extraction methods (acid precipitation and macroporous resin extraction), we show that the acid precipitation method is more efficient, and Landy medium is more prone for fermentation of lipopeptide and polyketide by the strain BEB17. The activity tests showed that the crude extracts from two extraction methods described above had a good inhibitory effect on banana fusarium wilt (Fusarium oxysporum f. sp. cubense race 4, Foc4). Furthermore, the crude extracts from macroporous resin extraction method display the best inhibitory effect compared to other treatments. When the concentration is 300 μg·mL^-1, the inhibition rate can reach 80.75%. This shows that this method of crude extract prepared by the active substance content is relatively higher. The further research shows that it can cause the mycelium of Foc4 swelling and deformity, and inhibit its spore germination. In addition, the Foc4 cell membrane were damaged by trypan blue staining.