牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究

Isolation, indentification and inhibitory activity of lipopeptides of endophytic bacteria from the root of Paeonia suffruticosa

摘要:

【目的】从牡丹(Paeonia suffruticosa Andr.)根部组织中分离鉴定内生细菌，测定拮抗菌株脂肽类活性物质的体外抑菌活性。【方法】采用平板对峙法筛选对牡丹灰霉病菌(Botrytis paeoniae Oadem)、牡丹炭疽病菌(Gloeosporium sp.)、牡丹黑斑病菌(Altenaria sp.)、牡丹黄斑病菌(Phyllosticta commonsii)有拮抗作用的内生细菌。基于形态特征、生理生化特性和16S rRNA基因序列同源性鉴定拮抗菌株。根据脂肽类抗菌物质合成相关基因序列对拮抗菌株进行基因扩增检测，采用酸沉淀法提取拮抗菌株的脂肽类物质，平板对峙法测定脂肽类物质的体外抑菌活性。【结果】从牡丹根部组织中共分离获得62株内生细菌，其中菌株Md31和Md33对4种病原菌均有较明显的抑制作用。Md31和Md33被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过对菌株Md31和Md33进行5个脂肽类合成功能基因bmyB、fenD、ituC、srfAA和srfAB的检测，序列同源性分析，表明两个菌株具有合成脂肽类物质的能力。菌株Md31和Md33的脂肽类粗提物对所测试的牡丹病原真菌均具有不同程度的抑制作用。【结论】获得了2株对牡丹病原菌有良好抑制效果的解淀粉芽孢杆菌Md31和Md33，2个菌株的脂肽类粗提物也具有较强的体外抑菌活性，该研究为牡丹内生细菌的进一步开发应用奠定了基础。

Abstract:

[Objective] To isolate and identify endophytic bacteria from the roots of Paeonia suffruticosa and evaluate antifungal activity of secondary metabolites of antagonistic strains in vitro. [Methods] The endophytic bacteria were screened against Botrytis paeoniae, Gloeosporium sp., Altenaria sp., Phyllosticta commonsii through the method of dual culture. The strains were indentified based on characteristics in morphology, physiology, biochemistry and phylogenetic analysis of 16S rRNA gene sequences. The lipopeptide biosynthetic genes fragments of antagonistic strains were amplified by polymerase chain reaction. The crude lipopeptides of strains were obtained through acid precipitate method. The inhibition ability of lipopeptides produced by antagonistic strains against pathogenic fungi was evaluated in vitro. [Results] The 62 endophytic bacterial isolates were isolated from the roots of Paeonia suffruticosa. The isolates Md31 and Md33 showed inhibitory activity against four pathogens. Md31 and Md33 were identified as Bacillus amyloliquefaciens. The genes fragments of bmyB, fenD, ituC, srfAA and srfAB were obtained in genomic DNA of the two strains. The crude lipopeptides prouduced by Md31 and Md33 were isolated from culture supernatants and exhibited inhibitory activity on mycelium growth of peony pathogens. [Conclusion] Two antagonistic B. amyloliquefaciens strains Md31 and Md33 were obtained, and their crude lipopeptides showed inhibitory activity against four peony pathogens in vitro, which will been provided a basis for the further development and application of peony endophytes.