一株扩展青霉拮抗菌的分离、鉴定及抑菌活性物质

Isolation, identification and characterization of an antagonistic bacterium against Penicillium expansum

摘要:

【目的】筛选有效抑制扩展青霉(Penicillium expansum)的拮抗菌，并鉴定其所产抑菌物质的主要种类及相对含量。【方法】从苹果表面分离到拮抗扩展青霉的菌株BA-16，经形态学、生理生化及16S rRNA基因序列分析对该菌进行鉴定；根据已知脂肽类抗生素合成相关基因序列设计3对特异性引物对菌株BA-16进行检测，对PCR产物克隆、测序和BLAST分析，采用酸沉淀法从菌株发酵液中制备出抑菌物质粗提液，对活性粗提物进行HPLC和MALDI-TOF-MS分析。【结果】经鉴定，BA-16被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)，所得PCR产物经测序和BLAST分析，证实BA-16带有sfp和fenB基因。HPLC和MS结果显示菌株发酵液中含有Fengycin和Surfactin两种脂肽类产物，Fengycin是拮抗扩展青霉的主要因素。【结论】本研究对于苹果采后青霉病的生物防治具有良好的应用开发前景。

Abstract:

[Objective] In order to obtain an antagonistic strain which can effectively inhibit the growth of Penicillium expansum and to identify the antifungal substances produced by this strain. [Methods] The antagonistic strain BA-16 that could inhibit Penicillium expansum growth was isolated from apple surface and then identified through phenotypic, physiological, biochemical and phylogenetic (16S rRNA gene) analyses. Three primers were designed on the basis of the relevant genes and PCR was done. After the PCR product was purified and sequenced, the obtained sequences were analyzed by BLAST. The crude anti-fungal extracts were prepared by using acidic precipitation method and the active antagonistic substances were purified and identified by HPLC and MALDI-TOF-MS analysis. [Results] The strain BA-16 was identified as Bacillus amyloliquefaciens. PCR detection and BLAST analysis revealed that strain BA-16 expressed the sfp and fenB genes. The HPLC and MS analysis results demonstrated that the fermentation broth of the strain contained fengycin and surfactin. Moreover, fengycin played an important role in inhibiting Penicillium expansum. [Conclusion] The strain BA-16 will be a potential biological control agent against blue mold decay in apples.