药用牡丹根部内生细菌的抑菌活性研究

Antimicrobial Activities and Mechanism of Endophytic Bacteria Isolated From Medicinal Plant Paeonia suffruticosa

以采自洛阳、铜陵和菏泽健康的"凤丹"(Paeonia ostii)根部组织为试材,采用表面消毒法进行内生细菌的分离,平板对峙法测定内生细菌对4种牡丹病原菌的拮抗作用;利用分子生物学的方法鉴定具有拮抗作用的菌株,同时进行拮抗菌株抑菌活性基因的扩增;通过酸沉淀的方法提取拮抗菌株的抑菌活性物质,测定其抑菌活性物质对牡丹灰霉病(Botrytis cinerea)的抑菌活性和抑菌机制,以期为牡丹内生细菌的进一步开发应用奠定理论基础。结果表明:从"凤丹"根部组织中共分离获得124株内生细菌,其中16株内生细菌对4种牡丹病原真菌均具有不同程度的抑菌活性,4个菌株Md1-14、Md2-17、Md2-35和Md3-14抑菌效果显著;鉴定结果表明Md1-14和Md3-14为苏云金芽胞杆菌(Bacillus thuringiensis)和假真菌样芽胞杆菌(B.pseudomycoides);Md2-17和Md2-35为贝莱斯芽胞杆菌(B.velezensis);扩增结果表明4个菌株中均具有合成抑菌活性物质的基因片段,拮抗菌株的抑菌活性物质对牡丹灰霉病菌具有显著的平板抑制作用,抑菌机制主要是导致灰霉病菌菌丝发生畸形、扭曲和局部膨大。

Endophytic bacteria strains were isolated fromPaeonia ostii roots in Luoyang,Tongling and Heze city by the method of surface-sterilized.The inhibition ability of isolates was evaluated against tree peony pathogens through the method of dual culture.The endophytic bacteria were identified based on phylogenetic analysis of 16 rRNA gene sequences.The antimicrobial activities gene fragments of endophytic bacteria were amplified with specific primers.The antimicrobial activities metabolites produced by endophytic bacteria were extracted by the method of acid precipitation.The inhibitory activity and mechanism of antimicrobial metabolites were studied against Botrytis cinerea.This study could provide the theoretical foundation for the development and utilization of endophytes resources from Paeonia ostii.The results showed that the 124 endophytic bacterial isolates were isolated from the roots of Paeonia ostii,and the 16 bacterial isolates showed inhibition ability on the mycelia of tree peony pathogens.Especially,the strains Md1-14,Md2-17,Md2-35 and Md3-14 showed strong inhibitory activity.Md1-14 and Md3-14 were identified as Bacillus thuringiensis and B.pseudomycoides,Md2-17 and Md2-35 were identified as B.velezensis.The antimicrobial activities gene fragments were obtained in genomic DNA of the four strains.The antimicrobial activities metabolites of four strains exhibited inhibitory activity on mycelium growth of Botrytis cinerea.The antimicrobial mechanism of antimicrobial metabolites was that the mycelia of Botrytis cinerea appeared deformity,distortion and local swelling.