中华蜂体内放线菌的分离、多样性及抗菌活性研究

【目的】探索药用昆虫中华蜂(Apis cerana cerana Fabricius)体内可培养放线菌的分离方法,研究其物种多样性及抗菌活性,挖掘更多微生物资源。【方法】选用7种分离培养基对中华蜂样品体内放线菌进行分离;通过采用16S rRNA PCR-RFLP和16S rRNA基因序列分析方法研究其多样性;选用4种致病菌对菌株进行抗菌活性初探。【结果】共分离得到180株放线菌,根据菌落的形态和细胞特征观察结果,从中选取84株作为代表菌株,其分属于3个目、4个科中的4个属,其中6株为潜在新种;最适的表面消毒方法:浓度为0.2%的Cl O2(二氧化氯)作为消毒剂,作用60 s;拮抗实验显示,31.0%、48.8%、27.4%、16.7%的代表菌株分别对大肠杆菌、枯草芽孢杆菌、玉米弯孢病菌、西瓜枯萎病菌有不同程度的抗菌活性,71.4%的代表菌株对至少一种病原菌有拮抗作用。【结论】分离方法的选择对昆虫体内放线菌的分离效果影响较大;中华蜂体内放线菌具有丰富的多样性,展现出很好的抗菌活性,表明其在发现新型生物活性物质中具有很大的潜力。

Isolation, diversity and antibiotic activity of actinomycetes from Apis cerana cerana Fabricius

[Objective] To study the diversity and antimicrobial activity of culturable actinomycetes isolated from Apis cerana cerana Fabricius, which will help to explore new microbial resources. [Methods] Seven media were used to isolate actinomycetes from Apis cerana cerana Fabricius samples. 16S rRNA PCR-RFLP analysis and 16S rRNA gene sequences analysis were utilized to study their genetic diversity. Pathogenic bacteria and fungus were used in antimicrobial test for exploring the antimicrobial activity of strains. [Results] Based on the morphology of the colonies and cells of the new isolates, 84 representative strains were selected from 180 isolated strains. They belonged to 3 orders, 4 families and 4 genera, and in which there were 6 strains identified as potential new novel species. The best surface disinfection method was by using 0.2% ClO2 for 60 s. The results of the antimicrobial test showed that 31.0%, 48.8%, 27.4% and 16.7% of the representative strains presented antimicrobial activity to Escherichia coli, Bacillus subtilis, Curvularia lunata and Fusarium oxysporum, and 71.4% of the representative strains presented antimicrobial activity to at least one pathogen. [Conclusion] The choice of the isolation method has a great impact on isolation result. There are great diverse actinomycetes inside Apis cerana cerana Fabricius and they have great antimicrobial activity, which show a great potential for researching new bioactive compounds.