缬草内生菌和根际放线菌的分离及安莎类抗生素的筛选

Isolation of endophytic and rhizospheric actinomycetes of Valerian and screening of ansamycins antibiotics

【目的】分离缬草内生菌及根际放线菌,筛选其中含安莎霉素类抗生素生物合成基因的菌株,挖掘其活性次级代谢产物资源。【方法】从缬草根、茎、叶及根际土壤中分离内生菌和根际放线菌,检测其对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、核桃溃疡病菌、苹果腐烂病菌、苹果轮纹病菌、油菜菌核病菌、小麦赤霉病菌、棉花枯萎病菌的抑菌效果。设计2种简并引物,用PCR方法从抑菌效果较好的菌株中筛选含安莎霉素类抗生素基因(AHBA)的菌株。分析目标菌株的最佳发酵培养基和最适发酵时长,对发酵分离物进行HPLC和质谱分析,检测其可能的次级代谢产物。【结果】试验共分离到145株菌,其中内生菌27株,占总菌株数的18.6%,根际放线菌118株,占总菌株数的81.4%;33株对病原菌有较好的抑菌效果,占总菌株数的22.8%。筛选到1株含AHBA基因的菌株AJ21,初步鉴定其为链霉菌。菌株AJ21最佳发酵培养基为SPY培养基,最佳发酵时间为6d。菌株AJ21发酵液经分离纯化、HPLC检测和质谱分析,初步确定其含有的安莎霉素类抗生素为放线菌素D。【结论】药用植物缬草内部及根际蕴含丰富的放线菌资源,具有良好的生物学活性。

【Objective】 This study separated endophytic and rhizospheric actinomycetes of Valerian, screened the strains with biosynthesis genes of ansamycin antibiotics,and obtained active secondary metabolites resources.【Method】 Endophytic and rhizospheric actinomycetes were isolated from roots,stems,leaves and rhizospheric soil of Valerian. Their anti-pathogenic effects on Escherichia coli,Staphylococcus aureus,Bacillus subtilis ATCC11562, Walnut ulcer pathogen,Valsa mali,Physalospora piricola,Sclerotinia sclerotiorum,Fusarium graminearum and Fusarium oxysporum were tested.Two degenerate primers were designed and strains with ansamycin antibiotic gene ( AHBA ) were screened by PCR from the strains with good anti-pathogenic effect.The optimum fermentation medium and optimum fermentation time of target strain were analyzed.The fermentation isolates were analyzed by HPLC and mass spectrometry,and the possible secondary metabolites were detected.【Result】 A total of 145 strains of actinomycetes were isolated.Among them,27 strains were endophytes,accounting for 18.6%,118 strains were rhizospheric actinomycetes,accounting for 81.4%,and 33 strains had good inhibitory effect on pathogenic bacteria,accounting for 22.8%.The strain AJ21 containing ansamycin antibiotic biosynthesis gene was identified as Streptomyces sp.The best fermentation medium for AJ21 was SPY medium and the best fermentation time was 6 days.The fermentation broth of the strain was isolated and purified.The ansamycin antibiotics contained in the fermentation broth were actinomycin D by HPLC and mass spectrometry.【Conclusion】 Valerian contains abundant actinomycetes with good biological activity in its interior and rhizosphere.