广东石豆兰内生细菌分离鉴定及抑菌活性

Isolation and Identification of Endophytic Bacteria in Bulbophyllum Kwangtungense Schltr for Antimicrobial Activities

目的:对广东石豆兰内生细菌分离鉴定及抑菌活性。方法:采用7种培养基从广东石豆兰四种外植体中分离内生细菌,根据菌落形态、个体形态及革兰氏染色结果排除重复菌株,并测定其16SrRNA基因序列,并构建系统进化树;使用黄瓜枯萎病原菌和棉花枯萎病原菌对内生菌进行抑菌筛选。结果:从广东石豆兰不同组织中分离到菌株由多到少分别为:假鳞茎>横走茎>根>叶;LB培养基获得菌株最多;经过分子鉴定得到39株内生菌,进化树显示它们分属于2门3目5科8属,其中芽孢杆菌属的菌株占据优势,占76.92%;发酵液对黄瓜枯萎病原菌和棉花枯萎病原菌具抑制作用各有13株,复筛阳性率为33.33%,有7株菌对两种病原菌均表现出抑制作用。结论:广东石豆兰含有较为丰富的内生菌资源,可能是抗菌生物活性次级代谢产物的有效来源。

Objective:Isolation and identification of endophytic bacteria in Bulbophyllum kwangtungense schltr for antimicrobial activities.Methods:The grinding solutes of 4 tissues from B.kwangtungense Schltr were spread onto 7 diverse isolation media.Duplicate isolates were eliminated by colony properties,individual morphology and results of gram staining.Based on 16S rRNA gene sequencing results,Neighhour-Joining phylogenetic tree was constructed to analyze the endophytic bacteria diversity.The antimicrobial activities of endophytic bacteria were detected with 2 testing strains,including Fusarium oxysporum f.sp.vasinfectum and Fusarium oxysporum f.sp cucumerinum.Results:The number of strains from different tissues ranged from more to less:pseudobulb>rhizome>root>leaf.The largest number of strains were isolated from LB medium.A total of 39 endophytic bacteria were gained by molecular identification,which were classified into 2 phyla,3 orders,5 families and 8 genera.Strains of bacillus dominated,accounting for 76.92%.The fermentation broth from 13 isolates(33.33%)showed antimicrobial activities to both pathogenic bacteria respectively,and 7 strains all inhibited both pathogenic bacteria.Conclusion:It is showed that a rich diversity of endophytes inside B.kwangtungense,and suggested that endophytic bacteria are promising sources of new natural secondary metabolites with favorable antimicrobial activities.