蛇足石杉内生细菌多样性

Diversity of endophytic bacteria isolated from Huperzia serrata

【目的】探索国家二级保护野生药用植物蛇足石杉内生细菌的物种和生理活性多样性,发现并收集药用植物内生菌资源。【方法】分别从四川和福建等不同生态环境采集蛇足石杉植株,运用纯培养手段,对经过表面消毒处理的蛇足石杉样品进行内生菌的分离、培养;根据菌株16S r RNA基因信息,计算从蛇足石杉不同区系分离获得的内生细菌间的Jaccard指数、多样性指数、优势度指数与均匀度指数等,分析内生菌物种多样性;应用6种筛选模型对分离得到的内生菌进行生理活性测定,初步评价蛇足石杉内生细菌的生理活性多样性和药用价值。【结果】从12份蛇足石杉植物样品中分离获得356株内生菌菌株,菌株16S r RNA基因序列信息显示,分离得到的蛇足石杉内生细菌隶属于放线菌门、厚壁菌门和变形菌门等3个门的26个科、41个属,来源于蛇足石杉地上和地下部位的菌株数目、多样性指数等无明显差异。从中发现了分属于拟无枝酸菌属(Amycolatopsis)、Angustibacter、节杆菌属(Arthrobacter)、短小杆菌属(Curtobacterium)、Frondihabitans、Glaciihabitans、Jatrophihabitans、Luteimicrobium、Massilia、Naumannella和Tardiphaga等11个属的11个潜在新物种,以及皮生球菌科(Dermacoccaceae)的1个新属。在抗菌活性筛选中,356株纯培养物抗粪肠球菌、抗肺炎克雷伯菌、抗耻垢分枝杆菌以及抗水稻白叶枯病菌的阳性率分别是9.0%、1.4%、2.2%、0.8%;抑制SS04生长的降血脂药物筛选模型上的阳性率是8.1%;抗HIV-1的初筛阳性率为4.5%。共计74株菌在一个或多个筛选模型中显示出活性,初筛总阳性率为20.8%。【结论】蛇足石杉内生细菌具有丰富的物种多样性和生理活性多样性,是进一步发掘新型天然产物的理想菌种资源。

[Objective]To explore the diversity of species and biological activities of the endophytes from Huperzia serrata that is a wild medicinal plant under state protection（category ii）,and to discover and collect endophytic bacteria from medicinal plants.[Methods]Huperzia serrata samples were collected from Sichuan and Fujian Provinces.Culture-dependent method was used to obtain endophytes from the surface-sterilized plant samples.The diversity of the isolates was analyzed according to the 16 S r RNA gene sequences information.Jaccard index,Shannon-wiener Index,Simpson Index and Pielou Index were calculated.Then six screening models were followed to study the physiological activities of the isolates,based on which we evaluated the diversity of biological activities of the endophytes from Huperzia serrata and their potential medicinal value.[Results] A total of 356 endophytic bacteria were purified from Huperzia serrata,and the analysis results of their 16 S r RNA gene sequences showed that they affiliated to 41 genera of 26 families in the phyla Actinobacteria,Firmicutes and Proteobacteria.The numbers and biodiversity indexes of endophytes from the aboveground part and belowground part of Huperzia serrata were approximately equivalent.Among them 11 potential novel species belonged to the genera Amycolatopsis,Angustibacter,Arthrobacter,Curtobacterium,Frondihabitans,Glaciihabitans,Jatrophihabitans,Luteimicrobium,Massilia,Naumannella and Tardiphaga,and 1 novel genus of the family Dermacoccaceae was discovered.The screening for anti-microbial results from these 356 isolates were as follows：the activity rates of against Enterococcus faecalis,Klebsiella pneumonia,Mycobacterium smegmatis and Xanthomonas campestris were 9.0%,1.4%,2.2% and0.8% respectively.Of them 4.5% exhibited activities on the screening model of statins-like antihyperlipidemics showing inhibition of Sporobolomyces salmonicolor SS04;8.6% of them had the activities of against HIV-1.In total,the fermentation broths from 74 strains exhibited activities on at least one screening model,the positive rate among the isolates was 20.8%.[Conclusion] This study demonstrated that the endophytic bacteria from Huperzia serrata were of great significant bio-diversity and antibiotic diversity,therefore,they could be an ideal microbial resource for further discovery of new natural products.