嗜酸丝状放线菌的选择性分离与多样性

Selective isolation and diversity of acidophilic filamentous actinomycetes from acidic soils

【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16SrRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为:土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸(各50mg/L)的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属:链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea)、韩国生工属(Kribbella)、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora)和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。

[ Objective] To establish effective methods for selective isolation of acidophilic filamentous actinomycetes from acidic soils, and to investigate their genus and species diversity. [ Methods] Four pretreatments and 5 media supplemented with different inhibitors were used for isolation. The best combination of methods was determined according to the number and ratio of actinomycete colonies on plates, and was applied to isolate actinomycetes from 17 acidic soil samples collected in Jiangxi Province. The isolates were grouped based on cultural characteristics. The micromorphology and pH growth range of each group were studied to choose the representative isolates, which were subsequently subjected to 16S rRNA gene analysis to investigate their diversity. [ Results] We found the best isolation approach involved Dispersion and Differential Centrifugation （DDC） pretreatment and GTV medium supplemented with cycloheximide, nalidixic acid nystatin （each at 50 mg/L）. 369 isolates were obtained,and were assigned to 10 color groups. 6.6% of the isolates were strictly acidophilic actinomycetes, 72.4% were neutrotolerant acidophilic actinomycetes, and 21.0% were acidotolerant ones. The 52 representative isolates belong to 12 recognized genera ： 30 of them fall within the genus Streptomyces, 6 belong to Micromonospora, 3 belong to Nocardia, 3 belong to Nonomuraea,2 belong to Kribbella, 2 belong to Microbispora, and the other 6 strains belong to Actinomadura, Amycolatopsis, Dactylosporangium, Lentzea, Planotetraspora and Streptacidiphilus, respectivley. The Streptomyces isolates formed 12 evolutionary groups in the Streptomyces 16S rRNA gene tree. [ Conclusion] The selective isolation approach established here is robust for isolating various acidophilic filamentous actinomycetes from soil. Acidic soils in Jiangxi Province harbor abundant and diverse acidophilic actinomycetes.