小溪自然保护区非盐环境土壤中嗜盐和耐盐菌多样性

Diversity of halophilic and halotolerant bacteria isolated from non-saline soil collected from Xiaoxi National Natural Reserve, Hunan Province

摘要:

摘要：【目的】研究湖南小溪国家级自然保护区普通非盐环境（ordinary non-saline environment）土壤样品中可培养嗜盐及耐盐细菌（含放线菌）多样性。【方法】采用纯培养法和基于16S rRNA基因序列的系统发育分析对样品中嗜盐及耐盐细菌多样性进行研究。【结果】用补充5%–20%（w/v）NaCl的MA、ISP 2、ISP 5、NA和HAA培养基从土壤样品中分离到114株细菌，其中8株为中度嗜盐菌，19株为轻度嗜盐菌，87株为耐盐菌。根据形态观察和部分生理生化实验结果去冗余，选取61个代表性菌株进行基于16S rRNA基因序列的系统发育多样性分析。结果表明，这些菌株属于细菌域（Bacteria）的3个大的系统发育类群（门；phylum）（Actinobacteria，Firmicutes，Proteobacteria）的16个科、18个属，代表了41个物种。多数菌株属于Firmicutes门（38株，62.3%）和Actinobacteria门（18株，29.5%）。大多数菌株与其系统发育关系最密切的已知物种的典型菌株之间存在一定的遗传差异（16S rRNA基因序列相似性为96.9%–99.8%），其中有7个菌株（JSM 070026, JSM 081004, JSM 081006, JSM 081008, JSM 083058, JSM 083085, JSM 084035）代表7个潜在新种（potential novel species）。【结论】研究结果表明，湖南小溪国家级自然保护区普通非盐环境土壤中存在较为丰富的可培养嗜盐及耐盐细菌多样性，并且潜藏着较多新的微生物类群（物种）。

关键词:关键词：普通非盐土壤;嗜盐和耐盐细菌;纯培养法;16S rRNA基因序列;系统发育分析

Abstract:

Abstract: [Objective] To investigate the diversity of cultivable halophilic and halotolerant bacteria isolated from ordinary non-saline soil samples collected from Xiaoxi National Natural Reserve (28°42′15′′–28°53′15′′ N, 110°6′50′′–110°21′35′′ E), Hunan Province, China. [Methods] Bacterial strains were isolated from the samples by using the conventional culture-dependent method and investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. [Results] We isolated 114 bacterial strains (8 moderately halophilic, 19 slightly halophilic, 87 halotolerant) from the samples on media (marine agar 2216, International Streotomyces Project medium 2 and 5, nutrient and humic acid agars) supplemented with 5% to 20% (w/v) NaCl. On the basis of morphological, physiological and biochemical characteristics, we selected 61 strains to perform a phylogenetic analysis based on 16S rRNA gene sequences. Results showed that 61 isolates represented 41 species, belonging to 18 genera (Actinomadura, Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, Erwinia, Halobacillus, Jeotgalibacillus, Microbacterium, Microbulbifer, Nocardia, Pseudomonas, Rheinheimera, Rhodococcus, Sphingomonas, Staphylococcus, Streptomyces, Yaniella) of 16 families (Alteromonadaceae, Bacillaceae, Brevibacteriaceae, Chromatiaceae, Dermabacteraceae, Enterobacteriaceae, Microbacteriaceae, Micrococcaceae, Nocardiaceae, Planococcaceae, Pseudomonadaceae, Sphingomonadaceae, Staphylococcaceae, Streptomycetaceae, Thermomonosporaceae, Yaniellaceae) in three phyla (Actinobacteria, Firmicutes, Proteobacteria). The most abundant and diverse isolates were within the phylum Firmicutes (38 strains; 62.3%) and the phylum Actinobacteria (18 strains; 29.5%). The phylogenetic distance matrix results suggested that there were obvious genetic divergences between most isolates and their closestly related type strains (16S rRNA gene sequence similarities ranged from 96.9% to 99.8%), and that, out of 61 isolates, at least 7 strains (JSM 070026, JSM 081004, JSM 081006, JSM 081008, JSM 083058, JSM 083085, JSM 084035) should represent 7 potential novel species within 6 characterized genera (Yaniella, Bacillus, Jeotgalibacillus, Sphingomonas, Rheinheimera, Microbulbifer). [Conclusion] The results presented here showed that there is abundant diversity of halophilic and halotolerant bacteria, as well as a number of novel species in non-saline soil collected from Xiaoxi National Natural Reserve, Hunan Province, China.

Key words:Keywords: ordinary non-saline soil; halophilic and halotolerant bacteria; culture-dependent method; 16S rRNA gene sequence; phylogenetic analysis