塔吉克斯坦哈特隆州土壤可培养细菌多样性分析

Diversity of culturable bacteria in soil sample from Hartlon,Tajikistan

采用纯培养、结合核糖体DNA扩增片段限制性酶切分析(16S r DNA PCR-RFLP)、RepPCR及16S r DNA序列测定和系统发育分析方法研究塔吉克斯坦哈特隆州土壤可培养细菌群落结构和种群多样性。从哈特隆州的农田、天然草地、林地及荒漠土壤中分离获得110个菌株,分布在厚壁菌门(Firmicutes)(80.9%)、变形杆菌门(Proteobacteria)(17.2%)和放线菌门(Actinobacteria)(1.85%)三个类群中,归属于芽孢杆菌(Bacillus)、寡养单胞菌(Stenotrophomonas)、短杆菌(Brevibacterium)、节杆菌(Arthrobacter)、棍状杆菌(Clavibacter)、短波单胞菌(Brevundimonas)、贪食菌(Variovorax)、假单胞杆菌(Pseudomonas)、芽孢八叠球菌(Sporosarcina)、赖氨酸芽孢杆菌(Lysinibacillus)、无色杆菌属(Achromobacter)和Rheinheimera共12个属中。其中Bacillus(71.8%)为该地区土壤可培养细菌绝对优势种群,Pseudomonas(10.9%)为次优势种群。对优势种群Bacillus的Rep-PCR基因组指纹图分析表明,63株分离物有17种不同的多态性指纹图谱,具有较为丰富的多样性特征。不同样地土壤中可培养细菌的种群分布、结构组成及多样性存在明显差异,与土壤环境密切相关。Shannon多样性指数、Simpson优势度和Pielou均匀度指数与土壤有机质、全氮、碱解氮及含水量呈显著正相关(p<0.05)。土壤速效钾含量也是显著影响细菌Shannon多样性、Simpson优势度的重要因素。研究不仅为后期功能菌株的筛选提供了基础,也为塔国不同土地利用方式下土壤环境评价提供了依据。

The republic of Tajikistan, located in Southeast Central Asian and next to Xinjiang, China on the east, is an inland alpine country. The harsh natural climate has produced a variety of complex ecosystems, and possessed unique rich biodiversity. In recent years, the effects of human activities greatly changed the ecological environment in Central Asia region, leading to a degradation growth of vegetation. The soil degradation and desertification made biodiversity strongly interfered in some areas. By using the technique of culture-dependent, amplified ribosomal DNA restriction fragment analysis （ 16S rDNA PCR-RFLP）, Rep-PCR, 16S rDNA sequencing and phylogenetic analysis, this paper firstly surveyed the soil microbial resources situation in Tajikistan to study the community structure and population diversity of soil culture bacteria in Hartlon, Tajikistan. One hundred and ten stains were isolated from farmland, natural grassland, forestland and desert in Hartlon. Using four restriction enzymes Hae III, Hinf I , Msp I and Hha I to digest 16S rDNA gene to analyze the Enzyme fingerprinting, results showed that the 110 stains clustered into 23 OTUS （Operational Taxonomic Units）. Phylogenetic analysis revealed that 110 stains soil cultivable bacteria in Hartlon could be distributed into three sorts： Firmicutes （80.9%）, Proteobacteria （ 17.2% ） and Actinobacteria （ 1.85% ） ,which belonged to 12 genus-- Bacillus, stenotro- phomonas , Brevibacterium , Arthrobacter , Clavibacter , Brevundimonas , Variovorax , Lysinibacillus , Sporosarcina , Pseudomonas, Achromobacter, and Rheinheimera. Therein, Bacillus which accounted for 71.8% was the dominate population in cultivable bacteria, followed by Pseudomonas which accounted for 10.9%. The Rep-PCR genomic fingerprinting analysis of Bacillus showed that there were 17 different poly-morphism fingerprints in 63 strains and had richer diversity. Population distribution and structural composition of soil cultivable bacteria had obvious differences in different sample plots. The species diversity trend showed： natural forestland〉 natural grassland〉 farmland〉desert, and natural grassland had the highest Shannon Index （2.538）, Simpson Dominance Index （0.890） and Pielou Evenness Index （0.906） ;In farmland, cherry and cotton intercropping had the highest soil bacteria Shannon Index （ 1.619）, Simpson Dominance Index （0.751 ） and Pielou Evenness Index （0.676）. Correlation analysis showed that, the soil physical and chemical properties were closely related to the cultivable bacteria; soil organic matter, total nitrogen, available phosphorus, available potassium and moisture content were the key physical and chemical factors that affected the diversity index significantly（P 〈 0.05）. This research not only studied the community structure and species diversity of soil cultivable bacteria in Tajikistan, analyzed its soil microbial properties, but also enriched microbial species and gene library. It laid the foundation to promote the protection of microbial resources, also provided scientific basis for studying the influence of microbiology and functional diversity on environmental changes, and monitoring early warning of soil health quality.