塔里木河中下游流域棉田及胡杨林土壤细菌群落结构及多样性研究

Structure of soil bacteria community and diversity in cotton field and Euphrates Community poplar forest in the middle and lower reaches of Tarim river basin

摘要:

【背景】新疆绿洲生态系统的大规模农垦开发对当地生态环境产生了一系列影响，评估农业活动对新疆地区原始生态土壤细菌群落的影响对合理利用土地、开发农业资源具有指导意义。【目的】通过研究塔里木河中下游流域受农垦开发影响的棉田及未经历土地开垦的胡杨林土壤细菌群落结构及多样性的变化，探究农垦开发活动对土壤微生态的影响。【方法】利用国标法测定塔里木河中下游流域绿洲棉田及原始胡杨林全氮、总盐、有机质、碱解氮、有效磷、速效钾、pH等土壤理化性质；提取土壤总DNA，通过PCR扩增建立文库，用新一代Illumina Hiseq 2500测序技术对土样DNA进行16S rRNA基因(V4区)高通量测序；利用生物信息学分析细菌群落的α多样性(包括Chao1、observed species、phylogenetic diversity (PD)、Shannon、Simpson和Good’s coverage指数)、β多样性在两地的变化，基于线性判别分析流程[linear discriminant analysis (LDA) effect size pipeline，LEfSe]分析在两地具有显著差异的细菌分类单元；结合土壤理化参数、群落功能预测分析可能影响两地细菌群落结构变化的主要因子。【结果】土壤基本理化性质分析：绿洲棉田与原始胡杨林相比，全氮、碱解氮、总盐、pH含量显著增加，土壤盐含量与全氮、碱解氮和pH呈显著正相关(P<0.05)；细菌多样性分析：α多样性指数Chao1、observed species和PD在棉田样品中均显著低于胡杨林样品，Shannon指数差异虽不显著，但是也表现出同样的变化趋势；细菌群落组成分析：两组样品中的优势细菌依次为α-变形杆菌纲、放线菌门、芽单胞菌门、拟杆菌门、酸杆菌门、γ-变形菌纲、β-变形菌纲、δ-变形菌纲、绿弯菌门、厚壁菌门、浮霉菌门、泉古菌门和硝化螺旋菌门(丰度>1%)；差异分析的结果显示，新疆绿洲棉田土壤中显著富集的微生物主要包括酸杆菌门、放线菌门、绿弯菌门、厚壁菌门、黄杆菌纲、α-变形菌纲、γ-变形菌纲中的一些物种，以及泉古菌门中的Candidatus Nitrososphaera；新疆原始胡杨林土壤中显著富集的微生物主要包括绿菌门、δ-变形菌纲以及变形菌门中的其他物种, 还包括泉古菌门中的Nitrosopumilus。主坐标分析(PCoA)结果显示，棉田生态系统的土壤细菌群落结构能够与胡杨林生态系统的群落结构明显分开，且前者的细菌群落结构一致性更高。功能预测结果显示，绿洲棉田的硝化作用显著高于原始胡杨林。冗余分析(RDA)结果表明全氮含量对细菌群落结构有显著影响。【结论】在长期耕种施肥等人为因素干扰下，绿洲棉田土壤细菌多样性和群落结构发生明显改变，细菌α多样性明显低于原始胡杨林，细菌β多样性差异减弱，同时一些与植物生长密切相关的细菌类群，如放线菌、硝化细菌等显著富集。该研究可为新疆绿洲棉田生态系统的开发利用现状提供数据参考。

Abstract:

[Background] The large-scale agricultural reclamation and development of oasis ecosystem in Xinjiang have ecological impacts on the environment. Thus, it is important to evaluate the effect of human activities on the soil biological characters, especially the soil bacterial community, to provide a guidance for rational utilization of land and sustainable development of agricultural resources in the future. [Objective] By comparing soil bacterial structure and diversity in cotton fields affected by agricultural reclamation, with those in poplar forests where ecological environment has largely been kept intact, we studied the effects of human activities on agricultural reclamation and development on soil microbial ecology. [Methods] The national standard method was used to measure the soil physical and chemical properties, including total nitrogen, total salt, organic matter, alkaline hydrolysis nitrogen, available phosphorus, available potassium and pH in the cotton fields and poplar forests in the lower and middle reaches of Tarim river basin. Total soil DNA was extracted, a library was established by PCR amplification, and 16S rRNA gene (V4 region) was high-throughput sequenced using Illumina Hiseq 2500 sequencing technology platform. Bioinformatics was used to analyze the changes of bacterial α-diversity (such as Chao1, observed species, phylogenetic diversity (PD), Shannon, Simpson and Good’s coverage indices) and β-diversity in the two areas, and redundancy analysis was used to find the main factors that may have affected the changes of the bacterial community structure in the both areas, as well as community function prediction results were also used to give an auxiliary interpretation. [Results] Basic physical and chemical properties of soil analysis: compared with the primary poplar forest in oasis cotton field, there were significant increases in total nitrogen, alkaline hydrolysis nitrogen, salt and pH, and significant positive correlation between soil salt content and total nitrogen, alkaline hydrolysis nitrogen and pH (P<0.05). Bacterial diversity analysis: the diversity index of chao1, observed species and PD in cotton field samples were significantly lower than those in poplar forest samples. The Shannon index difference was not significant, but it showed the same variation trend with the above three indexes. Bacterial community composition analysis: the dominant bacteria across the two places were Alphaproteobacteria, Actinomycetes, Gemmatimonadetes, Bacteroides, Acidobacteria, Gammaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Chloroflexi, Firmicutes, Planctomycetes, Crenarchaeota and Nitrospirae (>1%). According to the results of linear discriminant analysis, the significant enriched microorganisms in cotton field soil were Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Flavobacteria and some species of Alphaproteobacteria, Gammaproteobacteria, and one species (Candidatus Nitrososphaera) belonged to Crenarchaeota was also discovered in cotton field. However, in original poplar forest soil samples, the significant enriched microorganisms were Chlorobi, and Deltaproteobacteria. Some species in Proteobacteria and the Nitrosopumilus which belonged to Crenarchaeota was enriched in poplar forest soil. The results of principal coordinates analysis (PCoA) showed that soil bacterial community composition (BCC) of cotton field could be significantly separated from that of poplar forest, and the BCCs in cotton field ecosystem was more consistent. The results of functional prediction showed that nitrification function in cotton field was significantly surpassed the poplar forest. Redundancy analysis showed that the total nitrogen content had a significant effect on the variation of BCC. [Conclusion] Under the influence of long-term cultivation, fertilization and other human activities, the soil bacterial diversity and community composition of oasis cotton field have been significantly changed, the α-diversity of bacterial community was significantly lower than that of the poplar forest, the β-diversity of bacterial community was weakened, and some bacteria related to plant growth, such as Actinobacteria and nitrifying bacteria, have been significantly enriched. This result can provide useful information to utilize and develop Xinjiang oasis ecosystem filed in near future.