基于MiSeq平台的土壤微生物对长期免耕和秸秆还田响应的研究

No tillage and residue management has been worldwide used for reducing soil and water erosion, weed and disease control, and to improve soil infiltration and aeration at appropriate soil moisture content. The function of soil microbial community play a critical roles in soil sustainability and nutrient cycling linked to crop production. It acts as a buffer in the soil ecosystem due to their key role in soil processes including nutrient cycling, atmospheric nitrogen fixation, and the decomposition of organic materials and/or pollutants. Recent advances in sequencing technology, such as the development of pyrosequencing, is a promising new tool that will expand our understanding of the microbial community structure and diversity of soils quickly and more comprehensively than other molecular approaches currently in use. A long-term conservation tillage system experiment has been conducted in Longdong loess plateau of China since 2000, which focused on the effect of tillage and residue management on soil properties and crop productivity, and to reduce the soil and water erosion. There are four treatments: a) conventional tillage; b) conventional tillage with residue retention; c) no tillage; d) no tillage with residue retention. In this project, we applied high throughput pyrosequence technology based on MiSeq platform to study the influence of tillage and residue management on the diversity and functionality of soil microbial population in community and species level. Plant roots and rhizosphere soil will be determined. Soil physical and chemical properties would be measured. Soil microbial community distribution abundance, diversity and richness would also be evaluated at species level using pyrosequencing technology. Relationship between bacterial diversity indexes with soil tillage and residue management, and correlations among selected soil properties and soil microorganism diversity and construction would be analyzed. The results should help us to insight the relationship between soil microorganism with soil nutrition and environment conditions¸ thus to more completely understanding the soil ecosystem health and functioning under long-term conservation tillage. It also can help us to find plant growth-promoting rhizobacteria (PGPR) to improve crop production.

免耕和秸秆还田等保护性耕作在世界范围得到广泛应用，减少水土流失，改善了土壤结构。土壤微生物群落丰富的多样性和复杂的功能结构，在有机质分解、营养元素循环等农田生态系统过程中有重要作用,研究农田土壤微生物群落对长期保护性耕作的响应有重要意义。以建立16年的长期定位实验为平台，采用国际上最新的基于MiSeq平台的高通量焦磷酸测序分子生物学技术，分别在作物-土壤-微生物界面的种群和群落水平上，研究农田土壤微生物群落的结构与功能及时空分布和变异特征；解析作物、土壤营养和水分等环境因子对土壤微生物群落结构和多样性变化的影响，明确驱动土壤微生物群落变化的主控因子；揭示土壤微生物群落对长期保护性耕作的响应机理；探索和挖掘有益微生物功能群对作物生产力的调控潜势。本研究将丰富保护性耕作的基础研究理论，为农业可持续发展过程中，针对性地拟定土壤和农业生态系统管理以及保护策略提供理论依据。

本项目以陇东黄土高原雨养区长期保护性耕作农田为研究对象，在微生物-土壤-植物界面，研究了长期免耕和秸秆还田措施下农田营养元素和土壤酶的变化特征；采用BIOLOG方法，探讨了长期免耕和秸秆还田对土壤微生物碳源利用特征的影响；基于MiSeq平台的高通量测序技术，针对根际细菌总共检测出 13个细菌门，Protebacteria， Acidobacteria，Bacteroidetes，Acitinobacteria 和 Planctimycetes是优势门类，在该区土壤中所占比例超过85%，其中Protebacteria门比例超过30%；在细菌属水平上，检测到52种细菌。在门水平（phylum level）上共检测出5种真菌，其中Ascomycota，Basidiomycota和Zygomycota 是三个优势真菌门，当地农田土壤占有比例超过95%，其中 Ascomycota门比重超过75%；真菌属水平上共检测到55个真菌属，其结构和功能也不尽相同，既包括了致病菌（Fusarium，Alternaria和 Gibberella ），也包括了农田有益菌（Penicellium）。冗余分析（RDA）表明土壤微生物量氮、全磷和pH对土壤细菌群落结构影响显著；土壤全磷对土壤真菌群落的分布影响显著。该研究阐明了农田土壤微生物群落对长期免耕和秸秆还田的响应机制；采用PLS-DA分析、置换多元方差分析（PERMANOVA）、非度量多维分析（NMDS）和RDA等多种分析，明确了驱动土壤微生物群落结构和多样性变化的主控因子，结果将为土壤保护和农业可持续发展提供理论依据，亦丰富了保护性耕作的基础理论。