豆科植物根际氢氧化细菌的分离及促生作用机制研究

Hydrogen generated in the process of symbiotic nitrogen fixation by legumes and hydrogen-oxidizing bacteria that live dependent on hydrogen form the peculiar microbial community structure. It has important effects on microecological system in rhizosphere. However, on account of the peculiarity of hydrogen-oxidizing bacteria and the fact that most soil bacteria are remain unculturable, research on hydrogen-oxidizing bacteria is limited which makes the clarification of mechanisms of growth promoting effects is still in speculation stage. This project aims at exploring new hydrogen-oxidizing bacteria isolation methods and establishing hydrogen-oxidizing bacterial population screening and cultivation techniques. Specifically, considering the growth of hydrogen-oxidizing bacteria can be stimulated or induced when soil is rich in hydrogen either generated by nitrogen fixation by Hup- rhizobia or intentionally treated, leguminous crops – soybean and alfalfa are selected to be research materials in this project. We have established a gas-cycling incubation system through which both concentration and ratio of hydrogen can be controlled to isolate and identify hydrogen-oxidizing bacteria and then investigate the population diversity of hydrogen-oxidizing bacteria in rhizosphere in hope of discovering the ones belonging to novel physiological groups. Besides, we dedicate to investigate the effect of hydrogen generated by nitrogen fixation of Hup- rhizobia on the community structure of culturable soil microbes and the dynamic changes of dominant populations. Furthermore, we endeavor to uncover the dependence between bioactive substances of rhizosphere hydrogen-oxidizing bacteria, ACC deaminase and siderophore and plant growth promoting effect so as to supplement and perfect the mechanism of crops promoting effect by crop rotation with legumes. On the whole, this project will extend the research contents of biological nitrogen fixation from a novel perspective of microflora in rhizosphere soil and meanwhile enrich germplasm resources of soil microbes. It can also provide novel perspectives and theoretical basis for developing new approaches to increase crop production.

豆科植物共生固氮过程中释放的氢气及其依赖于氢气而生长的氢氧化细菌构成了豆科植物根际所特有的微生物群落结构，对土壤肥力和养分循环产生重要影响。由于氢氧化细菌的独特性和大多数土壤细菌的不可培养性，对这些氢氧化细菌的研究十分有限，其促生作用机制仍处猜测阶段。本项目以豆科作物大豆、苜蓿根际土壤为研究材料，通过接种Hup-根瘤菌的豆科作物固氮放氢和实验室条件下的土壤氢气处理，刺激土壤氢氧化细菌，采用气体循环培养体系，模拟土壤环境，建立新的氢氧化细菌的分离方法和培养技术，分离获得新的生理类群微生物；研究共生固氮作用中放氢效应对土壤中氢氧化细菌群落结构的影响以及这些氢氧化细菌产生的ACC脱氨酶等生物活性物质与作物生长的相关性，进而补充与完善豆科作物轮作增产效应的机制。该项研究将从根际土壤微生物区系的新视角丰富生物固氮的研究内容，并为丰富土壤微生物种质资源和开拓新型作物增产途径提供新思路和科学依据。

豆科植物共生固氮过程中释放的H2及其依赖于H2而生长的氢氧化细菌构成了豆科植物根际所特有的微生物群落结构，并对植物根际土壤微生物以及植物病原微生物生态系统产生有益影响。本项目以豆科作物大豆、紫花苜蓿以及西北地区广泛种植的其他多种豆科植物根际土壤为研究材料，通过豆科作物固氮过程中放氢和实验室条件下的土壤氢气处理，采用电解水制作氢气的气体循环培养体系，模拟自然环境中土壤生态特性，建立起了气体处理土壤系统装置、氢氧化细菌培养装置及氢气流速检测系统，建立根际氢氧化微生物分离、培养技术，分离获得具有氢氧化特性的生理类群微生物若干株；采用传统的和现代的微生物分类方法，观察获得的氢氧化菌株（群）培养特征和菌体形态，分析菌株生理生化特性和16SrRNA序列同源性，确定氢氧化细菌的分类地位。利用不同浓度氢气处理土壤样品采用宏基因组技术比较氢气处理土壤在物种多样性的差异，检测某些菌株的转录组，探索菌株中氢氧化酶表达与氢气存在的关系。通过项目的实施，建立了一套比较完整的氢气处理土壤系统及其较为先进的根际微生物分离、鉴定、培养技术平台，获得了具有氧化氢酶活性生理类群的微生物菌株；初步揭示了豆科作物根际氢氧化细菌种群生理生化特性以及对作物生长的影响的作用机理；发表研究论文6篇；申请专利4项；培养研究生6名。.我国从汉代起就将豆科作物用于轮作和间作。但随着经济的发展，我国农业为了应付快速增长的社会需求，大量地使用化肥以取得眼前的短期效益。化肥大量长期使用造成了土壤的退化，水系的污染，使得农业面临的困境进一步恶化。现代化的高产出农业急需一种不需要大量化肥，能改善土壤肥力并能支持可持续发展的技术。如把根瘤菌固氮时释放的氢气用来提高作物产量的技术能够运用在我国的农业生产中，即可提高作物单产，也能持续保持土壤肥力，为农业生产提供一条新的增产途径。