松辽平原主栽玉米内生固氮菌多样性及其与玉米品种和土壤肥力的关系研究

Mass production and utilization of chemical nitrogen fertilizers consumes a lot of energy and resources, and also lead to serious ecological and environmental problems，hindering the sustainable development of agriculture. Endophytic nitrogen-fixing bacteria and its diversity plays a important role for plant growth and nutrition, as long as disease resistance. Discovery and research of endophytic nitrogen-fixing bacteria open up a way to efficient use of biological nitrogen fixation in crops. Maize has an important strategic position in China’s grain security. Songliao Plain is China’s main maize producing areas, world's third-largest maize growing region. It has important significance for the sustainable development of agriculture and construction of ecological agriculture to exploit biological nitrogen-fixing and reduce uitilization of chemical nitrogen fertilizer in maize. In this project, a great quantity of endophytic nitrogen-fixing bacteria will be isolated from maize, and strain 16S rNDA and nifH sequences will be determined, and a data base of nifH sequences with strain identification will be established. Diversity of endophytic nitrogen-fixing bacteria in maize will be revealed by using experimental techniques of nifH high-throughput sequencing, DGGE and quantitative PCR. Furthermore, the influence of maize varieties and soil ecological factors on diversity of endophytic nitrogen-fixing bacteria in maize will be explored. Implementation of this project will provide a theoretical guide and technical support for making use of biological nitrogen fixation in maize.

我国化肥用量占世界三分之一。化学氮肥的生产和使用更是消耗大量能源，污染生态环境。禾本科作物内生固氮菌的发现和应用开辟了一条通过生物固氮作用为农作物提供氮素营养的途径。玉米作为三大作物之一在国民经济中起着举足轻重的作用，松辽平原是我国玉米主产区、世界第三大玉米种植带。本项目以松辽平原主栽玉米品种为研究对象，大量分离培养玉米内生固氮菌，测定菌株16S rDNA和nifH基因序列，建立“玉米内生固氮菌nifH序列与对应菌株分类信息数据库”；在此基础上以nifH为标记，通过高通量测序、变性梯度凝胶电泳、定量PCR等非培养技术揭示松辽平原主栽玉米品种内生固氮菌多样性、优势种群和活跃固氮菌群；明确玉米品种和土壤肥力因子与内生固氮菌多样性及其活跃固氮菌群的关系。项目实施将促进固氮菌资源研究和固氮微生物分子生态学发展，为生物固氮在玉米生产中的应用提供理论和技术支持。

肥料是农业基础生产资料，但化学氮肥的大量生产和使用消耗大量能源，污染生态环境，限制农业可持续发展。禾本科植物内生固氮菌的发现和应用开辟了一条通过生物固氮作用为农作物提供氮素营养的途径。本项目采用多种培养方法，分离、鉴定、保藏了玉米内生固氮菌1492株，分类地位涉及48属156种，该资源数量多，分类地位分布广泛，许多是国际上第一次发现，为固氮菌多样性研究和农业应用提供了物质保障。发现、发表了11个微生物新种，丰富了世界固氮微生物资源库，促进了固氮微生物资源研究。建立了“玉米内生固氮菌nifH基因序列与对应菌株分类信息数据库”，含有玉米内生固氮菌nifH基因序列476条，以及与此对应的476条16s RNA基因序列，覆盖物种35属142种，这是目前国际上唯一的固氮菌nifH基因数据库，对于固氮微生物资源研究具有重要意义。揭示了松辽平原主栽玉米内生固氮菌主要类群为Enterobacter、Skermanella、Pseudacidovorax、Pelomonas、Klebsiella、Bradyrhizobium、Raoultella、Azospirillum、Burkholderia、Blastochloris、Ensifer、Azohydromonas、Rhodopseudomonas、Desulfovibro、Derxia、Zoogloea、Azomonas、Mythylovirgula、Geobacter、Pseudomonas、Sideroxydans、Sphingomonas、Rheinheimera、Azorhizobium、Paenibacillus、Methylosinus、Methylocystis、Herbaspirillum、Polaromonas，其中前10位是优势菌群，Enterobacter和Pelomonas是固氮活跃菌群。品种和施肥对玉米内生固氮菌多样性的影响相对较小。