【作者】 张星星；

【导师】 陈文新； 田长富；

【作者基本信息】 中国农业大学 ， 微生物学， 2014， 博士

【摘要】 根瘤菌为土壤腐生细菌,在条件允许的情况下,与相匹配的豆科植物形成根瘤,建立共生固氮体系。根瘤菌剂在特定土壤条件下竞争结瘤能力差的现象严重制约了根瘤菌剂的推广应用。与大豆共生的慢生根瘤菌在世界范围广泛分布,市场上主要的大豆根瘤菌剂为这个属。本论文一方面用分子进化学和系统发育学方法研究了不同地理来源的大豆慢生根瘤菌(Bradyrhizobium)的遗传分化特征,为今后鉴别不同Bradyrhizobium根瘤菌剂打下了基础。(1)根据核心基因(SMc00019-truA-thrA)把东北、新疆、黄淮海和南方的76株大豆慢生根瘤菌代表菌株分为11个种群：B.japonicum, B.diazoefficiens, B. daqingense, B. elkanii, B. huanghuaihaiense, B. liaoningense, B. yuanmingense, Bradyrhizobium sp. Ⅰ, Bradyrhizobium sp. Ⅱ, Bradyrhizobium sp. Ⅲ和Bradyrhizobium sp. Ⅳ;(2) island基因(共生岛基因：nifA、nifH、 nodC、nodV、fixA、trpD和rhcC2)的核苷酸多态性和重组率显著低于off_island基因(共生岛外基因：SMc00019, thrA、truA、fabB、glyA、phyR、exoN和hsfA),除了B. elkanii、B. yuanmingense和Bradyrhizobium sp. Ⅱ的其它8个种群共有一个优势的island基因系统发育分支；(3) island基因在不同种群间的基因流水平与off_island基因的遗传分化水平有明显的相关性。另一方面,利用种水平的rpoB高通量测序方法,研究了我国三个大豆种植生态区(东北、黄淮海和南方)的土壤中土著大豆根瘤菌的生物地理分布特征,为针对不同土壤条件选择环境适应能力强的大豆根瘤菌剂提供了强有力的理论支持。(1)土壤微生物的α和β多样性均是按照不同生态区分布；(2)a多样性受到土壤环境因子pH, N、Orc、K, Na+、HCO3-、Cl-、P、 EC等的显著影响,p多样性与地理距离、土壤因子和地表植被(大豆或野大豆)具有明显的相关性；(3)不论土壤中还是根瘤中的根瘤菌均具有显著的生物地理分布特征,东北的优势种在土壤中是B.japonicum和Bradyrhizobium sp. Ⅲ,在根瘤中是Bradyrhizobium sp. Ⅲ;在南方,B.diazoefficiens在土壤和根瘤中均为优势种；黄淮海的土著大豆根瘤菌多样性最高(Bradyrhizobium sp. Ⅲ、B.japonicum、B. huanghuaihaiense、B. elkanii、S.sojae和S.fredii),根瘤中则仅有B. japonicum和S.fredii,其中S.fredii占优势；(4)土著大豆根瘤菌相对丰度的生物地理分布受到了土壤因子N、K、P、Orc和pH的影响,同时在不同生态区这种影响程度也不同。通过以上研究,可以很好地为不同生态区选择高效根瘤菌剂提供理论支持。例如：在东北选用Bradyrhizobium sp. Ⅲ和B.japonicum,在南方选用B. diazoefficiens,它们为各自生态区土壤中的优势种,且island基因都属于同一个优势island基因系统发育分支；在黄淮海则主要选择该区土壤中的优势快生大豆根瘤菌S.fredii。更多还原

【Abstract】 Rhizobia are soil bacteria that fix nitrogen after becoming established inside root nodules of appropriate legumes. Rhizobial inoculants could be outcompeted by indigenous rhizobia in certain soil conditions and the application of inoculants in agriculture is therefore severely affected. The soybean rhizobia belong to Bradyrhizobium were widely distributed in the world and as a principal inoculant for soybeans.This study which investigated genetic divergence levels among Bradyrhizobium strains nodulating soybeans in China using phylogenetics and evolutionary genetics, can lay a solid foundation for screening for effective inoculant for soybeans.(1) Based on three useful phylogenetic and taxonomic markers (SMc00019-thrA-truA),76representative Bradyrhizobium strains were identified as eleven genospecies:B. japonicum, B. diazoefficiens, B. daqingense, B. elkanii,B. huanghuaihaiense, B. liaoningense, B. yuanmingense, Bradyrhizobium sp. I, Bradyrhizobium sp. Ⅱ, Bradyrhizobium sp. Ⅲ and Bradyrhizobium sp. IV;(2) Seven genes inside the symbiosis island (island genes:nifA, nifH, nodC, nodV,fixA, trpD and rhcC2) showed contrasting lower level of nucleotide diversity and recombination rate than off_island genes (SMc00019, thrA, truA,fabB, glyA,phyR, exoN and hsfA). Moreover, eight genospecies (including B. japonicum, B. diazoefficiens, B. daqingense, B. huanghuaihaiense, B. liaoningense, Bradyrhizobium sp. I, Bradyrhizobium sp. Ⅲ and Bradyrhizobium sp. Ⅳ) formed a clade in the phylogeny of island genes.(3) There was a potential relationship between the gene flow of island genes and the divergence of off_island genes.On the other hand, we applied the pyrosequencing of rpoB amplicon to study biogeography and biodiversity of indigenous soybean rhizobia in three ecoregions of China (Northeast China, South China and HuangHuangHuai), which can offer theoretical guidance for selecting suitable strains as inoculant for soybeans.(1) The variation of bacterial diversity (a diversity) and community dissimilarities (β diversity) were attributable to spatial distribution.(2) a diversity of soil bacterial communities was significantly related to soil factors such as pH, N, Orc, K, Na+, HCO3-, Cl-, P, EC, and community dissimilarities was largely dependent of geographic distance, soil chemistry and aboveground plant.(3) soybean rhizobia, both in soil and root nodule, exhibit patterns of biogeography, the soybean rhizobia of most abundance in Northeast China was B. japonicum and Bradyrhizobium sp. Ⅲ in soil, whereas Bradyrhizobium sp. Ⅲ in root nodule; In South China, B. diazoefficiens was the dominant group both in soil and root nodule; The diversity of indigenous soybean rhizobia in soil of HuangHuaiHai was shown a high degree, including Bradyrhizobium sp.Ⅲ, B. japonicum, B. huanghuaihaiense, B. elkanii, S. sojae and S.fredii. B. japonicum and S.fredii were isolated from root nodule in HuangHuaiHai, and S.fredii was the main microsymbionts of soybean.(4) There were soil factors like N, P, K, Ore, and pH, that may affect the biogeographic distribution of indigenous soybean rhizobia, though the key factor may be different among three ecoregions.Taken together, these results could provide helpful guidance in selecting efficient rhizobial inoculants for different ecoregions. For example, Bradyrhizobium sp. Ⅲ and B. japonicum in Northeast China, and B. diazoefficiens in South China, these strains were dominant groups in each corresponding ecoregion, respectively and belong to the same epidemic clade in the phylogeny of island genes; S. fredii, the dominant group in HuangHuaiHai, can be a suitable inoculant strain in this ecoregion.更多还原