【作者】 郭慧娟；

【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】 无论是在酸性、碱性还是中性土壤中,目前国际上所使用的大豆根瘤菌剂主要是慢生根瘤菌(Bradyrhizobium)。但在偏碱性土壤中,中华根瘤菌属(Sinorhizobium)为与大豆共生的优势菌,且生长速度比慢生根瘤菌快,发酵成本低。虽然大豆快生根瘤菌在偏碱性土壤中有着广阔的应用前景,但其与不同大豆品种的共生特异现象限制了其在农业生产中的应用。本论文针对分离自新疆和黄淮海两个生态区的60株大豆快生根瘤菌代表菌株,分析了3个核心基因(SMc00019, truA, thrA)及不同复制子上的16个共生基因(chromosome:bacA, purL, nodM, gcvT, cysD, cobO, nodE; pSymB:exoA, exoY, phnC; pSymA:nodC, nodS, y4wE, nodDl, nodD2, rhcJ)的遗传分化和进化机制。这些菌株可以划分为5个种群,且不同的种群在不同的复制子上都有不同程度的遗传分化：(1) Sinorhizobium fredii种内多样性最高且分布最广,而S. sojae种内多样性最低且地理分布范围很窄；(2)对于染色体上的核心基因和共生基因及pSymB上的共生基因,基因间重组主要发生在种内而在种间很少,而pSymA上的共生基因可以在不同种群或菌株间进行水平基因转移,且pSymA上共生基因的核苷酸多态性水平及重组值低于染色体和pSymB上的基因。由此可见,大豆快生根瘤菌不同复制子上共生相关基因的进化历史是不相同的,这为逐步揭示大豆快生根瘤菌与大豆品种共生差异现象的形成机制打下了基础。在碱性土壤中,能否用大豆快生根瘤菌来代替慢生根瘤菌做为根瘤菌剂呢?本论文比较了分离自黄淮海地区的辽宁慢生根瘤菌B. liaoningense和费氏中华根瘤菌S. fredii在碱性土壤中的竞争结瘤能力及对土壤微生物群落的影响。利用基于rpoB种水平的高通量测序分析发现：(1)大豆开花初期土壤中的微生物群落α-和β-多样性在4个处理(H1,不接种不施肥对照；H2,接种S. fredii; H3,接种B. liaoningense; H4,不接菌施肥对照)比播种前相应的各处理(Q1-Q4)都发生了变化；(2)开花初期与播种前相比OTU百分比增加的有：Thiorhodovibrio, Thioalkalivibrio, Thauera, Sphingomonas, Sphingobium, Bradyrhizobium。降低的有：Nitrobacter, Mesorhizobium, Agrobacterium;(3) Sinorhizobium属在接种S.fredii处理前后的OTU百分比并没有明显变化,但在接种B. liaoningense的处理的开花初期比播种前高出了2倍；Bradyrhizobium属在接种B.liaoningense的处理在开花初期比播种前高出1.6倍,且P值(0.075)也接近显著性临界值；(4)尽管所检测土壤中含有丰度较高的Bradyrhizobium,却并没有发现已知的能够与大豆结瘤的Bradyrhizobium根瘤菌种。但B. liaoningense可以使S. fredii种群数量升高5.6倍(P=0.058)；另一方面对开花期大豆根瘤的占瘤率分析表明：在不接菌不施肥对照、施肥对照和接种S. fredii的处理中全部根瘤都是由S. fredii形成的,接种B. liaoningense的处理中S. fredii的占瘤率也高达94.1%。可见,在碱性土壤中接种根瘤菌B. liaoningense竞争不过土著S. fredii。因此建议在碱性土壤中接种竞争结瘤能力较强的S. fredii作为大豆根瘤菌剂,但是必须考虑S. fredii菌株的遗传分化与当地大豆品种的匹配性。更多还原

【Abstract】 Bradyrhizobium strains are widely used as inoculants for soybeans around the world in acidic, alkaline or neutral soil. But in alkalin soil, Sinorhizobium strains are the dominant microsymbionts of soybeans and Sinorhizobium strains are characterized by their faster growth rate than Bradyrhizobium and thus cost less in the fermentation.Sinorhizobium inoculants have a broad prospect in alkaline soil, but the symbiosis specificity of Sinorhizobium limited its application. In order to investigate the genetic differentiation and microevolutionary mechanisms of60Sinorhizobium strains from Xinjiang and Huanghuaihai, we selected three housekeeping genes (SMc00019, truA, and thrA) and16symbiosis-related genes (chromosome:bacA, purL, nodM, gcvT, cysD, cobO, nodE; pSymB:exoA, exoY, phnC; pSymA: nodC, nodS, y4wE, nodDl, nodD2, rhcJ). Five distinct species were identified and different levels of genetic differentiation were observed among these species or different replicons:(1) S.fredii has higher intraspecific diversity and wider distribution, but S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution.(2) Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons, this study laid a foundation to reveal the mechanism of symbiotic differences between Sinorhizobium strains and soybean cultivars.Can Sinorhizobium inoculants replace Bradyrhizobium inoculants in alkaline soil? The454pyrosequencing of rpoB amplicon was used to compare the microbial community structure and nodulation competition ability after inoculating S. fredii and B. liaoningense in alkaline soil. The results showed that:(1) a and β-diversity of microbial communities have changed in four treatments in flowering period (H1, no inoculation and no fertilizer control; H2, inoculating S.fredii; H3, inoculating B. liaoningense; H4, no inoculation and fertilizer control) as compared to those of corresponding soil samples collected before inoculation (Q1-Q4).(2) The abundance of OTUs has increased in genera of Thiorhodovibrio, Thioalkalivibrio, Thauera, Sphingomonas, Sphingobium, Bradyrhizobium and decreased in genera of Nitrobacter, Mesorhizobium and Agrobacterium.(3) As to the abundance of OTUs belonging to Sinorhizobium, H2did not cause significant difference compared to Q2whereas H3’s value is two times of Q3; H3aslo showed higher abundance of Bradyrhizobium than Q3(1.6times, p-value=0.075).(4) Although the abundance of Bradyrhizobium is higher than Sinorhizobium in all test soil samples, we did not find any defined Bradyrhizobium species which can nodulate with soybean. But the populations of S.fredii have increased by inoculating with B. liaoningense (5.6times, P=0.058). Nodule occupation ratio of S.fredii is94.1%in the treatment of inoculating B. liaoningense, and strains isolated from the other three treatments all belonged to S. fredii. Apparently, the nodulation competition ability of S. fredii with soybean is stronger than B. liaoningense in alkaline soil.In short, S. fredii is suggested to be used as the inoculants for soybeans in alkaline soils, given the compatibility of different S. fredii strains and soybean cultivars is well considered.更多还原