【作者】 杨文权；

【导师】 韦革宏；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2013， 博士

【摘要】 小冠花（Coronilla varia）原产于地中海地区，上世纪六七十年代从欧洲和美国引入我国，由于其根系发达、抗逆性强、营养价值高、花色优美等特点，可作为饲用、水土保持、公路护坡、观赏及绿肥植物等，在我国西北、华北、华东等很多地区都有引种栽培。但是，与小冠花共生的根瘤菌的多样性及分类地位没有进行系统的研究，土壤因子对其多样性的影响还不了解；而且，作为外来植物，小冠花是如何在引入地找到合适的根瘤菌并很快建立共生关系问题也亟待研究。针对以上问题，本试验对采自陕西中部6个地点的90株小冠花根瘤分离的细菌，先进行了回接，然后进行了表型分析（123项生理生化测定）和遗传型分析（所有供试菌株的16S rRNA、nodC、nifH PCR-RFLP，代表菌株的16S rRNA、recA、nodC、nifH基因全序列测定分析），并研究了小冠花根瘤菌多样性与土壤因子的关系，主要结论如下：1.90株供试菌株中有84株回接到了原宿主植物小冠花上。结瘤最多的植株有16个根瘤，最少的1个，平均结瘤6个。绝大多数根瘤着生在宿主植物的侧根上，少数着生在主根上或主根和侧根上都结瘤。根瘤为球状或短棒状，大小约0.4mm。颜色多为白色和粉色。2.对90株小冠花根瘤分离菌株的123项生理生化特性测定结果表明：供试菌株有较广泛的碳、氮源利用能力，有较强的抗逆性。其中，17.8%的菌株可在3.0%的NaCl上生长；大多数菌株都对磷雷霉素、青霉素及氨苄青霉素有一定的耐受性；所有供试菌株可在0.1%、0.2%的去氧胆酸钠和NaNO2上生长；多数菌株对大部分的染料有一定的耐受性；所有供试菌株均能在pH5.0-12.0的培养基上生长；12.2%的菌株可以在10℃、32.2%的菌株可以在40℃条件下生长。3.根据16S rRNA PCR-RFLP及16S rRNA、recA基因的全序列分析结果，除代表菌株CCNWSX661的聚类结果与16S rRNA不太一致外，其它均与16S rRNA序列揭示的系统发育关系一致。90株供试菌株分别位于Mesorhizobium、Rhizobium和Agrobacterium3个属中，对应M. sp.、M. alhagi、M. amorphae、M. caraganae、R.yanglingense和A. tumefaciens6个种，表现出了较高的多样性。其中，有4种基因型、共78株菌属于Mesorhizobium属，占所有供试菌株的86.7%，说明在陕西中部地区，小冠花主要与Mesorhizobium属的根瘤菌共生。4. nodC PCR-RFLP及其全序列分析表明，小冠花根瘤菌nodC基因共产生了5种基因型，分别位于Mesorhizobium和Rhizobium2个属中，其中，Mesorhizobium为主要的属，有4种基因型，占所有菌株的92.86%。系统发育树表明，该属根瘤菌的nodC基因可能是由原产地带入，通过基因水平转移进入到本地根瘤菌中，通过逐渐适应，最后朝着M. alhagi的nodC基因进化。5. nifH PCR-RFLP及全序列分析表明，小冠花根瘤菌niH基因共产生了4种基因型，分别位于Mesorhizobium、Agrobacterium和Rhizobium3个属的系统发育支上。16S rRNA的系统发育树与nodC、nifH的系统发育树不一致，表明小冠花根瘤菌的共生基因在不同种、属间可能存在水平转移。6.对6个采样地点小冠花根瘤菌的16S rRNA多样性与土壤有机质、pH、全氮、碱解氮、全磷、速效磷、全钾及速效钾的相关性分析表明，土壤pH与16S rRNA多样性呈显著正相关（P＜0.05），即在一定范围内，土壤pH越高，小冠花根瘤菌的16S rRNA多样性越高。表明在陕西地区，土壤pH可能对小冠花根瘤菌多样性有一定的影响。更多还原

【Abstract】 Coronilla varia, native to the Mediterranean region, was introduced to China fromEurope and America in the1960s-70s. Because of the developed root system, strongresistance, high nutritional value as well as its beautiful color, it has been widely used forerosion control, soil rehabilitation, pasturage, roadside planting, ornamental plants and greenmanure throughout the Northwest, North and Eastern China and other many places. However,the variety of rhizobia from root nodules of Coronilla varia hasn’t been studied systematically.It is not clear that which and how the soil factors affect its variety. Also, it is urgent thatCoronilla varia, as an exotic plant, how find the matched rhizobia to soon form the symbiosisin the introduced places.To deplore the problem above,90symbiotic rhizobial isolates from root nodules ofCoronilla varia, collected in6sites in the central part of Shaanxi, were characterized on thebasis of phenotypic and genetic analysis. They included123physiological and biochemicalindex,16S rRNA, nodC, nifH PCR-RFLP and the sequences analysis of16S rRNA, recA,nodC, nifH. Then, the rhizobial variety, their symbiotic evolution and the related soil factorswere studied. The main results were followed.1.84of90isolates could form nodules on the roots of Coronilla varia by nodulationtests on their original host plant. The most16, the least1, averaged six nodules were observedon each inoculated plant. Most of nodules located on the lateral roots, whereas, few were ontaproots or both taproots and lateral roots. The majority of nodules shaped in spherical andshort rod, with the size of about0.4mm, in white or pink color.2. The123physiological and biochemical index of90isolates from root nodules ofCoronilla varia showed they had the good ability of using the various carbon and nitrogensources, and the excellent resistance to the stresses.17.8%strains could grow on3.0%NaClmedium. Most of the isolates had fair tolerance to phosphorus mine mycin, penicillin andampicillin. All isolates could grow well on0.1%to0.2%deoxysodium cholate and NaNO2medium. They had fair tolerance to most dye. All isolates could grow on pH5.0-12.0medium. 12.2%and32.2%isolates could grow at10℃and40℃, respectively.3.16S rRNA PCR-RFLP and sequences analysis of16S rRNA, recA gene showed theconsistent phylogenetic relationships for all isolates except for the representative strainCCNWSX661.90isolates belonged to3genura, Mesorhizobium, Rhizobium andAgrobacterium and6species, M. sp., M. alhagi, M. amorphae, M. caraganae, R.yanglingense and A. tumefaciens. It showed the high species diversity of rhizobia from rootndules of Coronilla varia.78isolates, accounting for86.7%, had4genotypes, all belongingto Mesorhizobium. It indicated Coronilla varia mainly formed the symbiosis with rhizobiafrom Mesorhizobium.4. nodC PCR-RFLP and sequences analysis showed there were5genotypes of nodCgene of rhizobia from root nodules of Coronilla varia. They belonged to Mesorhizobium andRhizobium, but Mesorhizobium was the dorminant, including4genotypes and accounting for92.86%isolates. It indicated nodC gene of Mesorhizobium were probably from the nativehabitat through the lateral transfer and long-term adaptation, finally evolved towards M.alhagi.5. nifH PCR-RFLP and sequences analysis showed there were4genotypes of nifH geneof rhizobia from root nodules of Coronilla varia, belonging to Mesorhizobium,Agrobacterium and Rhizobium. The phylogenetic trees based on16S rRNA, nodC and nifHdisplayed obvious differences, implied that possible lateral transfer of the symbiotic genesoccurred between different species or genera.6. It was down that the correlation analysis of16S rRNA of rhizobia from root nodulesof Coronilla varia in6sampling sites and the soil physicochemical characteristics, includingcontent of the organic matter, total and available nitrogen, phosphorus, potassium and pH. Theresults showed that pH had significantly positive correlation with the diversity of16S rRNA(P＜0.05). Within a certain range, the diversity of16S rRNA gene tended to be higher as thepH of soil increased. It implied soil pH had stronger effect on variety of rhizobia fromCoronilla varia in Shannxi.更多还原