【作者】 潘峰；

【导师】 何绍江； 王平；

【作者基本信息】 华中农业大学 ， 微生物学， 2003， 硕士

【摘要】 从生长在湖北省几种不同土壤中2个不同品种饭豆(Vigna umbellata L.)的根瘤中分离、纯化并通过结瘤试验筛选出26株饭豆根瘤菌。以已知不同种属的8个根瘤菌参比菌株为对照，对这些菌株的多样性进行了系统的比较研究，并对其分类地位作了初步的探讨。 供试菌株的世代时间在2.70-11.45h之间，初步划分为快生与慢生两个类型。通过对这些菌株的表型特征(如培养特性、耐酸碱性、生长最终pH值、耐盐性、天然抗药性及碳源和氮源的利用)聚类分析所得数值分类树状图谱的分析，发现分离自不同类型土壤中的饭豆根瘤菌具有较大的多样性。在77％的相似水平上，饭豆根瘤菌与参比菌株形成了三大类群，群Ⅰ为慢生型菌群，群Ⅱ与群Ⅲ为快生型菌群；在80％相似水平上各群又可进一步划分为不同亚群，而且亚群中部分菌株的相似性与样品来源有相关性。 互接种族试验一方面证实了同一互接种族内的根瘤菌能够交叉感染形成根瘤，另一方面也发现饭豆根瘤菌(如CYY3302菌株)能够跨越分族界线与其它族植物形成根瘤。从而进一步证明了饭豆根瘤菌宿主范围的复杂性和饭豆根瘤菌群体组成的多样性。 快生型菌株质粒快速检测结果表明：供试的20个快生型根瘤菌株的质粒组成存在差异，表现为6个不同的质粒图谱类型。其质粒图谱类型与菌株的来源有关。 为了从遗传基础上分析供试菌株的多样性和系统发育关系，本研究采用了16S rDNA PCR-RFLP与16S-23S rDNA IGS PCR-RFLP的方法对一部分饭豆根瘤菌确定了在已知根瘤菌种、属的系统发育位置，并对表现特异的菌株的分类地位进行了推测。16S rDNA PCR-RFLP比较分析的结果表明：供试菌株在90％的相似水平上，树状图谱分为6个系统发育分支。分支Ⅴ中分离自汉川的3个菌株与R hizobium leguminosarum的模式菌株(USDA2370)有100％的相似性；分支Ⅵ中的10个菌株与Sinorhizobium fredii的模式菌株(USDA205)之间的相似性也达到100％。因此，可以认为它们分别与所对应的参比菌株应处于同一“种”水平上。但是，分支Ⅰ、分支Ⅱ和分支Ⅳ中的饭豆根瘤菌与供试的参比菌株有一定的差异，可能为其它已知种、属的根瘤菌，也可能在“种”水平上代表新的系统发育分支。 由16S-23S rDNA IGS PCR-RFLP分析可知，供试菌株依IGS大小可划分为三类。所有菌株在52％的相似性水平上聚在一起，再次肯定了饭豆根瘤菌是由慢生型菌群与快生型菌群这两大菌群组成。群Ⅰ中，在79％相似性的水平上分为ⅠA与ⅠB两个分支。群Ⅱ中，在62％相似性的水平上分为ⅡA与ⅡB两个分支，分支ⅡA在72％的相似性水平上进一步分为 IIAI＊、和 IlA3三簇；分支 IIB中的饭豆根瘤菌与参比菌株 US0A205聚在一起，表现的差异并不大。 上述表型与遗传标记的研究结果在较大尺度＿＿L对饭豆根瘤菌各类群的划分有较好的一致性。综合分析结果表明：供试的26株饭豆根瘤菌在不同相似水平上存在遗传多样性。其中山来自汉Jll 县的5株菌GAP2502、HCY5105、HCY5202、HCY5503与11CY5609 形成的菌群不同于其它菌群，因此可以推测这类菌群可能在“种”水平上代表sitzootum内的某一新的系统发育分支，这一推测为详尽地确定它们在根瘤菌中的分类地位提供了分子数据。 山 16 S rDNA部分序列分析结果可知，供试的 4个代表菌株分别位于不同的系统发育分支中。CYR4243与 Sinorhfzobf M fredl’i的模式菌株 USDA205的序列相似 性达到了99．S7％。HCYI 101与 nlzob。urn leg；im。nosarum中的三叶草生物型（by．trlfOlff）和豌豆生物型（by。z”ceae）这两个生物型的参比菌株亲缘关系最近，序列相似性为 100％。HCY5202与 Rhizobium g81egse亲缘关系最近，序歹相i 性为 99．S6％。CYY3302 与B elkanff的参比菌株 USDA86有最近的亲缘关系，序列相似性近似于 100％。 供试菌株的共生固氮有效性测定结果表明：9株从白蔓豆根瘤中分离的菌株均不能在红蛮豆上结瘤，显示此9个菌株均存在品系专一性。其余供试菌株在红蛮豆上可以结瘤。供试菌株在与红蛮豆的共生体系中，根瘤个数与植株干重呈现多样性，植株干重与所测定的固氮酶活之间相关性不明显，但植株干责与植株含氮量具有一定的相关性，因此能较好的反映各菌株的固氮能力。白蔓豆结痛试验表明，所选来自四个地区属于不同分类地位的6个代表菌株均可在白基Ij上纠痫。更多还原

【Abstract】 Twenty-six strains of rhizobia were isolated and purified from the nodules of 2 cultivars of rice bean (Vigna umbellata L.) collected from the different soils located at various ecological sites in Hubei province, China. These strains and 8 reference strains of other genus and species of rhizobia were comparatively studied by determining their biological characteristics. In addition, the phylogeny of these strains also was tentatively studied.The generation time of all strains tested was between 2.70 and 11.45 hours in YMA liquid medium, which showed great diversity. According to the result of acid-alkali endurance, salt stress, resistance of intrinsic antibiotics, utilization of carbon and nitrogen sources, the biodiversity of rhizobia isolated from rice bean was shown with the dendrogram through cluster analysis. At the similarity of 77%, all strains could be divided into 3 groups. Group I consisted of slow-growing strains. Fast-growing strains fell into groups II and III. The groupsIII were further divided into different subgroups at the similarity of 80%. Similarities of some strains clustered into a subgroup were related with geographical sampling sites.Results of host-plant range test shown that some strains of rhizobia isolated from Vigna umbellata L.could form nodules on Vigna, Arachis, Glycine and Phaseolus. it can be concluded that the rhizobia isolated from Vigna umbellata L. is not an independent cross-inoculation group.Six types of plasmid profiles of fast-growing strains were elucidated with Echardt’s method. The correlation of plasmid type and isolated place of strains were analysized.The genetic diversity and phylogeny of the isolated strains were further studied by using the methodlogy of 16S rDNA PCR-RFLP and 16S-23S rDNA IGS PCR-RFLP. The Result of 16S rDNA PCR-RFLP revealed that all strains could be divided into 6 groups at the similarity of 90%. 3 strains isolated from nodules of Vigna umbellata L var. glaber in Hanchuan fell into group V and were clustered with R.leguminosarum USDA2370 at the similarity of 100%. The similarities of all strains of group VI were up to 100% with Sinorhizobium fredii USDA205 and were in a same "species" level. Other strains were divided into I, II and IV group indicated different divergences with reference strains. It can be referred that these strains maybe respectively belong to other taxonomic positions in phylogenetic branch.All the strains tested were divided into three IGS types according to size of 16S-23S rDNA. At the similarity level of 52%, all the strains isolated from nodules of rice bean could be divided into two groups. Group I was composed of slow-growing strains and could be further divided into two branches: IA and IB, at the similarity of 79%. Group II was composed of fast-growing strains also could be further divided into 2 branches, IIA and IIB,at the similarity of 62%. The branch IIA consists IIA1, IIA2 and IIA3 clusters at the similarity of 72%. The strains of branch IIB were highly related with Sinorhizobium fredii USDA205.For some large groups, the results of 16S rDNA PCR-RFLP and 16S-23S rDNA IGS PCR-RFLP were in good agreement with that of numerical analysis of the phenotypic characteristics. From the above results, it can be concluded that the rhizobia strains isolated from the nodules of rice bean have genetic diversities. 5 strains from Hanchuan, including GAP2502, HCY5105, HCY5202, HCY5503 and HCY5609, had distinct difference with other strains. It indicated that these strains could represent a new phylogenetic branch of Rhizobium. the extensive analysis for the taxonomic position is under way for verification.The partial 16S rDNA sequences of representative strains CYR4243, CYY3302, HCY1101 and HCY5202 were analysized. The results showed that 4 strains respectively fell into different phylogenetic branches. CYR4243 shared 99.87% sequence homology with Sinorhizobium fredii type strain USDA205. HCY1101 was highly related with Rhizobium leguminosarum bv.trifolii and Rhizobium leguminosarum bv.viceae, HCY5202 s更多还原