Molecular Phylogenetic Analysis on indigenous strains of Sinorhizobium fredii in Taiwan

• Main Authors: Tseng, Wen-Sheng, 曾文聖
• Other Authors: Lin, Liang-Ping
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/01327719306356222809

博士 === 國立臺灣大學 === 農業化學研究所 === 90 === A total 17 strains of fast-growing soybean rhizobia collected in Taiwan, 5 strains of soybean Sinorhizobium fredii from China, were characterized genotypically at different levels of taxonomic resolution by com-puter-assisted analysis of nifDK gene region PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fin-gerprints with BOX, ERIC, and REP primers. In the analysis of IGS RFLPs with three enzymes, nine groups were found, whereas rep-PCR fingerprint-ing revealed an even greater genotypic diversity, with only four of the Tai-wan strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with nif RFLPs in computer-assisted cluster analysis of electrophoretic patterns. Genes for the small-subunit rRNA (16S ribosomal DNA) of nine Si-norhizobium fredii in Taiwan were amplified by the PCR, and the amplicons were cycle sequenced. Our results indicated that all of the strains of fast-growing soybean rhizobia collected in Taiwan and China that we were examined were closely related and were separated from Rhizobium. So we propose that the fast-growing soybean rhizobia collected in Taiwan and China were belonged to the same species of S. fredii. Then, the phylogenetic relationships of 117 strains of the genera Rhizobium, Agrobacterium, Si-norhizobium, and Mesorhizobium were studied by comparing sequences of their small-subunit rRNA. Comparative analysis of the sequence data con-firmed that the genera Sinorhizobium and Mesorhizobium belong to distinct phylogenetic lineages. The genera Rhizobium and Agrobacterium were found to be phylogenetically heterogeneous, and several subgroupings in which Rhizobium and Agrobacterium species were intermixed were evident. The present findings show that the genus and species definitions of these organ-isms are in need of revision. Different possibilities for this are discussed in the light of sequencing data. These nine S. fredii strains in Taiwan were compared by analyzing both the 16S-23S rDNA (rRNA) intergenic spacer (IGS) spacer and the nodD gene by PCR with sequence analysis. Three groups were identified when 16S-23S rDNA IGS analysis was carried out. We present a phylogenetic analysis of nine S. fredii strains in Taiwan and other members of the family Rhizobiaceae by compared with the nodD gene sequences. Phylogenetic analysis of the nodD gene sequences resolved 30 strains into three groups. These groups, A and B, fit within the Sinorhizobium lineage and are closely related to S. fredii and S. meliloti, respectively. The third group C appeared to belong to the Rhizobium species. These relations were confirmed by se-quencing a representative strain from each group. The correlations between the nodD sequences and the rDNA IGS sequences similarity matrices were relatively low; the groupings into nodD types did not correspond to the groupings into chromosomal types. Generally, strains classified into the same sequences group were clustered at levels of similarity of at 100% by previous other typing analysis. We designed a 30 bp probe from the conservative sequences of Rhizo-bium 16S rDNA gene sequences by comparative sequence analysis. The stepwise strategy in this study is to isolate total community DNA and use this DNA as a template for PCR amplification of 16S rRNA genes (rDNA) with domain-specific primers in blank, compost, chemical fertilizer soil. Followed by construction of a clone library for genes encoding 16S rDNA and rapid screening of the library by determining restriction fragment length polymorphisms (RFLPs) of the 16S rDNAs We detected the 187 16S rDNA sequences of clone library by hybridization with the probe. The sex 16S rDNA sequences can be obtained by dot blot hybridizations. The sex 16S rDNA sequces belong the fast-growning rhizobia or Agrobacterium by se-quencing and comparative sequence analysis... We analyzed 187 1.5kb SSU rDNAs amplified from extracted soil DNA, then defied phylotypes with the restriction enzymes AciI, RsaI and BstUI. All phylotypes were divided into 18 groups, the similarity is higher between blank soil and compost soil. Then, we compared the microbial diversity in blank, compost, chemical fer-tilizer soil The indices of blank, compost, chemical fertilizer soil are 0.990、0.986、0.962 for D value；and 0.979、0.977、0.931 for Evenness.