Taxonomia e diversidade genética de rizóbios microssimbiontes de distintas leguminosas com base na análise polifásica (Box-PCR e 16 S RNAr) e na metodologia de MLSA

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The term “rhizobia” is generically applied to bacteria capable of fixing the atmospheric nitrogen (N2) and converting it to a form assimilable by the plant, when in symbiosis with plants of the Leguminosae family. However, besides their ecological and economic importance, the rhizobia have not been subject of many studies. Based on the data obtained with the 16S ribosomal gene sequences, five rhizobial genera have been described so far, Rhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium (=Ensifer) and Mesorhizobium, all of them belonging to the alpha subclass of Proteobacteria. In addition, there are other bacteria described recently and also referred as rhizobia, due to be symbiosis of leguminous plants, and that belong to quite different genera, either in the alpha (Devosia, Methylobacterium) or in the beta (Ralstonia, Burkholderia) subclasses. Even though the 16S ribosomal gene is nowadays the molecule more used to estimate the phylogenetics relationships in prokaryotes, the high conservation in the nucleotide sequences of this gene, as observed among the different bacterial groups, is a limitation to its use to species determination. Therefore, several studies have been conducted to determine alternative methodologies to evaluate diversity genetic as well as to rhizobial phylogeny and taxonomy. A methodology widely used in the diversity studies has been the BOX-PCR technique. With this technique it is possible to obtain genomic fingerprints of bacterial strains. More recently, the MLSA (Multi Locus Sequence Analysis) methodology has been proposed as a new strategy to phylogenetic and taxonomic studies in prokaryotes. Due to the high genetic diversity of the tropical rhizobial strains still not well characterized, the aim of this study was to characterize the genetic diversity of rhizobial strains symbionts of several tropical leguminous plants of environmental and agricultural importance. Also, we have evaluated the potential utility of using the BOX-PCR and the MLSA technology in diversity, phylogeny and taxonomy studies. In a first study, the analysis of the dendrograma resulting from the BOXPCR of 68 SEMIAs isolated from 47 distinct legumes was possible to observe a low correlation (7, 6%) to the phylogenetic tree based in the 16S ribosomal gene sequences. However, when the polyphasic analysis using the BOX-PCR and the 16S ribosomal genesequences was used at a proportion of 2:8 (proportion for the relative weight of each analysis), it was possible to observe clusters with high similarities (90%) to the taxonomic groups obtained with the phylogenetic trees based in the 16S ribosomal gene sequences. In a second study using the MLSA methodology with the genes 16S RNAr, atpD, dnaK, glnII, recA and the ITS region in a collection of 169 Bradyrhizobium isolated of 43 distinct legumes, was possible to observe a division of the strains into two well defined clusters, where the first cluster included the type strains from B. japonicum, B. betae, B. liaoningense, B. canariense, B. yuanmingense and the B. japonicum USDA 110 strain, and the second cluster included the strains related to the B. elkanii USDA 76T. A high variability was observed among the atpD gene sequences analyzed, and five strains related to B. elkanii showed variability to this gene not detected before. Another important observation was that the cluster composed by the strains USDA 110, SEMIA 5080 and 6059, all isolated from soybean, were always clustered in all trees obtained from the six different housekeeping gene sequences analyzed. Also, these strains were always distinct from the cluster containing the B.japonicum USDA 6T. Therefore, it was observed that the polyphasic analysis, using the BOXPCRand the 16S ribosomal gene sequences, and the MLSA technique, allowed to access the genetic diversity as well as to infer the phylogenetic relationships of the rhizobia strains analyzed and also to identify possible new species of rhizobia.

ASSUNTO(S)

rizóbio genética vegetal taxonomia vegetal rhizobium plant genetics

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