Ecologia e taxonomia de rizóbios de mimosas do Brasil: diversidade e interação com seus hospedeiros de diferentes biomas

Abstract

Brazil is the main diversity center of mimosas in the world, with the Cerrado and Caatinga biomes concentrating about half of all the species described. Although these legumes establish symbiosis predominantly with Beta-rhizobia, members of the subclass Alpha-proteobacteria are also able to nodulate effectively. In Brazil, mimosas are predominantly nodulated by species of the genus Paraburkholderia, and to date, do not include the genus Cupriavidus and other alpha-rhizobia as native symbiotic partners. This thesis brings new biogeographic information about these interactions, and it is organized in two chapters: I. An ecological and taxonomic study of mimosas rhizobia of Amazon, Cerrado, Caatinga and Mata Atlântica biomes; and II. The description of a new species (Rhizobium altiplani) isolated from Mimosa pudica under an anthropic soil of Brasília within the Cerrado domain. The study was based on 724 bacteria derived from nodules of 16 invasive and endemic mimosas species collected from 25 study sites, where M. pudica constituted the main species studied. These isolates were characterized from phenotypic and genotypic attributes, where a dominance of Paraburkholderia species was demonstrated. However, for the first time, the presence of Cupriavidus nodulando mimosas species was observed spontaneously in three of the four biomes studied, while Rhizobium species were well represented in the Cerrado and Amazonian biomes. All collections were grouped from Box-PCR profiles or by comparing phenotypic characteristics profiles, and 188 bacteria were selected for submission to the Koch postulate using M. pudica as host plant. From this total, 106 rhizobia were able to form nodules with different size, color and leg-hemoglobin enzyme activity depending on the inoculated strains. Based on 16S rRNA gene sequences, 98 bacteria presented homologies with 16 alpha and beta-rhizobia species, consisting of P. tuberum, the main symbiont of mimosas in Brazil. The phylogenies of the 16S rRNA and recA genes showed that most of the isolates formed distinct clades for each species, being of low variability intraspecific, with the exception of the isolates of P. nodosa and P. diazotrophica species that showed differences among the populations of different localities. With respect to symbiotic genes (nifH and nodC), there was little evidence of horizontal gene transfer events, with most of the clusters similar to those observed in the conserved gene phylogenies. The strains of the genus Cupriavidus presented genes different from the type strains and similar to those found in Uruguay and Brazil, thus constituting a South American population. From this initial taxonomic study, five isolates belonging to the genus Rhizobium of the Federal District were distinct from any other type strain. This divergence was also confirmed by multilocus sequence analysis, genome comparison and phenotypic tests, and the isolation of a new species, Rhizobium altiplani, was confirmed. Finally, the R. altiplani type strain (BR 10423T) was transformed for expression of the GFP report gene and subjected to a cross inoculation experiment. The results showed that R. altiplani was able to nodulate Phaseolus vulgaris, Vigna unguiculata, Crotalaria juncea and Lupinus angustifolium and M. pudica, however, the effectiveness of nodulation occurred differently among host plants.