POLYPHASIC ANALYSIS OF BACTERIA RESEMBLING Agrobacterium ISOLATED FROM ROOT NODULES OF Phaseolus vulgaris AND Glycine max IN BRAZIL AND MOZAMBIQUE

Members of the genus Agrobacterium are Gram-negative, Alpha-Proteobacteria associated with soil and plant habitats, known mainly for their pathogenicity and use in biotechnology. The phylogenetic history and taxonomy of the genera Rhizobium and Agrobacterium is not well defined. The taxonomic science makes use of a series of data for the validation of a new taxon. We studied twenty isolates of nodules of common bean (Phaseolus vulgaris) and soybean (Glycine max) of the “Diazotrophic and Plant Growth Promoting Bacteria Culture Collection of Embrapa Soja” (WFCC Collection # 1213, WDCM Collection # 1054). In general, the bacteria were grown in modified YMA (yeast manitol agar) culture medium, for 72 h, at 28 ° C. The physiological tests concerning the use of carbon sources were carried out with the API 50CH kit (BioMérieux), and the antibiogram was performed on Petri dishes with disks of ten different antibiotics. Phenotypic properties were evaluated in YMA + Congo red, and YMA + bromothymol culture media. Several morphophysiological properties were different from those of the closest type strains. The extraction of the total DNA was performed with the DNeasy kit (Quiagem). For the genomic characterization, partial sequences of the 16S rRNA, and of the housekeeping genes gyrB (gyrase), glnII (glutamine synthetase), and recA (recombinant protein A) were obtained. The sequences were aligned and assembled with the program MEGA; in addition, housekeeping genes were concatenated for the multilocus sequencing analysis (MLSA). The phylogenetic tree with the 16S rRNA did not define clearly the species; however, the MLSA analysis pointed out that the strains did not group with any known species, being closer to Agrobacterium (=Rhizobium) radiobacter,Agrobacterium (=Rhizobium) pusense Agrobacterium salinitolerans and Agrobacterium arsenijevicii. Genomic fingerprinting by BOX-PCR indicated unique profiles for most of the strains. Although the strains have been isolated from legume nodules, nodulation ability was not confirmed in greenhouse tests, indicating that they probably lost their nodulation capacity, or that they function as bacteria helpers to improve legume nodulation, or even that they are opportunistic bacteria. Altogether, the results indicate that the strains might belong to new species, and to define that their genome will be sequenced to proceed with the genomic characterization.