ARTICLE IN PRESS - Embrapa · often-stressful tropical conditions in Brazil (Hungria and Stacey,...

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Soil Biology & Biochemistry 39 (2007) 1851–1864 Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N 2 with common bean (Phaseolus vulgaris L.) Fabiana Gisele Silva Pinto a,b,c,d , Mariangela Hungria a,c,d, , Fa´bio Martins Mercante e a Universidade Estadual de Londrina, Department of Microbiology, Cx. Postal 60001, 86051-990 Londrina, PR, Brazil b Universidade Estadual do Oeste do Parana´, Campus Cascavel, 85819-110 Cascavel, PR, Brazil c Embrapa Soja, Cx. Postal 231, 86001-970 Londrina, Parana´, Brazil d Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-MCT), Brazil e Embrapa Agropecua´ria Oeste, Cx. Postal 661, 79804-970 Dourados, MS, Brazil Received 30 October 2006; received in revised form 21 December 2006; accepted 2 January 2007 Available online 2 February 2007 Abstract Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N 2 fixation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bacterial genetic diversity; Biological nitrogen fixation; Common bean; Rhizobium etli; Rhizobium tropici 1. Introduction Common bean (Phaseolus vulgaris L.) represents the most important source of protein for the poorer popula- tions of Central and South America, with Brazil being the largest grower and consumer of the legume worldwide (Embrapa, 2006). Phaseolus vulgaris is considered native to the Americas, and domestication thought to have taken place separately in two major centers of genetic diversifica- tion: the Mesoamerican center or northern group (from Mexico to the northern region of South America), and the Andean center or southern group (from southern Peru to the north of Argentina); a third minor domestication center might exist in Colombia (Gepts, 1990; Kami et al., 1995). Archeological evidences indicate that common bean was spread by migration and trade among Indian populations throughout history (Freitas, 2006). An important feature of the common bean plant is its ability to establish a symbiotic partnership with specific bacteria, setting up the biological N 2 -fixation process in root nodules that may supply the plant’s needs for N. Five species of Rhizobium have been described as capable of nodulating—and in most cases fixing N 2 —with common bean: R. leguminosarum bv. phaseoli (Jordan, 1984), R. tropici (Martı´nez-Romero et al., 1991), R. etli bv. phaseoli (Segovia et al., 1993), R. gallicum (bv. gallicum and bv. phaseoli) and R. giardinii (bv. giardinii and bv. phaseoli) (Amarger et al., 1997). Furthermore, other genetically diverse isolates have been isolated from common bean nodules, possibly representing new species (Eardly et al., ARTICLE IN PRESS www.elsevier.com/locate/soilbio 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.01.001 Corresponding author. Embrapa Soja, Cx. Postal 231, 86001-970 Londrina, Parana´ , Brazil. Tel.: +55 43 33716206; fax: +55 43 33717100. E-mail address: [email protected] (M. Hungria).

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