Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems Luciana Grange 1 , Mariangela Hungria * Embrapa Soja, Cx. Postal 231, 86001-970, Londrina, PR, Brazil Received 25 March 2003; received in revised form 27 February 2004; accepted 1 March 2004 Abstract Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi- arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Apore ´ were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty- five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 8C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 8C, and not all R. tropici were able to nodulate Leucaena. q 2004 Elsevier Ltd. All rights reserved. Keywords: ERIC-PCR; Nitrogen fixation; RFLP; Rhizobium etli; Rhizobium tropici; Rhizobium leguminosarum; 16 S rRNA 1. Introduction Common bean (Phaseolus vulgaris L.), here referred to simply as bean, is widely cropped in Central and South America and in Africa, where it is an important source of protein for human consumption. Both Mesoamerica and the Andean region of South America are the centers of origin and/or domestication of P. vulgaris (Kaplan, 1965, 1980; Kaplan et al., 1973; Gepts, 1990), and an important secondary center of origin was identified in the southern Andes of Argentina, for the wild bean P. vulgaris var. aborigineus Burk (Baudet) (Gepts, 1990). At least six rhizobia are able to nodulate and in most cases fix atmospheric nitrogen with beans, including Rhizobium leguminosarum bv. phaseoli (Jordan, 1984), R. tropici (Martı ´nez-Romero et al., 1991), R. etli (Segovia et al., 1993), R. gallicum and R. giardinii (Amarger et al., 1997). Bradyrhizobium(Lange, 1961; Hungria et al., 1993), Rhizobium sp. OR 191 isolated from alfalfa nodules (Eardly et al., 1985), and R. mongolense (van Berkum et al., 1998) and other isolates with distinct phylogenic positions (Bromfield and Barran, 1990; Eardly et al., 1992, 1995) may well represent additional species. However, although P. vulgaris is able to perceive signals for nodulation from many rhizobia, most of the interactions are not effective (Michiels et al., 1998). Bean rhizobia from the Americas are believed to have been disseminated to other continents on seeds. 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.03.005 Soil Biology & Biochemistry 36 (2004) 1389–1398 www.elsevier.com/locate/soilbio 1 MSc at the Universidade Estadual de Londrina, Department of Genetics. * Corresponding author. Tel.: þ 55-43-3716206; fax: þ 55-43-3716100. E-mail address: hungria@cnpso.embrapa.br (M. Hungria).