Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity
Introduction
The diazotrophic bacteria collectively known as “rhizobia” are capable of nodulating and establishing symbiotic N2-fixing associations with many plant species of the Leguminosae family. Based on polyphasic analyses of phenetic and genetic properties, rhizobia are currently categorized into six genera – Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium [18], [37], [67]. Nevertheless, taxonomic changes have been proposed: first, since phylogenetic analysis based on the 16S rRNA gene shows that Rhizobium, Agrobacterium and Allorhizobium are closely related, they should be joined into a single genus, Rhizobium [74]; second, all Sinorhizobium species should be reclassified into the genus Ensifer [73]. In addition, some diazotrophic symbiotic bacteria isolated in recent years have been classified into non-traditional rhizobial genera, included in both the alpha-Proteobacteria (Methylobacterium, Devosia) and in the beta-Proteobacteria (Burkholderia, Cupriavidus) (=Ralstonia, =Wautersia) classes [18], [37], [67].
The usefulness of the 16S rRNA gene as a molecular marker for assessing phylogeny and taxonomy of prokaryotes has been broadly demonstrated [18], [27], [63], [66], [70], [71], [72], and the gene has also been applied to rhizobial taxonomy [18], [37], [51], [67], [68], [75], [76]. However, the high level of conservation documented in the 16S rRNA [21], [22], [42], [65], [69], and reports that genetic recombination and horizontal gene transfer may also occur among 16S rRNA genes [22], [62] suggest that this marker has shortcomings. On the basis of these observations, as well as to minimize their effects, other genes with a faster evolution rate than the 16S rRNA, but conserved enough to retain genetic information, have been proposed as alternative phylogenetic markers [38], [51], [54], [55]. Requisites are that these genes should be both broadly distributed among taxa and also be present in single copies within a given genome, and the current consensus is that at least five genes are necessary for reliable taxonomic classification [22], [54], [59], [77]. Applying this approach to bacterial taxonomy and elucidation of phylogenetic relationships, multilocus sequence analysis (MLSA) – using sequences of multiple protein-coding genes for genotypic characterization of diverse groups of prokaryotes – has been proposed [22]. MLSA has been used in studies with several genera of prokaryotes, including Burkholderia, Bacillus, Vibrio, Mycobacterium and Ensifer [22], [38], [59], [60]. Expectations are that these analyses will have a positive impact on how we perform taxonomic and biodiversity studies [60].
Common bean (Phaseolus vulgaris L.) represents the most important source of protein for low-income populations in Latin America and in Africa, and Brazil is its largest producer and consumer worldwide [13]. It is generally accepted that there are two major centers of genetic diversification of common bean, the Mesoamerican (Mexico, Colombia, Ecuador and northern Peru, probably the primary center), and the Andean center (southern Peru to northern Argentina). Although wild relatives of common beans are not indigenous to Brazil, beans have been cultivated there throughout recorded history [11], [17], [20], [32], [33]. The symbionts of common bean were first classified as Rhizobium phaseoli, based on the cross-inoculation-group concept [16]. In 1984, using a numerical taxonomy approach, they were reclassified into a new species, Rhizobium leguminosarum, which contains three biovars, named after their main host species – bv. viciae (Pisum sativum), bv. trifolii (Trifolium spp.) and bv. phaseoli (common bean) [31]. Advances in molecular biology technology as well as isolation of new strains from various parts of the world led to the description of several differences among common-bean rhizobia, such that they were pooled into two main groups, denominated type I and type II [9], [39], [40], [48]. In 1991, a new species, Rhizobium tropici, was described for the type II strains, with two subgroups, type IIA and type IIB [41]. Segovia et al. [50] then proposed that some rhizobia isolated from American soils and initially classified as type I should be reclassified into the new species Rhizobium etli, and two new species, Rhizobium gallicum and Rhizobium giardinii, were described by Amarger et al. [5], the latter forming non-N2-fixing nodules. Finally, a new biovar of Ensifer (=Sinorhizobium) meliloti characterized by tolerance to salinity was isolated from a Tunisian oasis, and termed biovar mediterranense [46].
Common bean is promiscuous in its symbiotic relationships [44] and its widespread and long-term cultivation in Brazil probably explains the large and diverse populations of its rhizobial symbionts detected in surveys. All described Rhizobium species have been found in Brazilian soils except for R. gallicum, as well as species of other genera such as Mesorhizobium and Ensifer and putative new species [23], [25], [26], [28], [29], [30], [45], [47], [57]. However, despite the socioeconomic importance of the legume in Brazil and the broad diversity of the rhizobial population, taxonomic and phylogenetic knowledge of indigenous isolates remains limited. The development of MLSA presents an opportunity to improve the identification of rhizobia in general and to determine the phylogenetic relationships among common-bean rhizobia, with emphasis on strains of R. tropici indigenous to Brazil.
Section snippets
Strains, culture conditions and DNA extraction
A total of 18 common-bean rhizobial strains were analyzed in this study, 12 indigenous to Brazil (detailed information given in Table 1) and six type/reference common bean Rhizobium strains, as follows: R. tropici strain CIAT 899T (type B) (=USDA 9030; =ATCC 49672; =UMR1899; =TAL 1797; =HAMBI 1163; =CM01; =SEMIA 4077; DSM 11418; BR 322), R. tropici type A strain CFN 299 (=USDA 9039; =LMG 9517; =UMR1026; =CENA 183), and R. etli bv. phaseoli strain CFN 42T (=USDA 9032; ATCC 51251; DSM 11541) were
MLSA
We used the MLSA approach to analyze 12 common-bean rhizobia indigenous to Brazil, chosen to represent large groups of similar strains and selected from a collection of over 850 isolates obtained from thirty-three states in three regions of the country: the northeast (close to Ecuador, high temperatures, low-fertility soils), the Cerrados (savannah, acidic soils, high temperatures and a yearly dry season of about 7 months), and the southern (milder temperatures and more fertile soils) regions
Acknowledgments
The work was partially supported by CNPq/MCT, projects Culture Collections (552393/2005-3 and MAPA 577933/2008-6), PROTAX (563960/05-1), and Research Productivity (300698/2007-0). F.L. Thompson is grateful for grants from CNPq, IFS, and FAPERJ. The authors thank Ligia M.O. Chueire, Pâmela Menna and Jesiane S.S. Batista (Embrapa Soja) for assistance in several steps of this work and Allan R.J. Eaglesham for helpful discussion in the manuscript.
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