Abstract
Common bean [Phaseolus vulgaris (Linnaeus)] is the key source of protein, carbohydrates and micronutrients for over 300 million people in the tropics. Like many legumes, P. vulgaris can fix atmospheric nitrogen in symbiosis with rhizobia, alleviating the need for the expensive and polluting N-fertilizers. The crop is known to nodulate with a wide range of rhizobia and, although Brazil is not a center of genetic origin/domestication of P. vulgaris, a variety of rhizobial species have been found as symbionts of the legume. Mato Grosso do Sul (MS) is one of the largest common bean producer states in Brazil, with reports of high yields and abundant natural nodulation. The objective of this study was to evaluate the diversity of 73 indigenous rhizobia isolated from common bean grown in 22 municipalities of MS. Great morphophysiological and genetic diversity was found, as indicated by the six and 35 clusters formed, considering the similarity level of 75 and 70%, respectively, for the phenotypic and rep-PCR dendrograms. Eleven representative isolates were selected for detailed genetic characterization using 16S rRNA and three protein-coding housekeeping genes, glnII, gyrB and recA. We identified species originated from the centers of origin/domestication of the legume, R. etli and R. phaseoli, species probably indigenous of Brazil, R. leucaenae and others of the Rhizobium/Agrobacterium clade, in addition to putative new species. The results highlight the great rhizobial diversity of the region.
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Acknowledgements
This study was idealized by Dr. Fabio Martins Mercante (1963-2016), an extraordinary Brazilian scientist who dedicated his career to studies of biological nitrogen fixation with common bean, giving a remarkable contribution to the area, including from the description of new species to the identification of elite strains for the crop. The authors thank the assistance of Renan A. Ribeiro and Jakeline R.M. Delamuta (Embrapa Soja) on the BOX-PCR, 16S rRNA and housekeeping gene analyses and Rubson N.R. Sibaldelli (Embrapa Soja) for confection of Fig. 1. Our research group is supported by the INCT-Plant-Growth Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-4, Fundação Araucária-STI, CAPES). M.R. Costa acknowledges a PhD fellowship from CAPES-Embrapa (15/2014) and M. Hungria a research fellowship from CNPq (300878/2015-0).
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Fábio Martins Mercante died before publication of this work was completed.
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Figure S1 - Phylogenetic tree based on glnII (307 bp) gene sequences of 11 rhizobial isolates sampled in Mato Grosso do Sul (in bold) and type strains (T). The tree was reconstructed by the maximum-likelihood method using the best model of sequence evolution and the robustness of branching was estimated with 1,000 bootstraps. Only confidence levels = 70% are shown at the internodes. The scale bar indicates 2 substitutions per 10-nucleotide positions; a–e represent the five clusters formed with the isolates. (PPTX 57.0 kb)
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Figure S2 - Phylogenetic tree based on gyrB (495 bp) gene sequences of 11 rhizobial isolates sampled in Mato Grosso do Sul (in bold) and type strains (T). The tree was reconstructed by the maximum-likelihood method using the best model of sequence evolution and the robustness of branching was estimated with 1,000 bootstraps. Only confidence levels = 70% are shown at the internodes. The scale bar indicates 1 substitution per 10-nucleotide positions; a–e represent the five clusters formed with the isolates. (PPTX 56.1 kb)
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Figure S3 - Phylogenetic tree based on recA (273 bp) gene sequences of 11 rhizobial isolates sampled in Mato Grosso do Sul (in bold) and type strains (T). The tree was reconstructed by the maximum-likelihood method using the best model of sequence evolution and the robustness of branching was estimated with 1,000 bootstraps. Only confidence levels = 70% are shown at the internodes. The scale bar indicates 1 substitution per 10-nucleotide positions; a–e represent the five clusters formed with the isolates. (PPTX 62.2 kb)
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Costa, M.R., Chibeba, A.M., Mercante, F.M. et al. Polyphasic characterization of rhizobia microsymbionts of common bean [Phaseolus vulgaris (L.)] isolated in Mato Grosso do Sul, a hotspot of Brazilian biodiversity. Symbiosis 76, 163–176 (2018). https://doi.org/10.1007/s13199-018-0543-6
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DOI: https://doi.org/10.1007/s13199-018-0543-6