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Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania

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Abstract

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

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Acknowledgements

This work was supported by the Executive Agency for Higher Education, Research, Development and Innovation Funding [PN-II-ID-PCE-2011-3-1011 funding grant; 292/2011]. The authors are grateful to Dr. Liliana Sfichi Duke for revising the English text and proof reading of the manuscript.

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  1. Rodica C. Efrose and Craita M. Rosu have contributed equally to this work.

Authors and Affiliations

  1. Department of Experimental and Applied Biology, NIRDBS-Institute of Biological Research Iasi, Lascar Catargi 47, 700107, Iasi, Romania

    Rodica C. Efrose, Craita M. Rosu, Catalina Stedel & Andrei Stefan

  2. Department of Biochemistry and Biotechnology, University of Thessaly, 41221, Larisa, Greece

    Catalina Stedel

  3. Department of Plant Science, University of Agricultural, Science and Veterinary Medicine “Ion Ionescu de la Brad”, Mihail Sadoveanu 3, 700490, Iasi, Romania

    Culita Sirbu

  4. Department of Biology, “Alexandru Ioan Cuza” University of Iasi, Carol I 22, 700505, Iasi, Romania

    Lucian D. Gorgan

  5. Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

    Nikolaos E. Labrou

  6. Laboratory of Molecular Biology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

    Emmanouil Flemetakis

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Rodica C. Efrose, Craita M. Rosu, Catalina Stedel, Andrei Stefan, Culita Sirbu, Lucian D. Gorgan, Nikolaos E. Labrou and Emmanouil Flemetakis declares that they have no conflict of interest.

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Efrose, R.C., Rosu, C.M., Stedel, C. et al. Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania. Antonie van Leeuwenhoek 111, 135–153 (2018). https://doi.org/10.1007/s10482-017-0934-3

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  • DOI: https://doi.org/10.1007/s10482-017-0934-3

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