Please cite this article in press as: A. Stefan, et al., Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables, Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.01.007 ARTICLE IN PRESS G Model SYAPM-25895; No. of Pages 9 Systematic and Applied Microbiology xxx (2018) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology j ourna l h omepage: www.elsevier.de/syapm Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables  Andrei Stefan a,1,2 , Jannick Van Cauwenberghe b,c,1 , Craita M. Rosu a , Catalina Stedel a,3 , Nikolaos E. Labrou d , Emmanouil Flemetakis e , Rodica C. Efrose a,∗ a Department of Experimental and Applied Biology, NIRDBS-Institute of Biological Research Iasi, Lascar Catargi 47, 700107 Iasi, Romania b Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n Col. Chamilpa, Cuernavaca, Morelos CP 62210, Mexico c Plant Conservation and Population Biology, Biology Department, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium d Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece e Laboratory of Molecular Biology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece a r t i c l e i n f o Article history: Received 28 September 2017 Received in revised form 18 January 2018 Accepted 22 January 2018 Keywords: Rhizobium leguminosarum symbiovar trifolii Trifolium sp. Multilocus sequence typing (MLST) Population genetic composition Diversity a b s t r a c t The identification and conservation of indigenous rhizobia associated with legume plants and their appli- cation as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diver- sity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phe- notypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic compo- sition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance. © 2018 Published by Elsevier GmbH.  Nucleotide sequence accession numbers: The GenBank accession num- bers for the sequences reported in the present study are KY587863–KY587907 (for 16S rRNA); KY587998–KY588042 (for IGS); KY587908–KY587952 (for atpD); KY587953–KY587997 (for glnII); KY587818–KY587862 (for recA); KY588089–KY588133 (for nodA) and KY588043–KY588088 (for nifH). Acces- sion numbers of the related reference strains are individually specified in the corresponding phylograms. ∗ Corresponding author. E-mail address: rodica.efrose@icbiasi.ro (R.C. Efrose). 1 Authors with equal contribution. 2 Present address: “Grigore Antipa” National Museum of Natural History, Kiseleff 1, 011341 Bucharest, Romania. 3 Present address: University of Thessaly, Department of Biochemistry and Biotechnology, Larisa 41221, Greece. Introduction ‘Rhizobia’ is a collective term used to designate alpha- and betaproteobacteria capable of establishing endosymbiotic mutu- alistic interactions with legumes and fixing atmospheric nitrogen. The rhizobia initiate the formation of new organs on the plant root, called root nodules, in which the bacterial cells divide and differ- entiate. The host plant provides carbon to the rhizobia in exchange for ammonia which is subsequently incorporated into organic com- pounds. As a direct consequence of this interaction, it is estimated that 33–46 Tg nitrogen per year are fixed in agricultural systems, decreasing the need for synthetic nitrogen fertilizers [15] and high- lighting the ecological and economic importance of this symbiosis in sustainable agriculture. Clovers (Trifolium spp.) are one of the most cultivated pasture legumes in temperate areas and are capable of establishing sym- https://doi.org/10.1016/j.syapm.2018.01.007 0723-2020/© 2018 Published by Elsevier GmbH.