Mesorhizobium mediterraneum and Mesorhizobium tianshanense – symbionts of sainfoin

1. Mesorhizobium mediterraneumand Mesorhizobium tianshanense – symbionts of sainfoin S.A. Khapchaeva (1), N.V. Punina(1,2), V.S. Zotov(1), S.V. Didovych(3), A.F. Topunov(1) (1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia;е-mail: hapchaeva.90@mail.ru (2) Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia; hin-enkelte@yandex.ru (3) Institute of Agriculture of Crimea, National Academy of Agrarian Sciences of Ukraine, Simferopol, Crimea, Ukraine; е-mail: sv-alex.68@mail.ru ABSTRACT The Onobrychis genus comprises a few agronomically important forage legume species, with sainfoin being the most widespread. The Onobrychis genus belongs to the Fabaceae family and Hedysareae tribe. It is widespread in temperate zones of North America, Europe, Mediterranean and Western Asia. There are a lot of specific and ecological varieties of the sainfoin in natural flora. The main cultivated species are O. viciafolia, O. antasiatica, O. arenaria. Recent research works have highlighted several additional beneficial properties of Onobrychisfor livestock, and this is mainly due to the nature of its particular secondary metabolites. Furthermore, it is known to enhance diversity and stability of agroecosystems, representing a valuable pollen and nectar source for honey production. In comparison to other legumes, the nitrogen fixation rate of Onobrychis genus has been measured in terms of both the amount of nitrogen fixed and expressed in terms of resultant increase in yield. Sainfoin is readily nodulated by many bacteria species such as Phyllobacterium, Rhizobium, andMesorhizobium, whichcan form the nodules on roots and can thus fix atmospheric nitrogen. However, they haven’t been examined in details. That is why the previous investigations of taxonomy of sainfoin's nodule bacteria did not give the whole notion about the simbiont’s biodiversity. So the investigation of phylogeny of sainfoin’ssymbionts by complex approach targeted to investigation of housekeeping and symbiotic parts of a genome is actual and perspective task. • RESULTS • The nucleotide sequences of the 16SrRNA for every investigated strain had been determined. Strains were referred into 2 species: M. tianshanense and M. mediterraneum, that firstly was found for sainfoinsymbionts. • Phylogeny by ITS-region revealed the presence of frequent recombination events between species. • The specific strain’s membership was confirmed by analysis of gyrB gene sequences. • By using of saAFLP analysis the significant interspecies diversity was showed. Investigated strains were divided into 6 genetically heterogeneous groups of strains. • The investigated strains were historically developed independently. Phylogeny of symbiotic genes (nodC and nifH ) correlate with host legume systematics. INTRODUCTION In prairie and foothill regions of Crimea for the first time 6isolates of symbiotic bacteria from sainfoin’s nodules were collected. These nodule bacteria were analyzed using fingerprinting saAFLP with rare-cutting restriction enzymes XmaJI,XbaI, and also by analysis of nucleotide sequences of internally transcribed spacer region between the 16S and 23S rRNA genes (ITS), 16S rRNA, gyrB, and symbiotic nodC (synthesis of oligomers of Nod factors) and nifH (synthesis of nitrogenase iron protein) genes. Fig 1. Phylogenetic tree constructed based on sequences16 S rRNAgene of 18 strains Mesorhizobium sp. using algorithm NJ, bootstrap value was 1000 replica MATERIALS AND METHODS Bacterial strains.6 rhizobial isolates were obtained from root nodules of legume plant – Onobrychis sp. growing in Crimea, Ukraine. DNA isolation.Total genomic DNAs of all rhizobial isolates were obtained after lysozyme-sodium dodecyl sulfate lysis followed by phenol-chloroform extraction and ethanol precipitation. PCR amplification 16S rRNA gene and 16-23S rRNA (ITS). The primers fD1 and rD1 were used to amplify 16S rRNA genes (Weisburg et al., 1991). For ITS amplification, the primers FGPS1490-72 (Normand et al., 1992) and FGPL 132 (Navarro et al., 1992) were used. PCR products were amplified, purified and directly sequenced. PCR amplification gyrB gene. GyrB was amplified with primers systems described earlier Yamamoto and Harayama (1995). The gene fragments were amplified, PCR-products were purified,were sequensed(Sanger et al., 1977). saAFLP.The AFLP procedure was performed as described by Valsangiacomo (1995), with some modifications. 80ngof DNA was digested with XmaJI, XbaI restriction enzymes and ligated with single adapter specific for restriction half-site. The restriction-ligation reaction was performed at 37°C for 2 h in a total volume of 20 μl (Zotov et al., 2012) PCR amplification nodC and nifH genes. NodC and nifH genes was amplified by PCR with primers systems nodCF`, nodCI` and nifHF, nifHI (Laguerre et al., 2001), respectively. Fig 3. Phylogenetic treeconstructed based on sequencesnifH and nodC genes of 54 strains Mesorhizobium sp.using algorithm NJ, bootstrap value was 1000 replica REFERENCES Baimiev A.K., Baimiev A.K., Gubaidullin I.I., Kulikovo O.L. and Chemeris A.V. // RussianJournal of Genetics (2007) 43: 587–590. CarboneroC.H., Mueller-HarveyI., BrownT.A., SmithL. // Plant Genetic Resources: Characterization and Utilization (2011) 9(1): 70–85. Gigova L., Petrova N., Mihaylova O., Drevon, J.J. // Microbios. (1997) 90: 137–150. HumeL.J.,WithersN.J., RhoadesD.A. // New Zealand Journal ofAgricultural Research (1985) Vol. 28: 337-348. Laguerre G., Nour S., Macheret V., Sanjuan J., Drouin P., Amarger N. // Microbiology (2001) 147, 981–993. ZotovV.S., Punina N.V., Khapchaeva S.A., DidovychS.V., Melnichuk T.N., Topunov A.F. // Russian Journal of Genetics: Applied Research (2013) Vol. 3, No. 2, pp. 102–113. Fig 2. Phylogenetic treeconstructed based on nucleotide sequences intergenic region 16-23SrRNA (ITS) of 78 strains Mesorhizobium sp.using algorithm NJ, bootstrap value was 1000 replica ACKNOWLEDGEMENT This research was made within the framework of contract about scientific cooperation between A.N. Bach Institute of Biochemistry RAS and Department of Microbiology, Institute of Agriculture of Crimea NAAS of Ukraine, by financial support of «Greenwide» company, and by target grant of Presidium RAS «Support of young scientists».