Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine V.S. Zotov (1), N.V. Punina...
1
Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine V.S. Zotov (1), N.V. Punina (1), S.A. Khapchaeva (1), S.V. Didovych (2), T.N. Melnichuk (2), A.F. Topunov (1) ABSTRACT The new taxonomic marker (hin-region) has been proposed which gives possibility for Rhizobium bacteria study on “species - group of strains” level. Using this marker the groups of Rhizobium strains were determined which could not be distinguished with other methods, and these results correlated with evolutionary similarity of bacteria. The developed approach for creating of marker systems affords to carry out effective inventory of inter- and intra- species genetic diversity of nodulating bacteria and to evaluate perspectives of their use in agriculture. The proposed marker system was used for description of Rhizobium bacteria samples isolated from different ecological-geographical regions of Ukraine and Russia. (1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia, е-mail: [email protected]; (2) Institute of Agriculture of Crimea of National Academy of Agriculture Sciences of Ukraine, Department of microbiology, Simferopol, Ukraine INTRODUCTION Legume family (Fabaceae) includes over 19,000 described species of plants that are usually able to enter into a symbiotic relationships with nitrogen- fixing bacteria forming the nodules on the roots. Bacteria of the genus Rhizobium is one of the representative symbiont of valuable crops, such as Pisum, Lens, Vicia faba, Trifolium, Phaseolus, Galega. Study of the taxonomic structure and biodiversity of this genus is very important due to the fact that knowledge of their genetic structure and polymorphism varietal plant lines reveals the evolutionary dynamics of the variability, and thus predicts the couple of micro- macrosymbiont most adapted to each other. This coordinated selection will allow creating the combinations of symbiont bacteria-plant with a more efficient process of nitrogen fixation for full development and the formation of high yield. At present, as a rule, taxonomy and evolutionary relationships of the order Rhizobiales are studied by comparison of the nucleotide sequences of 16S rRNA gene and MLSA analysis. However, this assessment does not always correspond to the dates obtained by other loci. Using universal primers systems, especially when we are working with a mixture of different microorganisms in one sample, makes MLSA analysis more time-consuming and limited due to the high divergence of specific markers. The adequately rhizobia’s isolates classification and taxonomy can’t always be realizing due to frequent interspecies recombination between different alleles of taxonomically important genes (ribosomal rRNA operon), and the horizontal transfer (sym-genes), that’s why we need to obtain additional genetic data. The genus Rhizobium is particular interest as a model object of this Order, that has a complex taxonomic structure, which is difficult to study by current methods. Previously, in details we’ve studied the genetic diversity of phytopathogenic bacteria of the genus Xanthomonas, affecting Brassica napus in the Moscow region and the North Caucasus region of Russia using hin- region PCR and saAFLP (XbaI) techniques (Zotov et al., 2010). In this study we used these techniques to research the genetic diversity of rhizobia from different eco-geographical zones of Ukraine. MATERIALS AND METHODS Bacterial strains. 73 rhizobial isolates was obtained from root nodules of legume plants (Pisum satіvum, Trifolium repens, T. pratense, Vicia faba, Phaseolus vulgaris, Galega orientalis) growing in different regions of Russia and Ukraine. 12 reference strains of Rhizobium sp. was obtained from cultures collections of Department of microbiology Institute of Agriculture of Crimea of NAAS and All-Russia Research Institute for Agricultural Microbiology RAAS, was investigated. DNA isolation. Total genomic DNAs of all Rhizobium strains were obtained after lysozyme- sodium dodecyl sulfate lysis followed by phenol-chloroform extraction and ethanol precipitation. saAFLP. The AFLP procedure was performed as described by Valsangiacomo (1995), with some modifications. 80 ng of DNA was digested with XmaJI (AvrII) restriction enzyme 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. Primer (Pr.CTAG2: GGCCGTGGTTTTAG- ctag) and PCR conditions were as described previously (Zotov et al., 2012). PCR amplification 16S rRNA gene and 16S-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 ITS_R (Zotov et al., 2012) were used. PCR products was amplified, purified and directly sequenced. Hin-region PCR. Hin-region was amplified by PCR with primers systems described earlier Zotov et al. (2012). PCR fragments were amplified, purified, directly sequenced and aligned using the Clustal W v. 1.75 (Thompson et al., 1994). Phylogenetic trees were constructed with Mega v. 3.1 (Kumar & Nei, 2006), using the neighbor-joining algorithms with bootstrap and genetic distances computed by Kimura’s 2-parameter model (Kimura, 1980). REFERENCES Weisburg W.G., Barns S.M., Pelletier D.A., Lane D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study // J. Bacteriol. Vol. 173. P. 697–703. Normand P., Cournoyer B., Nazaret S., Simonet P., 1992. Analysis of a ribosomal operon in the actinomycete Frankia // Gene. Vol. 111. P. 119–124. Valsangiacomo C., Baggi F., Gaia V., Balmelli T., Peduzzi R., Piffaretti J.-C. 1995. Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies // J. Clin. Microbiol. 33(7):1716-1719. Zotov V.S., Punina N.V., Ignatov A.N.43. et al., 2010. Elaboration and use of new approach to species and strain identification of phytopatological and nitrogen-fixing bacteria // Adaptation to Climate Change in the Baltic Sea Region: Contributions from Plant and Microbial Biotechnology. Program and Abstracts, Finland, P. 87. Zotov V.S., Punina N.V., Khapchaeva S.A., Didovich S.V., Melnichuk T.N., Topunov A.F. 2012. The new taxonomic marker of nodulation bacteria of Rhizobium genus and its evolution // Ekologicheskaya Genetika. St. St.Petersburg. 10(2):49-62 (in russian). CONCLUSION According to the results the collection of the nodule genus Rhizobium can be represented as follows: analysis of nucleotide sequences of 16S rRNA gene reveals of genera and species belonging isolates; test inoculation of host plants determines the specificity of the strains (i.e. biovars); nucleotide polymorphism intergenic region 16S-23S rRNA reflects the degree of intra- and inter- specific recombination; using technique saAFLP we can determine relationships between groups of strains within the proposed genotypes. efficiency of the symbiotic system can be evaluated with hin-region marker; The advantage of hin-region PCR analysis include: the ease and speed of execution, universality of marker system to study intra-generic relationships, high informativity and reproducibility, the ability to work with complex objects (such us plant tissue and soil). Hin-region PCR analysis can be used as a basis for fast and accurate screening of natural populations of the nodule genus Rhizobium by this features. Дендрограмма, построенная методом ближнего соседа (NJ) для 25 штаммов Bt, выделенных на территории Украины по суммарным результатам ERIC-, BOX-ПЦР и saAFLP для 36 полиморфных признаков. Значения бутстрепа расчитывались в 1000 повторениях. Schematic representation of the genomic DNA from rhizobia including reference (blue) and proposed by us (sand color) target markers: ribosomal gene cluster, restriction sites of endonuclease with an average cutting frequency, protein-coding genes, symbiotic genes and intergenic regions. 16S rRNA The nucleotide sequences of 16S rRNA gene analysis showed that isolates belonging to three species: the majority of isolates belonged to R.leguminosarum, one isolate to R.giardinii and 6 isolates did to R.galegae. However, the intraspecific polymorphism in this marker wasn’t identified. ITS The study of nucleotide sequences intergenic region 16S-23S rRNA (ITS) resulted to a significant intraspecific polymorphism. However, due to frequent recombination of this locus and its several genomic copies using of this region for taxonomic purposes is limited. R.leguminosarum bv. viciae 248b R.leguminosarum bv. viciae B-8 (its-L) R.leguminosarum bv. viciae К-29 R.leguminosarum bv. viciae 31 (its-L) R.leguminosarum bv. viciae 96-st R.leguminosarum bv. phaseoli FN-6 (its-L) R.leguminosarum bv. phaseoli FК-0 R.leguminosarum bv. phaseoli FN (its-L) R.leguminosarum bv. viciae 245а-st R.leguminosarum bv. trifolii КК (its-L) R.leguminosarum bv. trifolii КP012 (its-L) R.leguminosarum bv. trifolii КL91 (its-L) R.leguminosarum bv. viciae Y-1 (its-S ) Rhizobium leguminosarum LMG 14904 T (AF345271) R.leguminosarum bv. phaseoli 682 R.leguminosarum bv. trifolii 1326-st R.leguminosarum bv. viciae 261b-st R.leguminosarum bv. viciae B-9 (its-L) R.leguminosarum bv. phaseoli 105 Group of strains "R . leguminosarum LMG14904T" R.leguminosarum bv. viciae B-8 (its-S) R.leguminosarum bv. trifolii КК (its-S ) R.leguminosarum bv. viciae 31 (its-S ) R.leguminosarum bv. viciae 97-st R. leguminosarum bv. viciae Y-7 R.leguminosarum bv. trifolii КL91 (its-S ) R.leguminosarum bv. trifolii КP012 (its-S) R.leguminosarum bv. viciae B-9 (its-S ) Rhizobium gallicum R 602 T clone 1 (AF345267) Rhizobium gallicum R 602 T clone 2 (AF345266) Rhizobium mongolense USDA 1844 T (AF345273) R.leguminosarum bv. phaseoli FN-6 (its-S ) R.leguminosarum bv. phaseoli FА-4 R.leguminosarum bv. phaseoli FN (its-S ) Rhizobium etli LMG 17827 T (AF541974) Rhizobium etli CIAT 656 (NC 010994) G roup ofstrains Rhizobium hainanense US DA 3588 T (AF345269) Rhizobium tropici LMG 9503 T (AF345278) Rhizobium radiobacter LMG 140 T (AF541973) Rhizobium rhizogenes DS M 30148 T (AF345275) Rhizobium lusitanum P1-7 T (AY943641) Rhizobium undicola LMG 11875 T (AF345280) Rhizobium vitis LMG 8750 T (AF345279) R.giardinii FS Rhizobium giardinii H152 T (AF345268) G roup ofstrains "R .giardiniiH 152T" Rhizobium rubi DS M 6772 T clone 1 (AF345276) Rhizobium rubi DS M 6772 T clone 2 (AF345277) Rhizobium huautlense LMG 18254 T (AF345270) R.galegae 926st Rhizobium galegae LMG 6124 T (AF345265) G roup ofstrains "R .galegae LM G 6124T" 100 100 86 91 96 72 100 97 89 86 0.05 Hin - region Eight genotypes, 4 of which are the form of R.leguminosarum were identified on the basis of differences in the structure of the hin- regions nucleotide sequences. The level of genetic diversity within the identified genotypes was 96.2 ÷ 99.9%, and between them - 25.5 ÷ 89.9%. R . leguminosarum (20) - isolates from nodules T.repens, T.pratense and Vicia faba R. leguminosarum bv. viceae Y-7 R. leguminosarum bv. viceae 248b R. leguminosarum bv. viceae Y-9 R. leguminosarum bv. viceae 245а-st Rhizobium leguminosarum bv. trifolii WS M1325 (NC_ 012850) I/B Rhizobium etli 700-st Rhizobium etli CIAT652 (NC 010994) V/F Rhizobium leguminosarum bv. trifolii WS M2304 (NC_ 011369) R. leguminosarum bv. phaseoli 108** R. leguminosarum bv. phaseoli 105 R. leguminosarum bv. phaseoli FN II/C R. leguminosarum bv. phaseoli FК-0 - I/b R. leguminosarum bv. phaseoli 108* - I/b R. leguminosarum bv. phaseoli FК-6 - I/b R. leguminosarum bv. phaseoli FК-4 - I/b Rhizobium leguminosarum bv. viceae 3841 (NC_ 008380) R. leguminosarum bv. viceae 96-st R. leguminosarum bv. viceae 97-st I/B R. leguminosarum bv. viceae 261b-st R. leguminosarum bv. viceae 31 R. leguminosarum bv. viceae К-29 R. leguminosarum bv. trifolii 1326-st R. leguminosarum (26) - isolates from nodules Pisum and Phaseolus I/A III/D R. leguminosarum bv. phaseoli FN-6 IV /E R. giardinii bv. phaseoli FS R. galegae 913-st R. galegae 926-st R. galegae K-18 R. galegae 916-st R. galegae 912-st R. galegae K-3 V I/G 100 100 99 100 98 100 99 78 71 87 89 78 100 94 99 0.05 Previously identified genotypes of rhizobia biovar viciae had variety special specificity, that was established by studying separately all nodules per plant. R. leguminosarum bv. viceae 32-2 R. leguminosarum bv. viceae 28 R. leguminosarum bv. viceae 24 R. leguminosarum bv. viceae Y-4 R. leguminosarum bv. viceae 32 R. leguminosarum bv. viceae Y-3 R. leguminosarum bv. viceae 65 R. leguminosarum bv. viceae Y-1 R. leguminosarum bv. viceae Ch-14 R. leguminosarum bv. viceae Y-5 R. leguminosarum bv. viceae G-8 R. leguminosarum bv. viceae Y-2 R. leguminosarum bv. viceae Y-11 R. leguminosarum bv. viceae Y-12 R. leguminosarum bv. viceae P-2 R. leguminosarum bv. viceae 34 R. leguminosarum bv. viceae 31 R. leguminosarum bv. viceae K-29 R. leguminosarum bv. viceae 261b-st Isolates from nodules Pisum sativum R. leguminosarum bv. viceae 96-st R. leguminosarum bv. viceae 97-st R. leguminosarum bv. viceae 248b R. leguminosarum bv. viceae Y-9 R. leguminosarum bv. viceae 245a-st R. leguminosarum bv. viceae Y-7 R. leguminosarum bv. viceae B-6 R. leguminosarum bv. viceae B-16 R. leguminosarum bv. viceae B-9 R. leguminosarum bv. viceae B-8 R. leguminosarum bv. viceae B-25 R. leguminosarum bv. viceae B-17 R. leguminosarum bv. viceae B-18 R. leguminosarum bv. viceae B-15 Most of isolates from nodules Vicia faba 100 99 73 89 99 67 100 100 94 100 0.01 I/A I/B I/A+I/B I/A I/B Alternative phylogenetic marker - gyrB gene R . leguminosarum (55) R. leguminosarum bv. trifolii 1326st R. leguminosarum bv. viceae 31 R. leguminosarum bv. phaseoli 108* R. leguminosarum bv. phaseoli FК-6 R. leguminosarum bv. phaseoli FN R. leguminosarum bv. phaseoli 108** R. leguminosarum bv. phaseoli FК-4 R. leguminosarum bv. phaseoli FК-0 R. leguminosarum bv. phaseoli 105 R. leguminosarum bv. phaseoli FN-6 R. leguminosarum (65) Rhizobium radiobacter K84 (CP000628) Rhizobium rhizogenes IFO 13257 T (D14501) Rhizobium lusitanum P1-7 T (NR_ 043150) Rhizobium tropici CIAT 899 T (EU488752) Rhizobium hainanense I66 T (U71078) Rhizobium etli CIAT 652 (NC 010994) R. etli 700-st R . etli (1) Rhizobium sullae IS 123 T (Y10170) Rhizobium indigoferae CCBAU 71042 T (AF364068) Rhizobium yanglingense S H22623 T (AF003375) Rhizobium gallicum R602sp T (U86343) Rhizobium mongolense US DA 1844 T (U89817) Rhizobium loessense CCBAU 7190B T (AF364069) Rhizobium galegae ATCC 43677 T (D11343) R. galegae К-3 R. galegae 926-st R. galegae 916-st R. galegae 913-st R. galegae К-18 R. galegae 912-st R. galegae (6) Rhizobium huautlense SO2 T (AF025852) Rhizobium undicola LMG 11875 T (Y17047) Rhizobium vitis NCPPB3554 T (D01258) Rhizobium rubi IFO 13261 T (D01259) Rhizobium tumefaciens NCPPB2437 T (D01256) Rhizobium giardinii H152 T (U86344) R. giardinii FS R . giardinii (1) Mesorhizobium plurifarium LMG 11892 T (Y14158) Mesorhizobium amorphae ACCC 19665 T (AF041442) Mesorhizobium huakuii IFO 15243 T (D13431) Mesorhizobium mediterraneum UPM-Ca36 T (L38825) Mesorhizobium tianshanense US DA 3592 T (AF041447) Mesorhizobium chacoense PR5 T (AJ 278249) Mesorhizobium ciceri UPM-Ca7 T (U07934) Mesorhizobium loti LMG 6125 T (X67229) Mesorhizobium S inorhizobium meliloti LMG 6133 T (X67222) S inorhizobium kummerowiae CCBAU 71714 T (AF364067) S inorhizobium medicae A 321 T (L39882) S inorhizobium arboris HAMBI 1552 T (Z78204) Sinorhizobium kostiense HAMBI 1489 T (Z78203) S inorhizobium terangae LMG 6463 T (X68387) S inorhizobium saheli LMG 7837 T (X68390) S inorhizobium fredii ATCC 35423 (D01272) S inorhizobium xinjiangense IAM 14142 T (D12796) S inorhizobium Bradyrhizobium Bradyrhizobium japonicum DS M 30131 T (X87272) 99 70 72 99 95 93 98 99 99 99 98 96 97 98 71 97 99 97 99 89 86 98 97 99 84 99 0.01
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Transcript of Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine V.S. Zotov (1), N.V. Punina...
Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine
V.S. Zotov (1), N.V. Punina (1), S.A. Khapchaeva (1), S.V. Didovych (2),T.N. Melnichuk (2), A.F. Topunov (1)
ABSTRACTThe new taxonomic marker (hin-region) has been proposed which gives possibility for Rhizobium bacteria study on “species - group of strains” level. Using this marker the groups of Rhizobium strains were determined which could not be distinguished with other methods, and these results correlated with evolutionary similarity of bacteria. The developed approach for creating of marker systems affords to carry out effective inventory of inter- and intra-species genetic diversity of nodulating bacteria and to evaluate perspectives of their use in agriculture. The proposed marker system was used for description of Rhizobium bacteria samples isolated from different ecological-geographical regions of Ukraine and Russia.
(1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia, е-mail: [email protected];(2) Institute of Agriculture of Crimea of National Academy of Agriculture Sciences of Ukraine, Department of microbiology, Simferopol, Ukraine
INTRODUCTIONLegume family (Fabaceae) includes over 19,000 described species of
plants that are usually able to enter into a symbiotic relationships with nitrogen-fixing bacteria forming the nodules on the roots. Bacteria of the genus Rhizobium is one of the representative symbiont of valuable crops, such as Pisum, Lens, Vicia faba, Trifolium, Phaseolus, Galega. Study of the taxonomic structure and biodiversity of this genus is very important due to the fact that knowledge of their genetic structure and polymorphism varietal plant lines reveals the evolutionary dynamics of the variability, and thus predicts the couple of micro-macrosymbiont most adapted to each other. This coordinated selection will allow creating the combinations of symbiont bacteria-plant with a more efficient process of nitrogen fixation for full development and the formation of high yield. At present, as a rule, taxonomy and evolutionary relationships of the order Rhizobiales are studied by comparison of the nucleotide sequences of 16S rRNA gene and MLSA analysis. However, this assessment does not always correspond to the dates obtained by other loci. Using universal primers systems, especially when we are working with a mixture of different microorganisms in one sample, makes MLSA analysis more time-consuming and limited due to the high divergence of specific markers. The adequately rhizobia’s isolates classification and taxonomy can’t always be realizing due to frequent interspecies recombination between different alleles of taxonomically important genes (ribosomal rRNA operon), and the horizontal transfer (sym-genes), that’s why we need to obtain additional genetic data. The genus Rhizobium is particular interest as a model object of this Order, that has a complex taxonomic structure, which is difficult to study by current methods.
Previously, in details we’ve studied the genetic diversity of phytopathogenic bacteria of the genus Xanthomonas, affecting Brassica napus in the Moscow region and the North Caucasus region of Russia using hin-region PCR and saAFLP (XbaI) techniques (Zotov et al., 2010). In this study we used these techniques to research the genetic diversity of rhizobia from different eco-geographical zones of Ukraine.
MATERIALS AND METHODSBacterial strains. 73 rhizobial isolates was obtained from
root nodules of legume plants (Pisum satіvum, Trifolium repens, T. pratense, Vicia faba, Phaseolus vulgaris, Galega orientalis) growing in different regions of Russia and Ukraine. 12 reference strains of Rhizobium sp. was obtained from cultures collections of Department of microbiology Institute of Agriculture of Crimea of NAAS and All-Russia Research Institute for Agricultural Microbiology RAAS, was investigated.
DNA isolation. Total genomic DNAs of all Rhizobium strains were obtained after lysozyme-sodium dodecyl sulfate lysis followed by phenol-chloroform extraction and ethanol precipitation.
saAFLP. The AFLP procedure was performed as described by Valsangiacomo (1995), with some modifications. 80 ng of DNA was digested with XmaJI (AvrII) restriction enzyme 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. Primer (Pr.CTAG2: GGCCGTGGTTTTAG-ctag) and PCR conditions were as described previously (Zotov et al., 2012).
PCR amplification 16S rRNA gene and 16S-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 ITS_R (Zotov et al., 2012) were used. PCR products was amplified, purified and directly sequenced.
Hin-region PCR. Hin-region was amplified by PCR with primers systems described earlier Zotov et al. (2012). PCR fragments were amplified, purified, directly sequenced and aligned using the Clustal W v. 1.75 (Thompson et al., 1994). Phylogenetic trees were constructed with Mega v. 3.1 (Kumar & Nei, 2006), using the neighbor-joining algorithms with bootstrap and genetic distances computed by Kimura’s 2-parameter model (Kimura, 1980).
REFERENCESWeisburg W.G., Barns S.M., Pelletier D.A., Lane D.J., 1991. 16S ribosomal
DNA amplification for phylogenetic study // J. Bacteriol. Vol. 173. P. 697–703.
Normand P., Cournoyer B., Nazaret S., Simonet P., 1992. Analysis of a ribosomal operon in the actinomycete Frankia // Gene. Vol. 111. P. 119–124.
Valsangiacomo C., Baggi F., Gaia V., Balmelli T., Peduzzi R., Piffaretti J.-C. 1995. Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies // J. Clin. Microbiol. 33(7):1716-1719.
Zotov V.S., Punina N.V., Ignatov A.N.43. et al., 2010. Elaboration and use of new approach to species and strain identification of phytopatological and nitrogen-fixing bacteria // Adaptation to Climate Change in the Baltic Sea Region: Contributions from Plant and Microbial Biotechnology. Program and Abstracts, Finland, P. 87.
Zotov V.S., Punina N.V., Khapchaeva S.A., Didovich S.V., Melnichuk T.N., Topunov A.F. 2012. The new taxonomic marker of nodulation bacteria of Rhizobium genus and its evolution // Ekologicheskaya Genetika. St. St.Petersburg. 10(2):49-62 (in russian).
CONCLUSIONAccording to the results the collection of the nodule genus
Rhizobium can be represented as follows: analysis of nucleotide sequences of 16S rRNA gene reveals of
genera and species belonging isolates; test inoculation of host plants determines the specificity of the
strains (i.e. biovars); nucleotide polymorphism intergenic region 16S-23S rRNA reflects
the degree of intra- and inter-specific recombination; using technique saAFLP we can determine relationships between
groups of strains within the proposed genotypes. efficiency of the symbiotic system can be evaluated with hin-region
marker;The advantage of hin-region PCR analysis include: the ease and
speed of execution, universality of marker system to study intra-generic relationships, high informativity and reproducibility, the ability to work with complex objects (such us plant tissue and soil). Hin-region PCR analysis can be used as a basis for fast and accurate screening of natural populations of the nodule genus Rhizobium by this features.
Дендрограмма, построенная методом ближнего соседа (NJ) для 25 штаммов Bt, выделенных на территории Украины по суммарным результатам ERIC-, BOX-ПЦР и saAFLP для 36 полиморфных признаков. Значения бутстрепа расчитывались в 1000 повторениях.
Schematic representation of the genomic DNA from rhizobia including reference (blue) and proposed by us (sand color) target markers: ribosomal gene cluster, restriction sites of endonuclease with an average cutting frequency, protein-coding genes, symbiotic genes and intergenic regions.
16S rRNAThe nucleotide sequences of 16S rRNA gene analysis showed that isolates belonging to three species: the majority of isolates belonged to R.leguminosarum, one isolate to R.giardinii and 6 isolates did to R.galegae. However, the intraspecific polymorphism in this marker wasn’t identified.
ITSThe study of nucleotide sequences intergenic region 16S-23S rRNA (ITS) resulted to a significant intraspecific polymorphism. However, due to frequent recombination of this locus and its several genomic copies using of this region for taxonomic purposes is limited.
R.leguminosarum bv. viciae 248b R.leguminosarum bv. viciae B-8 (its-L) R.leguminosarum bv. viciae К-29 R.leguminosarum bv. viciae 31 (its-L)
R.leguminosarum bv. viciae 96-st R.leguminosarum bv. phaseoli FN-6 (its-L) R.leguminosarum bv. phaseoli FК-0 R.leguminosarum bv. phaseoli FN (its-L)
R.leguminosarum bv. viciae 245а-st R.leguminosarum bv. trifolii КК (its-L) R.leguminosarum bv. trifolii КP012 (its-L) R.leguminosarum bv. trifolii КL91 (its-L) R.leguminosarum bv. viciae Y-1 (its-S) Rhizobium leguminosarum LMG 14904 T (AF345271) R.leguminosarum bv. phaseoli 682 R.leguminosarum bv. trifolii 1326-st R.leguminosarum bv. viciae 261b-st R.leguminosarum bv. viciae B-9 (its-L)
R.leguminosarum bv. phaseoli 105
Group of strains "R. leguminosarum LMG14904T"
R.leguminosarum bv. viciae B-8 (its-S) R.leguminosarum bv. trifolii КК (its-S) R.leguminosarum bv. viciae 31 (its-S) R.leguminosarum bv. viciae 97-st R. leguminosarum bv. viciae Y-7
R.leguminosarum bv. trifolii КL91 (its-S) R.leguminosarum bv. trifolii КP012 (its-S) R.leguminosarum bv. viciae B-9 (its-S)
Rhizobium gallicum R 602 T clone 1 (AF345267) Rhizobium gallicum R 602 T clone 2 (AF345266)
Rhizobium mongolense USDA 1844 T (AF345273) R.leguminosarum bv. phaseoli FN-6 (its-S) R.leguminosarum bv. phaseoli FА-4 R.leguminosarum bv. phaseoli FN (its-S)
Rhizobium etli LMG 17827 T (AF541974) Rhizobium etli CIAT 656 (NC 010994)
Group of strains
Rhizobium hainanense USDA 3588 T (AF345269) Rhizobium tropici LMG 9503 T (AF345278)
Rhizobium radiobacter LMG 140 T (AF541973) Rhizobium rhizogenes DSM 30148 T (AF345275)
Rhizobium lusitanum P1-7 T (AY943641) Rhizobium undicola LMG 11875 T (AF345280)
Rhizobium vitis LMG 8750 T (AF345279) R.giardinii FS
Rhizobium giardinii H152 T (AF345268) Group of strains "R. giardinii H152T" Rhizobium rubi DSM 6772 T clone 1 (AF345276) Rhizobium rubi DSM 6772 T clone 2 (AF345277)
Rhizobium huautlense LMG 18254 T (AF345270) R.galegae 926st Rhizobium galegae LMG 6124 T (AF345265) Group of strains "R. galegae LMG6124T"
100
100
86
9196
72
100
97
89
86
0.05
Hin-regionEight genotypes, 4 of which are the form of R.leguminosarum were identified on the basis of differences in the structure of the hin-regions nucleotide sequences. The level of genetic diversity within the identified genotypes was 96.2 ÷ 99.9%, and between them - 25.5 ÷ 89.9%.
R. leguminosarum (20) - isolates from nodules T.repens, T.pratense and Vicia faba R. leguminosarum bv. viceae Y-7 R. leguminosarum bv. viceae 248b R. leguminosarum bv. viceae Y-9
R. leguminosarum bv. viceae 245а-st Rhizobium leguminosarum bv. trifolii WSM1325 (NC_012850)
I/B
Rhizobium etli 700-st Rhizobium etli CIAT652 (NC 010994) V/F
Rhizobium leguminosarum bv. trifolii WSM2304 (NC_011369) R. leguminosarum bv. phaseoli 108** R. leguminosarum bv. phaseoli 105 R. leguminosarum bv. phaseoli FN
II/C
R. leguminosarum bv. phaseoli FК-0 - I/b R. leguminosarum bv. phaseoli 108* - I/b R. leguminosarum bv. phaseoli FК-6 - I/b R. leguminosarum bv. phaseoli FК-4 - I/b Rhizobium leguminosarum bv. viceae 3841 (NC_008380) R. leguminosarum bv. viceae 96-st R. leguminosarum bv. viceae 97-st
I/B
R. leguminosarum bv. viceae 261b-st R. leguminosarum bv. viceae 31 R. leguminosarum bv. viceae К-29 R. leguminosarum bv. trifolii 1326-st
R. leguminosarum (26) - isolates from nodules Pisum and PhaseolusI/A
III/D R. leguminosarum bv. phaseoli FN-6IV/E R. giardinii bv. phaseoli FS
R. galegae 913-st R. galegae 926-st R. galegae K-18 R. galegae 916-st R. galegae 912-st R. galegae K-3
VI/G100
100
99
100
98
100
99
78
71
8789
78
100
94
99
0.05
Previously identified genotypes of rhizobia biovar viciae had variety special specificity, that was established by studying separately all nodules per plant.
R. leguminosarum bv. viceae 32-2 R. leguminosarum bv. viceae 28 R. leguminosarum bv. viceae 24 R. leguminosarum bv. viceae Y-4 R. leguminosarum bv. viceae 32 R. leguminosarum bv. viceae Y-3 R. leguminosarum bv. viceae 65 R. leguminosarum bv. viceae Y-1 R. leguminosarum bv. viceae Ch-14 R. leguminosarum bv. viceae Y-5 R. leguminosarum bv. viceae G-8 R. leguminosarum bv. viceae Y-2 R. leguminosarum bv. viceae Y-11 R. leguminosarum bv. viceae Y-12 R. leguminosarum bv. viceae P-2 R. leguminosarum bv. viceae 34 R. leguminosarum bv. viceae 31 R. leguminosarum bv. viceae K-29
R. leguminosarum bv. viceae 261b-st
Isolates from nodules Pisum sativum
R. leguminosarum bv. viceae 96-st R. leguminosarum bv. viceae 97-st
R. leguminosarum bv. viceae 248b R. leguminosarum bv. viceae Y-9
R. leguminosarum bv. viceae 245a-st R. leguminosarum bv. viceae Y-7
R. leguminosarum bv. viceae B-6 R. leguminosarum bv. viceae B-16 R. leguminosarum bv. viceae B-9 R. leguminosarum bv. viceae B-8 R. leguminosarum bv. viceae B-25 R. leguminosarum bv. viceae B-17 R. leguminosarum bv. viceae B-18 R. leguminosarum bv. viceae B-15
Most of isolates from nodules Vicia faba
100
9973
89
99
67
100
100
94
100
0.01
I/A
I/B
I/A+I/B
I/A
I/B
Alternative phylogenetic marker - gyrB gene
R. leguminosarum (55)
R. leguminosarum bv. trifolii 1326st R. leguminosarum bv. viceae 31 R. leguminosarum bv. phaseoli 108* R. leguminosarum bv. phaseoli FК-6 R. leguminosarum bv. phaseoli FN R. leguminosarum bv. phaseoli 108** R. leguminosarum bv. phaseoli FК-4 R. leguminosarum bv. phaseoli FК-0 R. leguminosarum bv. phaseoli 105
R. leguminosarum bv. phaseoli FN-6
R. leguminosarum (65)
Rhizobium radiobacter K84 (CP000628) Rhizobium rhizogenes IFO 13257 T (D14501) Rhizobium lusitanum P1-7 T (NR_043150) Rhizobium tropici CIAT 899 T (EU488752) Rhizobium hainanense I66 T (U71078)
Rhizobium etli CIAT 652 (NC 010994) R. etli 700-st R. etli (1) Rhizobium sullae IS 123 T (Y10170) Rhizobium indigoferae CCBAU 71042 T (AF364068) Rhizobium yanglingense SH22623 T (AF003375) Rhizobium gallicum R602sp T (U86343)
Rhizobium mongolense USDA 1844 T (U89817) Rhizobium loessense CCBAU 7190B T (AF364069)
Rhizobium galegae ATCC 43677 T (D11343) R. galegae К-3 R. galegae 926-st R. galegae 916-st R. galegae 913-st R. galegae К-18 R. galegae 912-st
R. galegae (6)
Rhizobium huautlense SO2 T (AF025852) Rhizobium undicola LMG 11875 T (Y17047)
Rhizobium vitis NCPPB3554 T (D01258) Rhizobium rubi IFO 13261 T (D01259)
Rhizobium tumefaciens NCPPB2437 T (D01256) Rhizobium giardinii H152 T (U86344)
R. giardinii FS R. giardinii (1) Mesorhizobium plurifarium LMG 11892 T (Y14158) Mesorhizobium amorphae ACCC 19665 T (AF041442) Mesorhizobium huakuii IFO 15243 T (D13431) Mesorhizobium mediterraneum UPM-Ca36 T (L38825) Mesorhizobium tianshanense USDA 3592 T (AF041447)
Mesorhizobium chacoense PR5 T (AJ278249) Mesorhizobium ciceri UPM-Ca7 T (U07934) Mesorhizobium loti LMG 6125 T (X67229)
Mesorhizobium
Sinorhizobium meliloti LMG 6133 T (X67222) Sinorhizobium kummerowiae CCBAU 71714 T (AF364067) Sinorhizobium medicae A 321 T (L39882) Sinorhizobium arboris HAMBI 1552 T (Z78204)
Sinorhizobium kostiense HAMBI 1489 T (Z78203) Sinorhizobium terangae LMG 6463 T (X68387)
Sinorhizobium saheli LMG 7837 T (X68390) Sinorhizobium fredii ATCC 35423 (D01272) Sinorhizobium xinjiangense IAM 14142 T (D12796)
Sinorhizobium
Bradyrhizobium Bradyrhizobium japonicum DSM 30131 T (X87272)
99
70
72
99
95
93
98
99
99
99
98
96
97
98
71
97
99
97
99
89
8698
97
99
84
99
0.01
