Mikrobiol. Z. 2020; 82(2):38-50.
doi: https://doi.org/10.15407/microbiolj82.02.038

Phenotypic and Genotypic Properties of Bradyrhizobia Nodulating Leguminous Plants
of the Glycine, Vigna and Lupinus Genera

D.V. Krutylo

Institute of Agricultural Microbiology and Agro-Industrial Manufacture, NAAS of Ukraine
97 Shevchenko Str., Chernihiv, 14027, Ukraine

Objective. To study phenotypic and genotypic features of nodule bacteria – microsymbionts of leguminous plants of the Glycine, Vigna and Lupinus genera. Methods. Serological affinity of 12 rhizobia strains was determined in agglutination reactions. Host specificity of strains was studied in vegetation experiments. RFLP analysis of rpoB gene was performed using MspI, HaeIII and NdeII restrictases. Sequencing of the 16S-23S rDNA intergenic spacer of rhizobia was performed on ABI 3130 Genetic Analyzer. Results. It was established that studied rhizobia strains differ significantly in serological properties and belong to 7 serogroups: КВ11, М8, 1967, 46, В1, В2 and 367а. Microsymbionts of soybean, cowpea and mung bean form the group of cross inoculation, however, are unable to infect lupine. Alternatively, lupine rhizobia B. lupini 367а and Bradyrhizobium sp. ЛД4, enter into symbiotic interactions with lupine, however, they do not nodulate soybean. B. japonicum 631 strain is capable of symbiosis with leguminous plants of different tribes: Phaseoleae and Lupineae. Based on RFLP analysis of rpoB gene, rhizobia were grouped into four clusters with following microsymbionts: I – cowpea, II – mung bean and soybean, III – slow-growing soybean nodule bacteria, IV – lupine rhizobia and intensive-growing soybean rhizobia. 16S-23S rDNA sequencing confirmed pertinence of soybean rhizobia to the B. japonicum species (USDA 4, USDA 6, USDA 123 genetic groups). This species also included mung bean microsymbionts (USDA 4 group). Bradyrhizobium sp. В11 isolate was obtained for the first time from cowpea nodules, and it was identified as a new B. diazoefficiens species (USDA 110 group). ITS regions of B. lupini 367а and Bradyrhizobium sp. ЛД4 rhizobia were found to be identical. B. japonicum 631 strain has a 100% similarity with B. lupini 367a strain according to 16S-23S rDNA, and with high degree of probability can be included to B. lupini species. Conclusions. Serological heterogeneity of rhizobia from root nodules of plants of the Glycine, Vigna and Lupinus genera has been demonstrated. It was established that microsymbionts of soybean, cowpea and mung bean belong to one group of cross inoculation. 16S-23S rDNA sequencing allowed to classify the studied strains as different genetic groups and identify them as B. japonicum, B. diazoefficiens and B. lupini. The serological grouping of nodule bacteria was found to coincide with the genetic (rpoB gene and ITS region) grouping, and their host specificity was related to species affiliation.

Keywords: Bradyrhizobium japonicum, B. diazoefficiens, B. lupini, RFLP analysis, 16S-23S rDNA, rpoB gene, soybean, cowpea, mung bean, lupine.

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  1. Jordan D. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int J Syst Evol Microbiol. 1982; 32(1):136–9. https://doi.org/10.1099/00207713-32-1-136
  2. de Souza JAM, Alves LMC, de Mello Varani A, de Macedo Lemos EG. The family Bradyrhizobiaceae. In: The Prokaryotes, Eds.: E. Rosenberg, EF. DeLong, S. Lory, E. Stackebrandt, F. Thompson. Springer: Berlin/Heidelberg, Germany; 2014.
  3. Kuykendall LD, Saxena B, Devine TE, Udell SE. Genetic diversity in Bradyrhizobium japonicum Jordan 1982 and a proposal for Bradyrhizobium elkanii sp. nov. Can J Microbiol. 2011; 38(6):501–5. https://doi.org/10.1139/m92-082
  4. Xu LM, Ge C, Cui Z, Li J, Fan H. Bradyrhizobium liaoningense sp. nov. isolated from the root nodules of soybeans. Int J Syst Bacteriol. 1995; 45:706–11. https://doi.org/10.1099/00207713-45-4-706
  5. Yao ZY, Kan FL, Wang ET, Wei GH, Chen WX. Characterization of rhizobia that nodulate legume species of the genus Lespedeza and description of Bradyrhizobium yuanmingense sp. nov. Int J Syst Evol Microbiol. 2002; 52:2219–30. https://doi.org/10.1099/ijs.0.01408-0
  6. Avontuur JR, Palmer M, Beukes CW, Chan WY, Coetzee MPA, Blom J, Stępkowski T, Kyrpides NC, Woyke T, Shapiro N, Whitman WB, Venter SN, Steenkamp ET. Genome-informed Bradyrhizobium taxonomy: where to from here? Syst Appl Microbiol. 2019. https://doi.org/10.1016/j.syapm.2019.03.006
  7. Stepkowski T, Banasiewicz J, Granada CE, Andrews M, Passaglia LM. Phylogeny and phylogeography of rhizobial symbionts nodulating legumes of the tribe Genisteae. Genes. 2018; 9(3):163. https://doi.org/10.3390/genes9030163
  8. Silva FV, Simões-Araújo JL, Silva Júnior JP, Xavier GR, Rumjanek NG. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant. Brazil J Microbiol. 2012; 43:682–91. https://doi.org/10.1590/S1517-83822012000200033
  9. Velázquez E, Valverde A, Rivas R, Gomis V, Peix Á, Gantois I, Igual JM, León-Barrios M, Willems A, Mateos PF, Martínez-Molina E. Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium. Antonie van Leeuwenhoek. 2010; 97:363–76. https://doi.org/10.1007/s10482-010-9415-7
  10. Delamuta JRM, Ribeiro RA, Ormeño-Orrillo E, Melo IS, Martınez-Romero E, Hungria M. Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov. Int J Syst Evol Microbiol. 2013; 63:3342–51. https://doi.org/10.1099/ijs.0.049130-0
  11. Peng GX, Tan ZY, Wang ET, Reinhold-Hurek B, Chen WF, Chen WX. Identification of isolates from soybean nodules in Xinjiang Region as Sinorhizobium xinjiangense and genetic differentiation of S. xinjiangense from Sinorhizobium fredii. Int J Syst Evol Microbiol. 2002; 52:457–62. https://doi.org/10.1099/00207713-52-2-457
  12. Chen WX, Wang ET, Li YB, Chen XQ, Li Y. Characterization of Rhizobium tianshanense sp. nov. moderately and slowly growing nodule bacterium isolated from an acid saline environment in Xinjiang, People’s Republic of China. Int J Syst Bacteriol. 1995; 45:153–9. https://doi.org/10.1099/00207713-45-1-153
  13. Fotev YuV, Sidorova KK, Novikova TI, Belousova VP. [Study of nodulation and nitrogen fixation in two cowpea Vigna unguiculata (L.) Walp. cultivars inoculated with different strains of Bradyrhizobium sp.] Vavilov Journal of Genetics and Breeding. 2016; 20(3):348–54. Russian. https://doi.org/10.18699/VJ16.099
  14. Tampakaki AP, Fotiadis CT, Ntatsi G, Savvas D. Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece. Syst Appl Microbiol. 2017; 40(3):179–89. https://doi.org/10.1016/j.syapm.2017.01.001
  15. Mpepereki S, Wollum AG, Makonese F. Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soil. Plant Soil. 1996; 186:167–71. https://doi.org/10.1007/BF00035071
  16. Peix A, Ramírez-Bahena MH, Flores-Félix JD, Alonso de la Vega P, Rivas R, Mateos PF, Igual JM, Martínez-Molina E, Trujillo ME, Velázquez E. Revision of the taxonomic status of the species Rhizobium lupini and reclassification as Bradyrhizobium lupini comb. nov. Int J Syst Evol Microbiol. 2015; 65:1213–19. https://doi.org/10.1099/ijs.0.000082
  17. Duran D, Rey L, Sanchez-Canizares C, Navarro A, Imperial J, Ruiz-Argueso T. Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariaejosephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain. Syst Appl Microbiol. 2013; 36:128–36. https://doi.org/10.1016/j.syapm.2012.10.008
  18. El Hilali I, El Jamali J, Thami Alami I, Filali Maltouf A. Characterization and biodiversity of a fast-growing rhizobacterial population nodulating lupine in Morocco. Int J New Tech Res. 2016; 12:27–37.
  19. Pudełko K. Diversity among field populations of bacterial strains nodulating lupins in Poland. Fragm Agron. 2010; 7:107–16.
  20. Krutylo DV, Nadkernychna OV, Kovalevska TM, Patyka VP. [Biological diversity of soybean nodule bacteria in soils of Ukraine]. Mikrobiol Z. 2008; 70(6):27–34. Ukrainian.
  21. Krutylo DV, Volkova IV. [Serological diversity of soybean nodule bacteria in Ukraine soils]. Agroecological Journal. 2012; 4:66–71. Ukrainian.
  22. Kots SYa, Morgun VV, Patyka VF, Datsenko VK, Krugova ED, Kyrychenko EV et al. [Biological nitrogen fixation: legume-rhizobial symbiosis]. Vol. 1. Kyiv: Logos, 2010. Russian.
  23. Kebot E, Meyer B. [Experimental immunology]. Moscow: Medicina Publ.; 1968. Russian.
  24. Hardy RWF, Holsten RD, Jackson EK, Burns RC. The acetylene-ethylene assay for nitrogen fixation: laboratory and field evaluation. Plant Physiol. 1968; 43(8):1185–1207. https://doi.org/10.1104/pp.43.8.1185
  25. Beringer JE. R1 transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974; 84:188–98. https://doi.org/10.1099/00221287-84-1-188
  26. Normand P, Ponsonnet C, Nesme X, Neyra M, Simonet P. ITS analysis of prokaryotes. Mol Microbial Ecology Manual. 1996; 5(3–4):1–12.
  27. Ponsonnet C, Nesme X. Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions. Arch Microbiol. 1994; 161:300–9. https://doi.org/10.1007/BF00303584
  28. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22:4673–80. https://doi.org/10.1093/nar/22.22.4673
  29. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol. 2013; 30:2725–29. https://doi.org/10.1093/molbev/mst197
  30. Nei M, Kumar S. Molecular evolution and phylogenetics. Oxford, UK: Oxford University Press; 2000.
  31. Martens M, Dawyndt P, Coopman R, Gillis M, De Vos P, Willems A. Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int J Syst Evol Microbiol. 2008; 58:200–14. https://doi.org/10.1099/ijs.0.65392-0
  32. Rivas R, Martens M, De Lajudie P, Willems A. Multilocus sequence analysis of the genus Bradyrhizobium. Syst Appl Microbiol. 2009; 32:101–10. https://doi.org/10.1016/j.syapm.2008.12.005
  33. Spaink HP, Kondorosi A, Hooykaas P. [The Rhizobiaceae: Molecular biology of model plant-associated bacteria]. St.-Petersburg: Biont; 2002. Russian.
  34. Safronova VI, Chizhevskaya EP, Belimov AA, Pavlova EA. [Taxonomy of microsymbionts of Hedysarum and Astragalus basing on ribosomal RNA genes sequencing]. Agricultural Biology. 2011; 3:61–4. Russian.
  35. Wang ET, Tian CF, Chen WF, Young JPW, Chen WX. Ecology and Evolution of Rhizobia: Principles and Applications. Singapore: Springer Verlag; 2019. https://doi.org/10.1007/978-981-32-9555-1
  36. Sikora S, Redzepović S. Genotypic characterization of soybean rhizobia. Food Technol Biotechnol. 2003; 41:61–7.
  37. Appunu C, N’Zoue A, Laguerre G. Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India. Appl Environ Microbiol. 2008; 74 (19):5991–6. https://doi.org/10.1128/AEM.01320-08
  38. Chidebe IN, Jaiswal SK, Dakora FD. Distribution and phylogeny of microsymbionts associated with cowpea (Vigna unguiculata) nodulation in three agroecological regions of Mozambique. Appl Environ Microbiol 2018; 84(2):1–25. https://doi.org/10.1128/AEM.01712-17
  39. Saeki Y. Characterization of soybean-nodulating rhizobial communities and diversity. In: Aleksandra S, editor. [Soybean–molecular aspects of breeding]. Rijeka: Intech; 2011. p. 163–84. https://doi.org/10.5772/14417
  40. van Berkum P, Fuhrmann JJ. Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. Int J Syst Evol Microbiol. 2000; 50(6):2165–72. https://doi.org/10.1099/00207713-50-6-2165
  41. Ahmad MH, Eaglesham ARJ, Hassouna S. Examining serological diversity of “cowpea” rhizobia by the ELISA technique. Archives of Microbiology. 1981; 130:281–7. https://doi.org/10.1007/BF00425941