Rhizobia: Culture Collections, Identification, and Methods of Preservation

  • Manvika SahgalEmail author
  • Vandana Jaggi
Part of the Soil Biology book series (SOILBIOL, volume 54)


Across the globe nitrogen is limiting primary productivity. Although fertilizer nitrogen could supplement soil nitrogen depletion, efforts should be to augment soil biological nitrogen fixation mediated by microbes. This could be addressed by conservation and sustainable use of rhizobia that can fix nitrogen in soil in association with legumes. Rhizobia commonly occur in soils but often fail to produce effective nodulation either because their population in soil is low or those present cannot effectively nodulate the particular legume. Rhizobia present in various ecosystems are considerably diverse; at present 130 species within 15 genera are reported. Rhizobial inoculation is almost always needed when certain new leguminous crops are introduced to new areas or when ineffective and incompatible rhizobia are present in the soils. For this, depository of authentic microbial gene pool is a must. Culture collection centers act as repository of valuable microbial strains. In the changing global scenario, these centers are becoming Biological Resource Centres (BRCs), carry out research, enhance value of strains, and control access to dangerous microorganisms. For adoption and popularizing use of rhizobium-legume technology, their isolation, identification, preservation, and deposition in biological banks are imminent. Moreover, there is a need to identify the compatible rhizobium-legume symbioses for sustainable agriculture.



The authors thank Indian Council of Agricultural Research (ICAR), and Ministry of Environment & Forests (MoEF), and GOI for financial assistance to MS. The senior author MS thanks Mariangela Hungria, Brazil for critical suggestions.


  1. Amarger N, Macheret V, Laguerre G (1997) Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Bacteriol 47:996–1006CrossRefGoogle Scholar
  2. Anonymous (2017) India to overtake China’s population in 7 yrs. A UN report published in Times of IndiaGoogle Scholar
  3. Australian Inoculant Research and Control Service, New South Wales Department of Agriculture Melbourne.
  4. Azevedo H, Lopes FM, Silla PR, Hungria M (2015) A database for the taxonomic and phylogenetic identification of the genus Bradyrhizobium using multilocus sequence analysis. BMC Genomics 16:S10CrossRefGoogle Scholar
  5. Ba S, Willems A, de Lajudie P, Roche P, Jeder H, Quatrini P, Neyra M, Ferro M, Prome JC, Gillis M, Boivin-Masson C, Lorquin J (2002) Symbiotic and taxonomic diversity of rhizobia isolated from Acacia tortilis subsp. Raddiana in Africa. Syst Appl Microbiol 25:130–145CrossRefGoogle Scholar
  6. Campos AK, de Andrade Mota M, de Araújo JV, Cecon PR (2004) Predatory activity, radial growth and sporulation of fungi predating Monacrosporium nematode submitted to cryopreservation. Sci Rural 34:465–469CrossRefGoogle Scholar
  7. Commonwealth Scientific and Industrial Research Organization’s (CSIRO) Cunningham Laboratory, Brisbane.
  8. Cummings SP, Gyaneshwar P, Vinuesa P, Farruggia FT, Andrews M, Humphry D, Elliott GN, Nelson A, Orr C, Pettitt D, Shah GR, Santos SR, Krishnan HB, Odee D, Moreira FMS, Sprent JI, Young JPW, James EK (2009) Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia. Environ Microbiol 11:2510–2525CrossRefGoogle Scholar
  9. Day JG, Stacey GN (eds) (2007) Cryopreservation and freeze-drying protocols, 2nd edn. Methods in molecular biology series 368. Humana, TotowaGoogle Scholar
  10. Denardin ND, Freire JRJ (2000) Assessment of polymers for the formulation of legume inoculants. World J Microbiol Biotechnol 16:215–217CrossRefGoogle Scholar
  11. Fernandes Júnior PI, Rohr TG, de Oliveira PJ, Xavier GR, Rumjanek NG (2009) Polymers as carriers for rhizobia inoculant formulations. Pesq Agropec Bras Brasília 44:1184–1190CrossRefGoogle Scholar
  12. Frank B (1889) Über die Pilzsymbiose der Leguminosen. Ber Dtsch Bot Ges. 7:332–346Google Scholar
  13. Fred EB, Baldwin IL, McCoy E (1932) Root nodule bacteria and leguminous plants. University of Wisconsin. Digitized19 Feb 2008Google Scholar
  14. Freire JRJ, Kolling I (1986) World culture collections of Rhizobium. World J Microbiol Biotechnol 2:231–235CrossRefGoogle Scholar
  15. Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GB (eds) (1981) Manual of methods for general bacteriology. American Society of Microbiology, WashingtonGoogle Scholar
  16. Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coenye T, Feil EJ, Stackebrandt E, de Peer YV, Vandamme P, Thompson FL, Swings J (2005) Re-evaluating prokaryotic species. Nat Rev Microbiol 3:733–739CrossRefGoogle Scholar
  17. Graham PH, Sadowsky MJ, Kersters HH, Barnet YM, Bradley RS, Cooper JE, De Ley DJ, Jarvis BDW, Roslycky EB, Strijdom BW, Young JPW (1991) Proposed minimal standards for the description of new genera and species of root-and stem-nodulating bacteria. Int J Syst Bacteriol 41:582–587CrossRefGoogle Scholar
  18. Haukka K, Lindstrom K, Young JPW (1998) Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America. Appl Environ Microbiol 64:419–426PubMedPubMedCentralGoogle Scholar
  19. Howeison JG, Dilworth MJ (2016) Working with rhizobia. Australian Centre for International Agricultural research, CanberraGoogle Scholar
  20. Hubálek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229CrossRefGoogle Scholar
  21. Hungria M, Ardley J, O’Hara GW, Howeison JG (2016) Preservation of rhizobia. In: Howeison JG and Dilworth MJ (eds) Working with rhizobia. Australian Centre for International Agricultural Research, Canberra, pp 61–72Google Scholar
  22. Johnson MD, Allen ON (1952) Cultural reactions of rhizobia with special reference to strains isolated from Sesbania species. Ant Leewenhoek 18:1–12CrossRefGoogle Scholar
  23. Jordan DC (1982) Notes: transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. Nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int J Syst Bacteriol 32(1):136–139CrossRefGoogle Scholar
  24. Jordan DC (1984) Family III. Rhizobiaceae Conn (1938). In: Krieg NR, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 1. Williams and Wilkins, Baltimore, pp 234–254Google Scholar
  25. Kampfer P, Glaeser SP (2012) Prokaryotic taxonomy in the sequencing era the polyphasic approach revisited. Environ Microbiol 14:291–317CrossRefGoogle Scholar
  26. Konstantinidis K, Tiedje JM (2005a) Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci USA 102:2567–2572CrossRefGoogle Scholar
  27. Konstantinidis KT, Tiedje JM (2005b) Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187:6258–6264CrossRefGoogle Scholar
  28. Leon-Barrios M, Lorite MJ, Donate-Correa J, Sanjuan J (2009) Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range. Syst Appl Microbiol 32:413–420CrossRefGoogle Scholar
  29. Lindström K, Young JP (2011) International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Agrobacterium and Rhizobium: minutes of the meeting, 7 September 2010, Geneva, Switzerland. Int J Syst Evol Microbiol 61:3089–3093CrossRefGoogle Scholar
  30. Lortet G, Mear N Lorquin J, Dreyfus B, de Lajudie P, Rosenberg C, Boivin C (1996) Nod factor thin-layer chromatography profiling as a tool to characterize symbiotic specificity of rhizobial strains: application to Sinorhizobium saheli, S. teranga, and Rhizobium sp. strains isolated from Acacia and Sesbania. Mol Plant–Microbe Interact 9:736–747CrossRefGoogle Scholar
  31. Maatallah J, Berraho E, Munoz S, Sanjuan J, Lluch C (2002) Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco. J Appl Microbiol 93:531–540CrossRefGoogle Scholar
  32. Menna P, Hungria M (2011) Phylogeny of nodulation and nitrogen fixation genes in Bradyrhizobium: supporting evidence for the theory of monophyletic origin, and spread and maintenance by both horizontal and vertical transfer. Int J Syst Evol Microbiol 61:3052–3067CrossRefGoogle Scholar
  33. Menna P, Barcellos FG, Hungria M (2009) Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes. Int J Syst Evol Microbiol 59:2934–2950CrossRefGoogle Scholar
  34. Mnasri B, Mrabet M, Laguerre G, Aouani ME, Mhamdi R (2007) Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti. Arch Microbiol 187:79–85CrossRefGoogle Scholar
  35. Nandasena KG, O’Hara GW, Tiwari RP, Sezmis E, Howieson JG (2007) In situ lateral transfer of symbiosis islands results in rapid evolution of diverse competitive strains of mesorhizobia suboptimal in symbiotic nitrogen fixation on the pasture legume Biserrula pelecinus L. Environ Microbiol 9:2496–2511CrossRefGoogle Scholar
  36. Nour SM, Cleyet Marel JC, Normand P, Fernandez MP (1995) Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Int J Syst Bacteriol 45:640–648CrossRefGoogle Scholar
  37. OECD (2007) Promoting pro-poor growth: policy guidance for donors. OECD Publishing.
  38. Ormeño-Orrillo E, Servín-Garcidueñas LE, Rogel MA, González V, Peralta H, Mora J, Martínez-Romero J, Martínez-Romero E (2015) Taxonomy of Rhizobia and Agrobacteria from the Rhizobiaceae family in light of genomics. Syst Appl Microbiol 38:287–291CrossRefGoogle Scholar
  39. Peterson TA, Russelle MP (1991) Alfalfa and the nitrogen cycle in the Corn Belt. J Soil Water Conserv 46:229–235Google Scholar
  40. Quispel A (1988) Hellriegel and Wilfarth’s discovery of (symbiotic) nitrogen fixation one hundred years ago. In: Bothe H, de Bruijn FJ, Newton WE (eds) Nitrogen fixation: one hundred years after. Gustav Fisher, Stuttgart, pp 3–12Google Scholar
  41. Radeva G, Jurgens G, Niemi M, Nick G, Suominen L, Lindstrom K (2001) Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis. Syst Appl Microbiol 24:192–205CrossRefGoogle Scholar
  42. Ramírez-Bahena MH, García Fraile P, Peix A, Valverde A, Rivas R, Igual JM, Mateos PF, Martínez Molina E, Velázquez E (2008) Revision of the taxonomic status of the species Rhizobium leguminosarum (Frank 1879) Frank 1889AL, Rhizobium phaseoli Dangeard 1926AL and Rhizobium trifolii Dangeard 1926AL. R. trifolii is a later synonym of R. leguminosarum. Reclassification of the strain R. leguminosarum DSM 30132 (=NCIMB 11478) as Rhizobium pisi sp. nov. Int J Syst Evol Microbiol 58:2484–2490CrossRefGoogle Scholar
  43. Rivas R, Laranjo M, Mateos PF, Oliveira S, Martínez-Molina E, Velázquez E (2007) Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum. Lett Appl Microbiol 44:412–418CrossRefGoogle Scholar
  44. Rogel MA, Ormeño-Orrillo E, Martinez Romero E (2011) Symbiovars in rhizobia reflects bacterial adaptation to legumes. Syst. Appl Microbiol 34:96–104. Scholar
  45. Romeiro RS (2001) Methods in plant bacteriology. UFV, Viçosa, p 279Google Scholar
  46. Sahgal M, Sharma A, Johri BN, Prakash A (2004) Selection of growth promotory rhizobia for Dalbergia sissoo from diverse soil ecosystems of India. Symbiosis 36:83–96Google Scholar
  47. Sanz T, Fernández MA, Salvador A, Muñhoz J, Fiszman SM (2005) Thermogelation properties of methylcellulose (MC) and their effect on a batter formula. Food Hydrocoll 19:141–147CrossRefGoogle Scholar
  48. Segovia L, Young JPW, Martinez-Romero E (1993) Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov. Int J Syst Bacteriol 43:374–377CrossRefGoogle Scholar
  49. Silva C, Vinuesa P, Eguiarte LE, Souza V, Martinez-Romero E (2005) Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes. Mol Ecol 14:4033–4050CrossRefGoogle Scholar
  50. Sly LI (2010) Biodiversity and the role of microbial resource centres. In: Brown AG (ed) Proceedings of conference “biodiversity and world food security: nourishing the planet and its people”. Conducted by the Crawford Fund for International Agricultural Research, Parliament House, Canberra, Australia, 30 August–1 September, 2010, pp 60–67Google Scholar
  51. Smith D (2003) Culture collections over the world. Int Microbiol 6:95–100CrossRefGoogle Scholar
  52. Stackebrandt E, Frederiksen W, Garrity GM, Grimont PAD, Kampfer P, Maiden X, Nesme MCJ, Rossello-Mora R, Swings J, Truper HG, Vauterin L, Ward AC, Whitman WB (2002) Report of the adhoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52:1043–1047PubMedGoogle Scholar
  53. Terpolilli J, O’Hara GW, Tiwari RP, Dilworth MJ, Howieson JG (2008) The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021. New Phytol 179:62–66CrossRefGoogle Scholar
  54. Tian CF, Wang ET, Wu LJ, Han TX, Chen WF, Gu CT, Gu JG, Chen WX (2008) Rhizobium fabae sp. nov., a bacterium that nodulates Vicia faba. Int J Syst Evol Microbiol 58:2871–2875CrossRefGoogle Scholar
  55. Tilman D, Reich PB, Knops J, Wedin D, Mielke T, Lehman C (2001) Diversity and productivity in a long-term grassland experiment. Sci 294:843–845CrossRefGoogle Scholar
  56. Tindall BJ, Rosselló-Móra R, Busse H-J, Ludwig W, Kämpfer P (2010) Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60:249–266CrossRefGoogle Scholar
  57. Trewavas A (2001) Urban myths of organic farming. Nature 410:409–410CrossRefGoogle Scholar
  58. Van Elsas JD (2001) Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv.2 in irrigation water. Can J Microbiol 47:842–854CrossRefGoogle Scholar
  59. Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy; a consensus approach to bacterial systematics. Microbiol Mol Biol Rev 60:407–438Google Scholar
  60. Villegas MC, Rome S, Maure L, Domergue O, Gardan L, Bailly X, Cleyet- Marel JC, Brunel B (2006) Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti. Syst Appl Microbiol 29:526–538CrossRefGoogle Scholar
  61. Vinuesa P, Leon-Barrios M, Silva C, Willems A, Jarabo-Lorenzo A, Perez-Galdona R, Werner D, Martinez-Romero E (2005) Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int J Syst Evol Microbiol 55:569–575CrossRefGoogle Scholar
  62. Wang ET, Rogel MA, Garcia-De los Santos A, Martinez-Romero J, Cevallos MA, Martinez-Romero E (1999) Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis. Int J Syst Bacteriol 49:1479–1491CrossRefGoogle Scholar
  63. Weir BS (2016) The current taxonomy of rhizobia. NZ Rhizobia website. Last updated: 10 Jan 2016
  64. Willems A, Coopman R, Gillis M (2001) Comparison of sequence analysis of 16S–23S rDNA spacer regions, AFLP analysis and DNA–DNA hybridizations in Bradyrhizobium. Int J Syst Evol Microbiol 51:623–632CrossRefGoogle Scholar
  65. World Directory of Culture Collections, sixth version (2014)

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of MicrobiologyG. B. Pant University of Agriculture and TechnologyPantnagarIndia

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