Advertisement

Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria

  • Nisha Tak
  • Garima Bissa
  • Hukam S. Gehlot
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2057)

Abstract

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.

Key words

Legume root-nodules Rhizobia DNA fingerprinting Housekeeping and symbiotic genes Phylogeny Host-range 

Notes

Acknowledgments

The authors are thankful to the following funding agencies for financial assistance: University Grants Commission, New Delhi; DBT, Govt. of India; DST-SERB; and Crawford Fund Award-ATSE, Australia conferred to HSG and NT for advance training at CRS, Murdoch University, Perth, WA. We sincerely acknowledge Prof. Ann Hirsch, University of California, Los Angeles, USA and American Society for Microbiology for the ASM-IUSSTF Indo-US Research Professorship award to NT for GFP-related studies.

References

  1. 1.
    Lewis G, Schrire B, Mackinder B, Lock M (eds) (2005) Legumes of the world. Royal Botanic Gardens, KewGoogle Scholar
  2. 2.
    Sprent JI (2009) Legume nodulation: a global perspective. Wiley-Blackwell, OxfordCrossRefGoogle Scholar
  3. 3.
    Azani N, Babineau M, Bailey CD, The Legume Phylogeny Working Group (LPWG) et al (2017) A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 66:44–77CrossRefGoogle Scholar
  4. 4.
    Sprent JI, Ardley JK, James EK (2013) From north to south: a latitudinal look at legume nodulation processes. S Afr J Bot 89:31–41CrossRefGoogle Scholar
  5. 5.
    Sprent JI, Ardley JK, James EK (2017) Biogeography of nodulated legumes and their nitrogen-fixing symbionts. New Phytol 215:40–56CrossRefGoogle Scholar
  6. 6.
    Andrews M, Andrews ME (2017) Specificity in legume-rhizobia symbioses. Int J Mol Sci 18:705CrossRefGoogle Scholar
  7. 7.
    Gyaneshwar P, Hirsch AM, Moulin L et al (2011) Legume-nodulating betaproteobacteria: diversity, host range, and future prospects. Mol Plant Microbe Interact 24:1276–1288CrossRefGoogle Scholar
  8. 8.
    Peix A, Ramírez-Bahena MH, Velázquez E et al (2015) Bacterial associations with legumes. Crit Rev Plant Sci 34:17–42CrossRefGoogle Scholar
  9. 9.
    Shamseldin A, Abdelkhalek A, Sadowsky MJ (2017) Recent changes to the classification of symbiotic, nitrogen-fixing, legume-associating bacteria: a review. Symbiosis 71:91–109CrossRefGoogle Scholar
  10. 10.
    Herridge DF (2008) Inoculation technology for legumes. In: Dilworth MJ, James EK, Sprent JI, Newton WE (eds) Nitrogen-fixing leguminous symbioses. Springer, Dordrecht, pp 77–109Google Scholar
  11. 11.
    Baldwin IL, Fred EB (1929) Nomenclature of the root-nodule bacteria of the Leguminosae. J Bacteriol 17:141–150PubMedPubMedCentralGoogle Scholar
  12. 12.
    Le Quéré A, Tak N, Gehlot HS et al (2017) Genomic characterization of Ensifer aridi, a proposed new species of nitrogen-fixing rhizobium recovered from Asian, African and American deserts. BMC Genomics 18:85Google Scholar
  13. 13.
    Mahato NK, Gupta V, Singh P et al (2017) Microbial taxonomy in the era of OMICS: application of DNA sequences, computational tools and techniques. Antonie Van Leeuwenhoek 110:1357–1371CrossRefGoogle Scholar
  14. 14.
    Richardson AE, Viccars LA, Watson JM et al (1995) Differentiation of Rhizobium strains using the polymerase chain reaction with random and directed primers. Soil Biol Biochem 27:515–524CrossRefGoogle Scholar
  15. 15.
    Stępkowski T, Moulin L, Krzyżańska A et al (2005) European origin of Bradyrhizobium populations infecting lupins and serradella in soils of Western Australia and South Africa. Appl Environ Microbiol 71:7041–7052CrossRefGoogle Scholar
  16. 16.
    Martens M, Dawyndt P, Coopman R et al (2008) 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 58:200–214CrossRefGoogle Scholar
  17. 17.
    Gehlot HS, Panwar D, Tak N et al (2012) Nodulation of legumes from the Thar desert of India and molecular characterization of their rhizobia. Plant Soil 357:227–243CrossRefGoogle Scholar
  18. 18.
    Gehlot HS, Tak N, Kaushik M et al (2013) An invasive Mimosa in India does not adopt the symbionts of its native relatives. Ann Bot 112:179–196CrossRefGoogle Scholar
  19. 19.
    Tak N, Gehlot HS, Kaushik M et al (2013) Genome sequence of Ensifer sp. TW10; a Tephrosia wallichii (Biyani) microsymbiont native to the Indian Thar Desert. Stand Genomic Sci 9:304–314CrossRefGoogle Scholar
  20. 20.
    Panwar D, Tak N, Gehlot HS (2014) Nodulated native legumes in an arid environment of Indian Thar Desert. In: Fulekar MH, Kale RK (eds) Recent trends in life sciences. IK International Publishing House Pvt. Ltd, India, pp 284–306Google Scholar
  21. 21.
    Sankhla IS, Meghwal RR, Tak N et al (2015) Phenotypic and molecular characterization of microsymbionts associated with Crotalaria medicagenia: a native legume of the Indian Thar desert. Plant Archives 15:1003–1010Google Scholar
  22. 22.
    Gehlot HS, Ardley J, Tak N et al (2016) High-quality permanent draft genome sequence of Ensifer sp. PC2, isolated from a nitrogen-fixing root nodule of the legume tree (Khejri) native to the Thar Desert of India. Stand Genomic Sci 11:43CrossRefGoogle Scholar
  23. 23.
    Tak N, Awasthi E, Bissa G et al (2016) Multi locus sequence analysis and symbiotic characterization of novel Ensifer strains nodulating Tephrosia spp. in the Indian Thar Desert. Syst Appl Microbiol 39:534–545CrossRefGoogle Scholar
  24. 24.
    Choudhary S, Meghwal RR, Sankhla IS et al (2017) Molecular characterization and phylogeny of novel diverse nitrogen fixing microsymbionts associated with Vachellia (Acacia) leucophloea in arid and semi arid regions of Rajasthan. Indian For 143:266–278Google Scholar
  25. 25.
    Sankhla IS, Tak N, Meghwal RR et al (2017) Molecular characterization of nitrogen fixing microsymbionts from root nodules of Vachellia (Acacia) jacquemontii, a native legume from the Thar Desert of India. Plant Soil 410:21–40CrossRefGoogle Scholar
  26. 26.
    Rathi S, Gaur S, Tak N et al (2017) Genetically diverse root nodule bacteria associated with Alysicarpus vaginalis from alkaline soil of Rajasthan, India. Plant Archives 17:495–505Google Scholar
  27. 27.
    Choudhary S, Tak N, Gehlot HS (2018) Phylogeny and genetic diversity assessment of Ensifer strains nodulating Senegalia (Acacia) senegal (L.) Britton. In arid regions of Western Rajasthan, India. Microbiology 87:127–142CrossRefGoogle Scholar
  28. 28.
    Sankhla IS, Meghwal RR, Choudhary S et al (2018) Molecular characterization of microsymbionts associated with root nodules of Crotalaria burhia Buch.-ham. Ex Benth., a native keystone legume species from Thar Desert of India. Indian J Exp Biol 56:373–384Google Scholar
  29. 29.
    Ojha A, Tak N, Rathi S et al (2017) Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita, important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India. Syst Appl Microbiol 40:334–344CrossRefGoogle Scholar
  30. 30.
    Somasegaran P, Hoben HJ (1994) Handbook for rhizobia: methods in legume-rhizobium technology. Springer-Verlag, New YorkCrossRefGoogle Scholar
  31. 31.
    Howieson JG, Dilworth MJ (eds) (2016) Working with rhizobia. Australian Centre for International Agricultural Research, CanberraGoogle Scholar
  32. 32.
    Morón B, Soria-Díaz ME, Ault J et al (2005) Low pH changes the profile of nodulation factors produced by Rhizobium tropici CIAT899. Chem Biol 12:1029–1040CrossRefGoogle Scholar
  33. 33.
    Weisburg WG, Barns SM, Pelletier DA et al (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703CrossRefGoogle Scholar
  34. 34.
    Yanagi M, Yamasato K (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett 107:115–120CrossRefGoogle Scholar
  35. 35.
    Gaunt MW, Turner SL, Rigottier-Gois L et al (2001) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol 51:2037–2048CrossRefGoogle Scholar
  36. 36.
    Turner SL, Young JPW (2000) The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. Mol Biol Evol 17:309–319CrossRefGoogle Scholar
  37. 37.
    Stępkowski T, Czaplińska M, Miedzinska K et al (2003) The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria. Syst Appl Microbiol 26:483–494CrossRefGoogle Scholar
  38. 38.
    Chaintreuil C, Boivin C, Dreyfus B et al (2001) Characterization of the common nodulation genes of the photosynthetic Bradyrhizobium sp. ORS285 reveals the presence of a new insertion sequence upstream of nodA. FEMS Microbiol Lett 194:83–86CrossRefGoogle Scholar
  39. 39.
    Haukka K, Lindström 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
  40. 40.
    Zhang XX, Turner SL, Guo XW et al (2000) The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity. Appl Environ Microbiol 66:2988–2995CrossRefGoogle Scholar
  41. 41.
    Laguerre G, Nour SM, Macheret V et al (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology 147:981–993CrossRefGoogle Scholar
  42. 42.
    Perret X, Broughton WJ (1998) Rapid identification of Rhizobium strains by targeted PCR fingerprinting. Plant Soil 204:21–34CrossRefGoogle Scholar
  43. 43.
    Howieson JG, Loi A, Carr SJ (1995) Biserrula pelecinus L.-a legume pasture species with potential for acid, duplex soils which is nodulated by unique root-nodule bacteria. Aust J Agric Res 46:997–1009CrossRefGoogle Scholar
  44. 44.
    Cheng HP, Walker GC (1998) Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti. J Bacteriol 180:5183–5191PubMedPubMedCentralGoogle Scholar
  45. 45.
    Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652CrossRefGoogle Scholar
  46. 46.
    Cappuccino JG, Sherman N (2007) Microbiology: a laboratory manual. Dorling Kindersley Pvt. Ltd, License of Pearson Education, New Delhi, IndiaGoogle Scholar
  47. 47.
    Cheng HR, Jiang N (2006) Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 28:55–59CrossRefGoogle Scholar
  48. 48.
    Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  49. 49.
    Yates RJ, Howieson JG, Nandasena KG et al (2004) Root-nodule bacteria from indigenous legumes in the north-west of Western Australia and their interaction with exotic legumes. Soil Biol Biochem 36:1319–1329CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Nisha Tak
    • 1
  • Garima Bissa
    • 1
  • Hukam S. Gehlot
    • 1
  1. 1.BNF and Microbial Genomics Lab., Department of Botany, Center of Advanced StudyJai Narain Vyas UniversityJodhpurIndia

Personalised recommendations