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

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


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 



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.


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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

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