Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants

J Microbiol Biotechnol. 2024 Mar 28;34(3):570-579. doi: 10.4014/jmb.2310.10025. Epub 2023 Dec 30.


Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

Keywords: Biological nitrogen fixation; indole acetic acid; nitrogen-fixing bacteria; phylogenetic; root nodulation.

MeSH terms

  • Bacteria / genetics
  • Fabaceae* / microbiology
  • Glycine max
  • Legumins
  • Nitrogen / metabolism
  • Nitrogen Fixation
  • Nitrogen-Fixing Bacteria* / genetics
  • Nitrogen-Fixing Bacteria* / metabolism
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics
  • Rhizobium* / genetics
  • Rhizobium* / metabolism
  • Root Nodules, Plant / metabolism
  • Root Nodules, Plant / microbiology
  • Symbiosis / genetics
  • Vegetables


  • Legumins
  • RNA, Ribosomal, 16S
  • Nitrogen