• fluorescent tagging;
  • confocal laser scanning microscopy;
  • infection thread;
  • Rhizobium legume specificity;
  • root nodule endophytes;
  • Vigna radiata


Legumes develop symbiotic relationships with Rhizobium by a complex exchange of signals. Despite the high specificity between symbiotic partners, the presence of non-rhizobial bacteria in root nodules has been reported. To investigate how these rhizobacteria enter root nodules, fluorescently tagged Pseudomonas fluorescens and Klebsiella pneumoniae were co-inoculated with host-nodulating Ensifer adhaerens to Vigna radiata seedlings and root hair infection was monitored using confocal microscopy at 5 days post inoculation. Pseudomonas fluorescens and K. pneumoniae invaded the root hair only when co-inoculated with E. adhaerens. Recovery of inoculated tagged strains and confirmation through CLSM and 16S rRNA gene sequencing confirmed that the test rhizobacteria occupied nodules. We hereby report with the help of confocal microscopy that rhizobacteria migrate along the length of host-nodulating rhizobial strain and become localized in root nodules. We further report isolation of eight non-rhizobial bacterial genera, predominantly Bacillus spp. and Paenibacillus spp., from nodules of field-grown V. radiata.