Facilitated colonisation of the diazotroph Azorhizobium caulinodans in rice roots detected through GFP-labelling

Rice being a non-legume, does not possess the capacity to carry out biological nitrogen fixation in symbiosis with rhizobia. Azorhizobium caulinodans is a micro-symbiont of Sesbania rostrata with several unique and beneficial characteristics that makes it a potential candidate to induce biological nitrogen fixing associations with the rice plant.  Maximum colonisation of rice by the bacterium is important for high benefit. This study was aimed at finding the maximum colonisation levels of A. caulinodans detected by GFPlabelling of the bacterium. A. caulinodans was labelled with green fluorescent protein to facilitate accurate and real time observations of the bacterium in and around the root hairs. GFPlabelling was carried out by inserting the gfp-gene-containing plasmid pBBR5-hem-gfp5-S65T into A. caulinodans ORS 571, with a helper plasmid (pRK2013) by tri-parental-mating. The bacterium was observed to colonise rice roots by bright green fluorescence emitted by the bacterium under blue light. The fluorescence intensities reflecting the degree of colonisation of the bacteria were quantified using Zen light image analysis software and were statistically analysed. Highest colonisation was observed when 5 mL of the labelled Azorhizobium was applied twice a week, and the observations were made 15 days after application of the inoculum in the presence of the  flavonoid naringenin.