Use of green fluorescent protein to visualize rice root colonization by Azospirillum irakense and A-brasilense

To visualize rice root colonization by two Azospirillum species, A. irakense KBC1 was equipped with a plasmid expressing the enhanced green fluorescent protein (EGFP) and A. brasilense Sp7 was equipped with a plasmid expressing the enhanced yellow fluorescent protein (EYFP). In both cases, intensive fluorescence was observed under the epifluorescent microscope. Striking differences for association with roots of rice seedlings were observed between the two species: (i) A. irakense cells attached faster than A. brasilense to rice roots following inoculation; (ii) A. irakense attached to rice roots as vibroid cells, while A. brasilense occurred as rounded cyst-like cells; (iii) A. irakense cells were mainly found on root hairs, whereas A. brasilense cells were mainly concentrated on root surfaces. The two Azospirillum species obviously do not compete with each other for colonization of rice roots. These results demonstrate that the two Azospirillum species differ in their mode of rice root colonization. As the two Azospirillum species are extensively studied for unravelling mechanisms of plant root colonization and plant growth promotion, labelling with fluorescent protein is a useful additional tool for these studies.