Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria

Micropropagated sugarcane plants have been used in Brazil for almost three decades. Besides the improvement in plant health, micropropagated sugarcane carries no endophytic plant growth-promoting bacteria. The Brazilian inoculation technology to reintroduce diazotrophic bacteria in micropropagated sugarcane plantlets revealed a synergistic-like effect in PGP-bacteria mixed inoculations. The infection model of single diazotrophic bacteria species in sugarcane was studied in detail, but still many questions remain open. In this study we used a combined fluorescence in situ hybridization (FISH) and a cultivation based approach (MPN) to evaluate the colonization of sugarcane plantlets by mixed inocula. The highest colonization for three out of the five species studied was obtained with a mixed inoculum, when the Azospirillum amazonense showed an increase by almost 100 times in colonization and Herbaspirillum spp. and Burkholderia tropica was determined at 10⁷ cells per gram root fresh weight. All of the inoculated bacterial species could be detected using the FISH probes 12 h after bacterial inoculation. The FISH results confirmed the MPN counts and showed differences in the population numbers and colonization behavior of particular bacterial inoculum strains in the different mixed inocula. A putative antagonistic effect among the inoculated H. seropedicae and H. rubrisubalbicans strains was observed using FISH, as well as the better competitiveness of B. tropica as compared to the A. amazonense strain. The observed data probably reflect also specific interactions with the sugarcane variety used in this particular inoculation system, and may not be generalized as a rule. This is the first study about the competition for sugarcane colonization in a mixed bacterial inoculum.