Quantifying and visualizing Nitrospirillum amazonense strain CBAmC in sugarcane after using different inoculation methods

Background and Aims Nitrospirillum amazonense strain CBAmC is a nitrogen-fixing bacterium that has been used for sugarcane inoculation. Despite its beneficial effects, however, little is known about the establishment and colonization of this microorganism in its inoculated host plant. Furthermore, alternative inoculation methods need to be tested in the sugarcane crop. The objective of the present work was to quantify and visualize CBAmC in sugarcane cultivar RB867515 after using three inoculation methods. Methods Utilizing one node as propagation material, three bacterial inoculation methods were tested under greenhouse conditions. For up to 60 days after inoculation (DAI), qPCR was used to quantify the bacterial loads in the plant or substrate while FISH-CLSM was used to confirm the establishment and colonization profile of CBAmC in different plant tissues. Results FISH-CLSM indicated that the CBAmC strain could establish itself on different tissues of sugarcane, especially roots. qPCR revealed variable bacterial loads in the plants inoculated according to the three methods tested. In addition, little or no bacterial loads were detected in the substrate a few days after inoculation, regardless of the method used. Conclusions N. amazonense CBAmC proves to be a bacterium that preferentially associates with the surface of the root tissues of sugarcane propagated by one node, instead of colonizing internal plant tissues or remaining in the soil. The inoculation method consisting of immersion of the mini-setts in bacterial suspension proved to be more efficient than the inoculation methods of applying the suspension onto the post-sprouting substrate or to the lowest leaf.