Infection of mottled stripe disease-susceptible and resistant sugar cane varieties by the endophytic diazotroph Herbaspirillum

Leaves of a mottled stripe disease-susceptible cultivar (B-4362) and of a mottled stripe disease-resistant cultivar (SP 70-1143) of sugar cane (interspecific hybrids of Saccharum) were inoculated with the diazotrophic endophytes, Herbaspirillum rubrisubalbicans or Herbaspirillum seropedicae, via injection into the apex of the stem. At 7 and 20 d.a.i., H. seropedicae could be isolated only from a small necrotic area around the point of inoculation, where there was considerable degradation of host cells, and was not detected in any other part of the leaves. This suggested a hypersensitive response by the host to this bacterium, and no disease symptoms formed on either cultivar. By contrast, H. rubrisubalbicans could be re-isolated from throughout the infected leaves of both cultivars at both harvests and produced widespread disease symptoms on the leaves of cv. B-4362. Symptoms consisted of necrotic regions near the point of inoculation, and red stripes and red patches along the vertical axis of the leaves, where the bacteria had spread in the primary and secondary veins. The xylem-conducting elements in diseased regions of leaves were filled with bacteria and, at the edges of disease symptoms, the vessels were filled with a gum which stained blue-green with toluidine blue. This material probably contained phenolic compounds,and was produced as a host defence response. Leaves of cv. SP 70-1143 only developed small red stripes near the point of inoculation. These symptoms did not spread along the leaves, and the infected xylem vessels were never seen to be completely full of bacteria. Instead, the vessels contained encapsulated bacterial colonies attached to secondary wall deposition; these colonies were surrounded by blue-green material that might have been host-defence gums. In cv. B-4362, bacteria were abundant in the intercellular spaces of mesophyll adjacent to infected xylem, and also filled sub-stomatal cavities. Immunogold labelling using polyclonal antisera raised against H. rubrisubalbicans gave a weak signal with the bacteria in cv. SP 70-1143, showing that few binding sites were available to the antibodies. By contrast, bacteria in cv. B-4362 reacted strongly with the antibody, suggesting that they had a denser coating of immunoreactive mucus. In the later stages of infection of cv. B-4362, lysed bacteria were seen within degraded plant cells surrounded by a matrix of plant gums and bacterial mucus. This matrix reacted strongly to the H. rubrisubalbicans antibodies. Immunogold labelling using antibodies against nitrogenase component II showed that nitrogenase was expressed by bacteria in the early stages of infection of cv. B-4362, but not in later stages, or by bacteria infecting cv. SP 70-1143.