Herbaspirillum, an endophytic diazotroph colonizing vascular tissue in leaves of Sorghum bicolor L. Moench

Leaves of Sorghum bicolor were examined at 5 d and 14 d after inoculation with the N2-fixing endophytic bacteria Herbasporillum seropedicae and Herbaspirillum rubrisubalbicans. Plants inoculated with H. rubrisubalbicans expressed symptoms of 'red stripe disease' i.e. red stripes along the secondary veins of the leaf blade close to the inoculation point and spreading up the leaves. Infected leaves showed dense colonization by H. rubrisubalbicans in regions showing red stripe symptoms at 5 d after inoculation. The infection was confined within the vascular system, in particular, the protoxylem and associated lacunae, which were often completely filled with bacteria, with some of the latter expressing nitrogenase. The bacteria were recognized using H. rubrisubalbicans-specific antibodies and immunogold labelling, which also showed that the antibody reacted with material on the surface of the bacteria, and that this mucus was released into the lumen of the xylem. At 14 d after inoculation, disease symptoms were slightly more severe, with both meta- and protoxylem being even more heavily colonized in parts of the leaf showing red stripes. However, a strong host defence response was also apparent at this stage, with gums lining the walls of the vessels and enclosing the bacteria, although the latter were still actively dividing. At the edges of visible disease symptoms, plant gums filled the xylem; bacteria had formed distinct colonies within these gums, with some of the colonies associated with the xylem walls. Plants inoculated with H. seropedicae either did not express the disease or showed very mild symptoms close to the inoculation point. In the latter case, H. seropedicae were localized within protoxylem vessels and the metaxylem was partly occluded with plant-derived gums. By contrast with H. rubrisubalbicans, H. seropedicae was also localized in leave at 14 d without disease symptoms and did not always appear to elicit a host response, i.e. they colonized the walls of metaxylem, with the xylem vessels themselves remaining unoccluded and largely free of gums. The fine line separating plant pathogens, endophytes and symbioses is discussed in light of these results.