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 inoculat ion with the N-2-fixing endophytic bacteria Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans. Plants inoculated with H. rubris ubalbicans 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 coloni zation by H. rubrisubalbicans in regions showing red stripe symptoms a t 5 d after inoculation. The infection was confined within the vascula r system, in particular, the protoxylem and associated lacunae, which were often completely filled with bacteria, with some of the latter ex pressing nitrogenase. The bacteria were recognized using H. rubrisubal bicans-specific antibodies and immunogold labelling, which also showed that the antibody reacted with material on the surface of the bacteri a, 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 respon se was also apparent at this stage, with gums lining the walls of the vessels and enclosing the bacteria, although the latter were still act ively dividing. At the edges of visible disease symptoms, plant gums f illed the xylem; bacteria had formed distinct colonies within these gu ms, with some of the colonies associated with the xylem walls. Plants inoculated with H. seropedicae either did not express the disease or s howed 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 wi th H. rubrisubalbicans, H. seropedicae was also localized in leaves at 14 d without disease symptoms and did not always appear to elicit a h ost response, i.e. they colonized the walls of metaxylem, with the xyl em vessels themselves remaining unoccluded and largely free of gums. T he fine line separating plant pathogens, endophytes and symbioses is d iscussed in light of these results.