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.