plant surface microbiology.pdf

27 Applications of Quantitative Microscopy in Plant Surface Microbiology 505
of incubation, and more detailed microscopy to be performed with a cleaner,
phase-transparent background. These added features have significantly
improved the signal-to-noise ratio of image quality, making it possible to
accurately quantify many of the pre-infection and post-infection events
occurring on root hairs in vivo at single bacterial cell resolution, including
rhizobial attachment to root hairs (phenotype Roa) of constant length by
phase-contrast microscopy (Dazzo et al. 1976; Dazzo 1982). The results of
studies using this quantitative microscopical counting technique revealed
important spatio-temporal aspects of the Roa phenotype, including its distinct
cellular orientations/patterns/phases of adhesion, the positive relationship
of certain patterns of attachment to host specificity, the importance of
cell-surface glycoconjugates and saccharide-binding host lectins to symbiont
recognition, the inhibition of symbiont recognition and infection by combined
nitrogen, and the manipulation of rhizobial genes affecting cell surface
components and rhizobial attachment to host root hairs (Dazzo and Hubbell
1975; Dazzo et al. 1976, 1978, 1984; Dazzo and Brill 1978, Sherwood et al. 1984;
Rolfe et al. 1996). This same modification of the slide culture technique also
made it possible to quantitate clover root hair infection. Thus, quantitative
microscopy of the infection process resulted in the discovery of potent, stimulating
infection-related biological activities of various purified rhizobial
components required for primary host infection by R. leguminosarum bv.
trifolii, including its clover lectin-binding acidic heteropolysaccharides and
corresponding oligosaccharide repeat unit fragments which retained their
affinity for the clover lectin, its clover lectin-binding lipopolysaccharide glycoform,
and its diverse family of membrane chitolipooligosaccharides that
modulate cell wall architecture and growth physiology of these target differentiated
host cells (Abe et al. 1984; Dazzo et al. 1991, 1996). Further applications
of this modified Fåhraeus slide technique to study the R. leguminosarum
bv. trifolii-white clover symbiosis have utilized real-time video
microscopy and digital image analysis of track-reconstructions to define the
quantitative influence of root secretions on rhizobial motility in situ in the
aqueous, external clover root environment (Dazzo and Petersen 1989), and of
cells and purified lectin-binding lipopolysaccharide of Rhizobium on cytoplasmic
streaming in root hairs indicating activation of their cytoskeleton
activity (Dazzo and Petersen 1989, Dazzo et al. 1991).Another modification of
the Fåhraeus slide technique was to culture seedlings vertically and flat on
small agarose-solidified plates with a portion of their roots covered with the
same nitrogen-free medium and small coverslips. This modification plus the
customized construction of a “horizontal growth station” created the opportunity
to perform real-time and time-lapse video microscopy of seedling
roots grown axenically and geotropically with as little as 10 ml volumes of bacterial
test solutions. Applications of this technique resulted in the detection
and quantitation of symbiosis-related growth responses of clover root hairs to
minute quantities of several different types of bioactive metabolites made by