plant surface microbiology.pdf

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Frank B. Dazzo
to introduce polyunsaturation of the N-acyl fatty acid moiety, in O-acetylation
and in sulfation of CLOS Nod factors, are still capable of inducing
ENOD20 (a marker of cortical cell activation) and (most importantly) eliciting
cortical cell divisions in this legume host (Vernoud et al. 1999). This result
is fully consistent with our studies described earlier that defined the minimal
structural requirements for uptake and bioactivity of rhizobial CLOS analogs
in legume roots, including induction of alfalfa and white clover cortical cell
divisions (Philip-Hollingsworth et al. 1997), contrary to the dogma indicating
that those structural features of CLOS dictate host specificity in the S.
meliloti-alfalfa symbiosis. Finally, a third powerful approach to detect target
mRNA is based on staining tissue sections for in situ PCR-amplified antisense
riboprobes. This approach has recently been used to detect a novel
Enod [dd23b] in white clover roots induced within 6 h after inoculation with
wild-type R. leguminosarum bv. trifolii or the corresponding purified wildtype
CLOS (Crockard et al. 2002).
3 Quantitation of Symbiotic Interactions Between
Rhizobium and Legumes by Image Analysis
The value of quantitative microscopy for plant surface microbiology can be
enhanced even further when coupled with computer-assisted digital image
analysis (Hollingsworth et al. 1989; Orgambide et al. 1996). This fast-growing
technology utilizes the digital computer to derive numerical information
regarding selected image features. Although image analysis technology cannot
add anything that is not already present, its ability to extract the maximum
amount of data from the image, as well as to quickly store, retrieve, and
electronically transmit that data makes it an invaluable research tool for the
microscopist. Computer-assisted microscopy has been used to enhance developmental
morphology studies of the Rhizobium-legume symbiosis since 1989
(Dazzo and Petersen 1989). Here, I highlight a few examples of new information
on the Rhizobium-legume symbiosis derived from microscopical studies
utilizing digital image analysis, and later illustrate how we have opened new
ground in plant surface microbiology by development and implementation of
innovative image analysis software tailored to studies of in situ microbial
ecology.
3.1 Definitive Elucidation of the Nature of Rhizobium Extracellular
Microfibrils
The extracellular microfibrils made by R. leguminosarum bv. trifolii in pure
culture were isolated and shown by chemical analysis to consist of microcrystalline
cellulose (Napoli et al. 1975a). However, the nature of the microfibrils