plant surface microbiology.pdf

titative data about the community composition. In addition, the bacterial
diversity can be investigated using PCR-amplification of phylogenetic marker
genes combined with subsequent electrophoretic fingerprint analysis or
cloning and sequencing studies. These approaches can be supplemented by a
general microbial structural and functional diversity analysis using community
phospholipid fatty acid and substrate utilization pattern analysis, respectively.
2 In Situ Studies of Microbial Communities Using Specific
Fluorescence Labeling and Confocal Laser Scanning
Microscopy
A detailed understanding of the ecology of bacterial populations requires in
situ information about the localization of the colonization sites at specific
areas on root surfaces and also about neighboring populations. Therefore,
true in situ studies need to be performed and these must include an identification
of the bacteria on a phylogenetic level and also information about their
in situ activity. Since soil and plant surfaces are very complex in microstructure
and optical appearance, special microscopic techniques have to be
applied. Confocal laser scanning microscopy enables us to circumvent to a
great degree disturbing autofluorescence from out-of-focus-planes by performing
optical sections (xy and xz scans) through the sample (Hartmann et
al. 1998). It has been demonstrated that CSLM studies combined with the
application of specific fluorescent probes considerably improve microbial
ecology studies in the rhizosphere (Schloter et al. 1993; Aßmus et al. 1995).
The confocal pinhole cuts off all out-of-focus fluorescence to reach the amplifiers.
The application of several lasers with different excitation wavelengths in
combination with differently fluoro-labeled probes allow the simultaneous
analysis of different populations and/or activities (Amann et al. 1995; Stoffels
et al. 2001). If possible, nested approaches with overlapping probe specificities
should be used to improve the fidelity of the in situ identification, e.g., by fluorescence
in situ hybridization. In addition, the use of the green fluorescent
protein (GFP) as a structural and functional autofluorescence marker has
successfully lightened up the biology and ecology of diverse biota, including
bacteria, fungi, protozoa and plants (Lorang et al. 2001).
2.1 Fluorescence in Situ Hybridization
24 Microbial Community Analysis in the Rhizosphere 451
Root samples are fixed overnight at 4 °C in 3 % paraformaldehyde containing
PBS (phosphate-buffered saline, composed of 0.13 M NaCl, 7 mM Na 2HPO 4
and 3 mM NaH 2 PO 4 [pH 7.2]). Root pieces are washed in PBS, mixed with
0.3 % agarose, dropped onto glass slides and dried at room temperature.