plant surface microbiology.pdf

3.5 Community Analysis by Fatty Acid Pattern and Community Level
Physiological Profile Studies
The overall microbial diversity in environmental habits can be assessed by
cultivation independent biomarker analysis, different from the phylogenetic
ribosomal genes or other genetic markers. As is the case in chemotaxonomic
studies, the fatty acid patterns are used for this purpose. In one type of analysis,
the fatty acid methyl esters (FAME) are obtained from the fatty acids after
saponification of 5 g of soil or root with adhering soil in methanoic NaOH (at
100 °C, 30 min; Dunfield and Germida 2001). Alternatively, the lipids are
extracted from 5 g of soil with methanol:chloroform (2:1), the phospholipids
are separated by chromatography, and finally hydrolyzed to liberate the phospholipid
fatty acids (PLFA; White and Ringelberg 1998). The PLFA analysis
has the advantage of giving insight into the living community, because PFLA
are efficiently hydrolyzed in dead biomass, while the direct FAME analysis
may contain fatty acids from dead organisms too. The GC-MSanalysis finally
provides much information on the diversity of this biomarker (Zelles 1997;
White and Ringelberg 1998). Using the FAME analysis, Germida et al. (1998)
investigated the diversity of root-associated bacterial communities in canola
and wheat, and Dunfiled and Germida (2001) compared the bacterial communities
in the rhizosphere and endorhizosphere of field-grown genetically
modified varieties of canola (Brassica napus).An example of a recent application
of the PFLA approach in rhizosphere studies is the investigation of the
microbial community response in the rhizosphere of Spartina alterniflora to
changing environmental conditions by Lovell et al. (2001).
An investigation targeting the analysis of the functional abilities of a complex
community is the substrate utilization profile assays using the Biolog R -
plates. Baudoin et al. (2001) applied this approach recently to characterize the
functional microbial diversity in different rhizosphere compartments of
maize plants. The differences between the rhizosphere and nonrhizosphere
soil samples were more pronounced in 4-week-old compared to 2-week-old
plants. In addition, adhering soil from different root zones (ramification, root
hair-elongation, root tip) revealed dissimilar community level physiological
profiles (CLPP). However, this approach needs to be regarded as reflecting the
potential rather than the in situ-activity of most culturable microbes, because
these are known to respond and contribute most to the activity at the incubation
conditions of the CLPP-assay (Garland et al. 1997).
4 Conclusions
24 Microbial Community Analysis in the Rhizosphere 463
Using a polyphasic approach including cultivation-dependent and different
cultivation-independent methods, it could be shown that a high proportion of
culturable bacteria is present in the rhizoplane when a variety of appropriate