plant surface microbiology.pdf

24 Microbial Community Analysis in the Rhizosphere 461
(up to 10-mm long) pleomorphic cells hybridized with a set of FLUOS-labeled
oligonucleotide probes directed against the domain Bacteria and the TRITClabeled
oligonucleotide probe Rhi1247 directed against Rhizobium (Table 1).
Obviously, these large cells were bacteroids originating from crushed nodules
and were missing when the nodules had been removed before the application
of the extraction procedure.
3.4 Community Analysis by (RT) PCR-Amplification of Phylogenetic
Marker Genes, D/TGGE-Fingerprinting and Clone Bank Studies
The differentiated rhizosphere compartments can also be used to isolate
rRNA and genomic DNA following previously described protocols (Felske et
al. 1996; Miethling et al. 2000).A further purification of the DNA extracts, e.g.,
with the Wizard DNA clean-up (Promega, Madison, WI), may be necessary,
before PCR can be applied. For amplification, the highly conserved bacterial
16S rRNA primers U968-GC and L1346 are used.Amplification of 16S rDNA is
performed as described by Felske et al. (1996) using the following PCR-program:
1 cycle at 94 °C for 5 min, 35 cycles at 94 °C for 90 s (denaturation), 61 °C
for 40 s (annealing), 70 °C for 40 s (extension), and a single final extension at
70 °C for 5 min. Amplification of 16S rRNA as well as denaturing temperature
gradient gel electrophoretic (D/TGGE) separation of the PCR-products of
DNA and RNA is performed as described by Miethling et al. (2000).
D/TGGE profiles represent the frequency distribution of PCR-amplified
segments of rDNA or rRNA separated due to their melting behavior in the
electric field of a temperature gradient gel. The resulting profiles represent the
frequency distribution of the most prominent community members in a first
approximation (Muyzer and Smalla 1998). Since the ratio of 16S rDNA and
16S rRNA is dependent on cellular activity (Wagner 1994), comparisons of
TGGE patterns derived from 16S rRNA and 16S rDNA amplicons can provide
interesting information about the active members of the community. Variations
in the relation of band intensities (rRNA/rDNA) indicated shifts in the
relative activity of the respective dominant DNA sequences. In particular, the
composition of the communities are changing along the gradient from bulk
soil to the rhizoplane/endorhizosphere (Mogge et al. 2000, Wieland et al.
2001). Additional sequences show higher evenness visible by larger band formation
in the rhizoplane/endorhizosphere compartment, which is clearly different
from all the other examined habitats. In this compartment, a larger
fraction of the community seems to be active, as deduced from the fraction of
bands common to the rDNA and rRNA patterns of the communities. Using
the same methodological approach, Wieland et al. (2001) have demonstrated
recently that the TGGE-patterns of 16S rRNA did not change during the plant
development in the bulk soil, whereas some pattern variation could be correlated
to plant development in the rhizosphere and rhizoplane habitats. On the