plant surface microbiology.pdf

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Anton Hartmann et al.
(see above) and washed in phosphate buffer is suspended in 20 ml 0.1 %
sodium-cholate buffer (Macdonald 1986). The suspension is treated in a
Stomacher 80 at the highest speed for 4 min to disrupt polymers. After transfer
into Erlenmeyer flasks, 0.5 g of polyethylene glycol 6000 (Sigma, Deisenhofen)
and 0.4 g of cation change polystyrene beads (chelex 100: Sigma,
Deisenhofen) are added and the suspension is stirred at 50 rpm/min for 1 h at
4 °C. The stomacher/stirring procedure is repeated three times, whereby the
roots are transferred to “fresh” 0.1 % sodium cholate buffer with PEG 6000
and chelex 100 after each extraction step (compartment IIIa-c). Finally,
aliquots of the obtained suspensions are combined. Root and soil particles are
removed by filtration through gauze (40-mm mesh width) and subsequent filtration
through 5-mm syringe filters (Sartorius No. 17549, Göttingen, Germany).
In the case of Medicago sativa grown in sandy loam, this approach yielded
total counts of 3.3x10 9 to 6.5x10 8 /g root dry weight from the first to the third
treatment, while hybridizing bacteria remained constant at 1.5x10 8 /g root dry
weight (Mogge et al. 2000). It was calculated that about 88 % of the bacteria
had been desorbed from the rhizoplane by this technique. This result was confirmed
by in situ studies of roots applying confocal laser scanning
microscopy. The roots usually harbor large numbers of phylogenetically different
bacteria, belonging, e.g., to the a-, b- and g-subclasses of proteobacteria.
However, after the third extraction step, no bacteria could be detected any
more on the root surface (20 root pieces of 2–3 cm length were scanned).
The suspensions obtained from bulk soil (I), ectorhizosphere (II), and rhizoplane/endorhizosphere
(IIIa-c: merged suspension) can be used for cultivation
and dot blot-hybridizations (see Sect. 3.2). DAPI-staining and FISH can
be applied for counting total and hybridizing bacteria in the three compartments
collected on polycarbonate filters (see Sect. 3.3). PCR-amplification of
16S rDNA and subsequent electrophoretic fingerprinting of the amplification
products as well as clone bank studies can be performed with these fractions
too (see Sect. 3.4). In addition, these compartments can be investigated for
structural and functional microbial diversity by community fatty acid analysis
and community level physiological profiling (see Sect. 3.5).
3.2 Community Analysis by Cultivation and Dot Blot Studies
Serial dilutions (0.85 % NaCl) from bulk soil (compartment I), ectorhizosphere
(compartment II), and rhizoplane/endorhizosphere (compartment III)
suspensions (Fig. 1) were plated onto agar media containing different nutritional
levels (Table 2). The selection of media used for the isolation of soil and
ectorhizosphere-associated bacteria was made to allow the growth of oligotrophic,
slow growing strains as well as fast growers. Minimal media were
suggested because of the sensitivity of soil bacteria to salts (NaCl) or organic