plant surface microbiology.pdf

24 Microbial Community Analysis in the Rhizosphere 457
reporter cells monitor in situ conditions, the tests are performed ex situ. For
this purpose, a separation of the bacteria from the soil was accomplished by
applying formaldehyde (1 %)-fixed extracts to density gradient centrifugation
with Nycodenz (Nycomed Pharma, Oslo, Norway) with a density of
1.3 g/ml. After a centrifugation step (10,000xg, 30 min, 4 °C) the bacteria on
the top of the Nycodenz layer were used for further analysis (Unge et al.
1999).
Monitoring of in situ bacterial growth activity in the plant rhizosphere is
suggested by Ramos et al. (2001) using ribosome content and synthesis rate
measurements.
3 Ex Situ Studies of Microbial Communities After
Separation of Rhizosphere Compartments
For the desorption of bacteria from surfaces, Campbell and Greaves (1990b)
recommended the use of a stomacher. Sodium cholate and the ion exchange
resin beads Dowex A1 or Chelex 100 were recommended for the treatment of
soil particles or root pieces by Macdonald (1986) or Hopkins et al. (1991),
respectively, to obtain the bacteria adsorbed. Herron and Wellington (1990)
developed a method to extract streptomycete spores from soil particles and
used polyethylene glycol (PEG) 6000 for reducing hydrophobic interactions.
Each extraction protocol for root-associated bacteria has to be optimized for
the system under investigation with the appropriate controls to prove its success.
Mogge et al. (2000) described a standardized protocol for the differentiation
of the rhizosphere compartments ectorhizosphere and rhizoplane/
endorhizosphere and the extraction of the adsorbed bacteria from the rhizoplane
of Medicago sativa europae. This procedure used the recommendations
by Macdonald (1986) and Herron and Wellington (1990) in a modified form.
FISH in combination with CLSM was applied for the proof of desorption efficiency
in root surface studies.
3.1 Recovery of Bacteria from Bulk Soil, Ecto- and Endorhizosphere
Roots are carefully separated from the soil using sterile tweezers. The soil
should be rather dry at the time of harvest to facilitate the separation of roots
from the adhering soil. All steps are conducted with sterile solutions on ice.
Bulk soil (compartment I) and root-attached soil particles which have been
collected by shaking the roots (ectorhizosphere: compartment II) are suspended
1:9 (w/v) in 0.01 M phosphate buffer (Na 2HPO 4/KH 2PO 4, pH 7.4) and
dispersed for 1 min at the highest speed in a Stomacher 80 (Seward Medical,
UK). To extract rhizoplane and endorhizosphere bacteria (compartment III),
1 g (fresh weight) of roots that have been cleaned from adhering soil particles