plant surface microbiology.pdf

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Anton Hartmann et al.
to 13, 26 and 35 % as compared to 4.2, 8.5 and 0.8 % respectively in the ectorhizosphere
as was shown by using the probes ALF1b, BET42a and GAM42
respectively to group the isolates obtained. No differences were found for isolates
of the Cytophaga-Flavobacteria group, which were only a minor portion
in both compartments (3.5 %).
Quantitative population analyses in soil and rhizosphere environments
were also conducted by using strains carrying unique selectable markers. This
was aimed to enumerate one particular introduced strain in the presence of a
large excess of other microbes. Since the usually suitable selectable markers
are missing in wild-type strains, spontaneous or transposon-induced
mutants, which are, e.g., resistant to an antibiotic, are frequently used for
selective plating assays. However, these mutants may be less fit than the wild
type and, therefore, the results of the surveys are biased. De Leij et al. (1998)
demonstrated such effects on environmental fitness in several mutants of
Pseudomonas fluorescens SBW25, constructed by site-directed genomic insertions
of marker genes. Recently, Hirano et al. (2001) selected a site in the gacScysM
intergenic region in Pseudomonas syringae pv. syringae B728, in which
the insertion of an antibiotic resistance marker cassette did not affect the fitness
of the bacterium in the field. They concluded that carefully selected
intergenic regions, which are suitable for the integration of specific marker
cassettes, exist in any bacterium.
3.3 Community Analysis by Fluorescence in Situ Hybridization on
Polycarbonate Filters
Bacterial suspensions (extract of the rhizosphere compartments, Fig. 1) are
fixed overnight at 4 °C with 3 % formaldehyde and concentrated in three parallels
onto 0.2-mm polycarbonate filters (100-ml aliquots). Dehydration of cells
is performed with 50, 80 and 96 % ethanol for 3 min each. For details on the
FISH protocol see Sect. 2.1. The slides are finally mounted with Citifluor AF1
to reduce photobleaching. A Zeiss Axiophot 2 epifluorescence microscope
(Zeiss, Jena, Germany) equipped with filter sets F31–000, F41–001 and
F41–007 (Chroma Tech. Corp., Battleboro, VT, USA) can be used for the enumeration
of bacteria on filters. Total cell counts (DAPI) and hybridizing bacteria
using a set of domain-specific probes (Table 1) are determined by evaluating
at least 10 microscopic fields with 20–100 cells per field.
In the case of the M. sativa roots, the extraction method was also applied to
the rhizoplane/endorhizosphere of roots inoculated with Sinorhizobium
meliloti as well as to inoculated roots after the nodules had been removed
with a sterile scalpel. During the three repeated stomacher/stirring-treatments,
nodules cracked and S. meliloti-bacteroids were released (Mogge et al.
2000). Figure 2E shows a representative photomicrograph of bacteria concentrated
on polycarbonate filters after extraction of roots with nodules. Large