M. Lin et al. Applied Soil Ecology 15 2000 211–225 213
the dominant members of soil microbial communities Heuer and Smalla, 1997b; Duineveld et al., 1998.
Furthermore, the metabolic potential of microbial communities can be assessed via community level
physiological profiling CLPP using the Biolog GN microtiter plate setup Garland and Mills, 1991; Gar-
land, 1997. In spite of its obvious drawbacks Smalla et al., 1998, this method has been widely used as
a rapid and powerful community-level approach to study the potential activity of microbial populations
in natural environments Winding, 1994; Zak et al., 1994; Garland, 1997; Heuer and Smalla, 1997b; Hitzl
et al., 1997; Insam, 1997; Knight et al., 1997. Combi- nation of the two methods thus allows a simultaneous
functional and structural assessment of the impact of the release of inoculant strains into soils Akkermans
et al., 1995; Smalla et al., 1998.
This study aimed to assess the fate and impact of A. faecalis strain A1501R in soil cropped with
rice in microcosms. To achieve this objective, the autecology of the inoculant strain was monitored by
different methods. Furthermore, the impact of the A. faecalis
strain on the soil and rhizosphere microbiota was assessed by comparing the PCR–DGGE profiles
and Biolog GN metabolic fingerprints of microbial communities between soil samples inoculated or not
with strain A1501R.
2. Materials and methods
2.1. Bacterial strain and selection of a rifampicin-resistant mutant
The A. faecalis inoculant strain, denoted A1501, was isolated from paddy soils in south China in 1980
You et al., 1995. It was identified as A. faecalis us- ing traditional taxonomic tests You et al., 1995, but
recent molecular evidence based on the 16S riboso- mal RNA gene sequence obtained in our laboratories
Genbank accession number AF143245 suggests it might in fact be closely related to fluorescent pseu-
domonads closest relative Pseudomonas stutzeri. Spontaneous rifampicin-resistant mutants of A. fae-
calis
A1501 were isolated on LB medium containing 100 mg of rifampicin per milliliter. One mutant clone
of A1501, denoted strain A1501R, that showed a growth rate similar to that of the wild-type strain, was
selected. The mutation in A1501R was stable without reversion to wild-type after more than 30 genera-
tions in LB medium, as well as in soil during 60-day microcosm experiments. Strain A1501R was grown
overnight at 30
◦
C in LB medium supplemented with 50 mg of rifampicin per milliliter, washed twice and
resuspended in sterile demineralized water to obtain a cell density of the order 10
10
–10
11
cells per milliliter for further microcosm experiments.
2.2. Soil microcosm experiment A microcosm experiment was carried out to inves-
tigate the fate and effect of A. faecalis strain A1501R in soil. Flevo silt loam FSL soil was taken from a
field microplot at the Institute for Plant Protection IPO-DLO Wageningen, the Netherlands. Washed
bacterial cells in sterile demineralized water, or ster- ile demineralized water control, were thoroughly
mixed through portions of the FSL soil, which were subsequently used to fill replicate plastic pots 100 g
of treated soil per pot. The initial inoculum density was about 10
7
CFU per gram of dry soil. Seeds of rice Oryza sativa L. japonica Zhongzuo 9037 were
germinated at 30
◦
C. Three-day-old seedlings were transferred to the pots two seedlings per pot, and
pots were flooded using sterile distilled water. The pots were placed in a growth chamber 80 relative
humidity with 16 h of light 26
◦
C and 8 h of dark- ness 18
◦
C. The height of the water layer on top of the soil was adjusted with distilled water based on the
growth of the rice seedlings. The water content in the soil was, thus, at 100 of the saturation level.
Replicate soil microcosms were sampled at regular times, i.e. 3 h time 0 and 2, 5, 15, 23, 30, 40 and
60 days following incubation. Bulk and rhizosphere soils were separated as described van Overbeek et
al., 1997. Samples were processed for the assessment of total bacterial and strain A1501R CFU counts, for
total community DNA extraction and for CLPP, as outlined below.
2.3. Enumeration of bacterial populations The numbers of culturable bacteria in soil were
determined by using selective or non-selective plate counts. Strain A1501R was enumerated by direct
plating onto LB agar containing 50 mg ml
− 1
of
214 M. Lin et al. Applied Soil Ecology 15 2000 211–225
rifampicin, which was sufficient to inhibit the growth of indigenous microorganisms; no colonies were
found on rifampicin-containing plates when samples from uninoculated soil were studied. Total bacterial
counts were obtained on 10 strength tryptic soy broth agar 0.1×TSA. For enumeration of bacterial
populations in soil, 10 g soil samples were suspended in 95 ml of sterile 0.1 sodium pyrophoshate PPi
solution and 10 g of gravel, in 250 ml flasks. The flasks were shaken for 10 min at 180 rpm. The sus-
pensions were serially diluted in 0.1 sodium PPi, after which aliquots were plated onto LB medium
containing 50 mg of rifampicin plus 100 mg of cyclo- heximide per milliliter, and on 0.1×TSA containing
cycloheximide 100 mg ml
− 1
. The plates were incu- bated for 48 h at 28
◦
C prior to colony enumerations. Counts were expressed as CFU per gram of dry soil
De Leij et al., 1995; van Overbeek et al., 1997. 2.4. Soil DNA extraction and PCR amplification
Total soil community DNA was extracted and puri- fied from duplicate 2 g bulk or rhizosphere soil sam-
ples as described in protocols developed in our labora- tories Smalla et al., 1993; van Elsas and Smalla, 1995;
van Elsas et al., 1997. This included cell lysis via bead beating with 100–110 mm diameter glass beads,
phenol extraction and further purification by CsCl and potassium acetate precipitation and Wizard spin col-
umn clean-up. The purified soil DNA was dissolved in a final volume of 100 ml of 10 mM Tris–EDTA buffer
pH, 8.0.
PCR amplification was performed with 1 ml of soil DNA in a 50 ml reaction mixture by touch-down
PCR 3 min at 94
◦
C; 1 min at 94
◦
C, 1 min at 64
◦
C, and 3 min at 72
◦
C [two cycles; repeating this cy- cle with decreasing annealing temperatures of 2
◦
C every two cycles down to 56
◦
C]; 1 min at 94
◦
C, 1 min at 54
◦
C, and 3 min at 72
◦
C [30 cycles]; and 10 min at 72
◦
C. This protocol is similar to those de- scribed previously van Elsas and Wolters, 1995. The
touch-down temperature cycling scheme improved the quality of PCR products as compared to a pre-
viously used fixed thermal cycling scheme Rosado, pers. comm.. The nucleotide sequences of the 16S
ribosomal RNA based conserved bacterial primers used were as follows Heuer and Smalla, 1997a;
Heuer et al., 1999: forward primer 968f GC-Clamp- 5
′
-AACGCGAAGAACCTTAC-3
′
and reverse primer 1401r 5
′
-CGGTGTGTACAAGGCCC-3
′
. The for- ward primer had a 40-nucleotide GC-rich sequence
GC clamp at its 5
′
-end. The primer pair amplified the 16S rDNA region corresponding to positions
968–1401 E. coli numbering of the majority of sequences present in the ribosomal database.
2.5. Construction of a specific probe based on the 16S rDNA variable region V6
The V6 variable region of the 16S rDNA of A. faecalis
A1501 was amplified by PCR with primers to the conserved regions around positions
971–1057 of E. coli. The nucleotide sequences of these primers were as follows: forward primer 971f
5
′
-GCGAAGAACCTTACC-3
′
, and reverse primer 1057r 5
′
-CATGCAGCACCTGT-3
′
Heuer et al., 1999. The around 86-bp PCR product about 57
variable bases was cloned and sequenced and the sequence obtained compared to database sequences
using a BLAST homology search via the Internet NCBI, Altschul et al., 1990. With the exception of
sequences from two organisms described as Pseu- domonas
spp., the sequence revealed to be specific for strain A1501. It was directly used as a probe in
dilution dot blot assays with soil DNA, as well as on blots of DGGE profiles.
2.6. Hybridizations with the DIG labeled specific V6 probe
The strain A1501 specific V6 probe was first la- beled with the random primer DNA labeling mix
Boehringer Mannheim, Germany, using the proto- col of the manufacturer. It was kept frozen until used
in hybridization experiments.
To check the specificity of the probe, 86 strains were isolated from high-dilution plates prepared from
soil samples inoculated with A1501R, according to differences of morphology or color of colonies. PCR
products 968f and 1401r primers, generated with this collection as well as with strain A1501R, were
transferred to nylon membranes using a dot blot ap- paratus according to procedures described previously
Sambrook et al., 1989. Furthermore, bulk soil DNA obtained from both uninoculated and inoculated soils
at different time points during microcosm incubation
M. Lin et al. Applied Soil Ecology 15 2000 211–225 215
was dotted onto membranes in a threefold dilution scheme. Finally, selected DGGE gels were denatured,
fixed and blotted onto nylon membranes, and the re- sulting filters used for hybridization analysis accord-
ing to standard procedures Sambrook et al., 1989.
Hybridizations, washes and detection with the DIG-labeled V6 probe were carried out at high strin-
gency using the protocol described in the Boehringer Mannheim protocol.
2.7. Denaturing gradient gel electrophoresis analysis DGGE was performed with a PhorU gradient sys-
tem Ingeny, Leiden, the Netherlands. The PCR products were applied directly onto 6 wt.vol poly-
acrylamide gels in 0.5×TEA 20 mM Tris–acetate pH 7.4, 10 mM acetate, 0.5 mM Na
2
EDTA with gradients from 45 to 65 denaturants. Hundred per-
cent of denaturants is 7 M urea plus 40 formamide Muyzer et al., 1996. The gels were run at 60
◦
C 100 V for 16 h. After electrophoresis, the gels were
incubated for 1 h in 10 ml SYBR Green I nucleic acid gel staining solution Molecular Probes, Leiden, the
Netherlands, after which they were photographed using a 302 nm UV transilluminator.
The molecular profiles obtained were analyzed using the Molecular Analyst fingerprinting software
BioRad, Veenendaal, Netherlands as well as manu- ally.
2.8. Biolog GN substrate utilization patterns On days 0, 2, 5, 15, 23, 30 and 40, CLPP patterns
were determined using the Biolog system, which de- tects the utilization of 95 specific carbon sources by
bacterial communities in microplates Garland and Mills, 1991; Garland, 1997. A modified protocol was
used, as follows: 3 g dry weight of soil was added to 30 ml of sterile one-quarter strength Ringer’s solution
in a 50 ml plastic tube containing 2 g of gravel. This slurry was shaken at 200 rpm for 2 h, then kept for
3 min. Five milliliter sample was removed and added to 45 ml of sterile one-quarter-strength Ringer’s so-
lution and centrifuged for 15 min 3500g. The pellet was washed once with 0.85 NaCl and resuspended
in 50 ml of 0.85 NaCl. The numbers of cultur- able bacteria in suspension were determined using
unselective or selective containing 50 mg of ri- fampicin per milliliter LB agar. The suspension was
placed in a sterile tray with a magnetic stirrer, which kept it homogeneous during inoculation of the mi-
crotiter plates. For each assessment, three replicate plates were inoculated. After inoculation with 150 ml
of bacterial suspension per well containing about 10
4
CFU, the plates were shaken at 120 rpm 25
◦
C. The plates were checked at regular times during 65 h
of incubation for color development, using the Biolog microplate reader. In addition, selected wells were
analyzed for the number of total and strain A1501R CFU as described above. The measured extinction
values were automatically corrected by incorporating the extinction value of the substrate-free reference
well E0 according to the Biolog instruction manual Biolog, Hayward, CA. The readings obtained at set
times, i.e. 37–48 h, when average well color devel- opment AWCD was maximal Heuer and Smalla,
1997b — were corrected for AWCD Garland and Mills, 1991; Garland, 1997, and corrected values
were used in comparative analyses. The data obtained with a selection of 32 discriminative substrates were
used in the full analyses.
2.9. Statistics and cluster analyses All experiments were performed in triplicate,
whereas samples from duplicate pots were subjected to DNA extraction and PCR–DGGE analysis. Data
were analyzed by analysis of variance and differences were considered significant at p0.05. The DGGE
banding patterns were compared using phenetic meth- ods with the Dice coefficient of similarity NT-SYS
program. Dendrograms were constructed using the neighbor joining unweighted pair group method with
mathematical averages UPGMA.
3. Results
