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
3.1. Fate of culturable A. faecalis A1501R and total bacterial counts in soil planted with rice seedlings
As shown in Fig. 1, the population density of strain A1501R introduced into soil increased gradually at the
beginning of the experiment. Strain A1501R reached maximum numbers of about 10
8
CFU per gram of dry
216 M. Lin et al. Applied Soil Ecology 15 2000 211–225
Fig. 1. Survival of strain A1501R following introduction into FSL soil microcosms:
h
total bacterial numbers CFU in uninoculated control soils;
total number of bacteria CFU in inoculated soils; A1501R CFU counts in bulk soil;
j
A1501R CFU counts in rhizosphere soil. Bars indicate standard deviations of triplicate systems.
soil after 2 days in bulk soil or after 15 days in rhizo- sphere soil, and then showed a gradual decline. The
inoculant finally kept a roughly stable population size in bulk soil, of between 10
6
and 10
7
CFU per gram of dry soil, during the later part of the 60 day incubation
period. The numbers of inoculant CFU in rhizosphere soil were largely similar to those in corresponding bulk
soil samples. They were significantly higher only at one time point, i.e. 15 days after inoculation Fig. 1.
Total counts of culturable bacteria in bulk and rhi- zosphere soil samples of the FSL soil were between
10
8
and 10
9
CFU per gram of dry soil, and changed very little over time Fig. 1. In addition, there was
no clear rhizosphere effect on the total bacterial CFU counts. Moreover, inoculation with strain A1501R did
not result in significant changes in the total numbers of culturable bacteria, which remained at about 10
9
CFU per gram of dry soil, irrespective of the presence of the inoculant strain.
3.2. Molecular analysis of strain A1501R fate in bulk soil using a V6 probe
The sequence of the variable V6 region of 16S rDNA from strain A1501R was compared with se-
quences of the NCBI database Altschul et al., 1990. Out of over 4000 sequences, only two 16S rRNA
sequences 0.05 were homologous Fig. 2. Both sequences belonged to organisms classified as Pseu-
domonas spp. The next-nearest strains Pseudomonas
alcaligenes and P. stutzeri already had 3 sequence
divergence, and all other strains had 6 or more se- quence divergence. These included strains of Vibrio
spp. and of different pseudomonads P. stutzeri, P. putida
, P. aeruginosa and P. mendocina. In addition, a FastA analysis with selected sequences Fig. 2
showed high divergence with sequences from Es- cherichia coli
, P. testosteroni, Agrobacterium tume- faciens
and Bacillus subtilis Lane, 1991. Thus, the V6 sequence of strain A1501 was about 72 homol-
ogous with that of E. coli, 60 with P. testosteroni, 42 with A. tumefaciens and 58 with B. subtilis.
The V6 probe was, therefore, promising for use in the specific detection of strain A1501R in soil. To test
for reactions with soil-derived strains, 86 strains were isolated from inoculated soil samples 40 days after
strain A1501 introduction, and their 968f-1401r PCR products generated via PCR. Dot blot hybridizations
of these products were then carried out with the strain A1501R specific V6 probe. In addition to the posi-
tive control, only five strains showed positive signals Fig. 3A. Among these, three strains that gave rise to
strongly hybridizing products 50–100 of intensity of positive control, were rifampicin-resistant and had
M. Lin et al. Applied Soil Ecology 15 2000 211–225 217
218 M. Lin et al. Applied Soil Ecology 15 2000 211–225
Fig. 3. A Dot blot hybridization of PCR amplicons obtained with DNA of 86 strains isolated from A1501R-inoculated soil
with the strain A1501R-specific V6 probe. Arrow indicates pos- itive clones Pseudomonas spp. N2 and N3; + left intense
dot=positive control product generated with total genomic DNA of strain A1501R; right faint dot=negative control. B Dot
blot hybridization of bulk soil DNA extracted over time from the microcosm study with the strain A1501R-specific V6 probe. For
each rank, a threefold dilution scheme was used, from the top to the bottom. Rank 1–4: inoculated soil respectively, from days
0, 15, 30 and 40; Rank 5: A1501R genomic DNA upper well: 1000 ng; Rank 6–9: control soil respectively, from days 0, 15, 30
and 40; Rank 10: negative control; Rank 11: A1501R genomic DNA upper well: 500 ng; Rank 12: strain N2 genomic DNA.
a Biolog GN profile identical to that of strain A1501R. They were thus identified as the inoculant strain. Two
remaining strains that showed weak signals 10 of positive control; Fig. 3A were rifampicin-sensitive.
These two strains showed identical substrate uti- lization profiles in Biolog GN plates, where they
showed no close match to strain A1501R or to any strain of the Biolog database not shown. They were
tentatively identified as “Pseudomonas” spp. The strains produced amplicons that comigrated in DGGE
with the A1501R specific amplicon, suggesting their 986-1401 16S rDNA regions were highly similar
not shown.
The V6 probe was used in a dilutiondot blot ap- proach with DNA obtained from uninoculated and
inoculated bulk soils from the microcosm study Fig. 3B. The results revealed weak background signals in
DNA obtained from uninoculated soils on days 0 and 40 slots 6 and 9, whereas this background was not
detected at other time points slots 7 and 8. DNA of one cross-reacting strain slot 12 also reacted with the
probe. On the other hand, the inoculated soil samples consistently showed strong signals in several dilutions
of the soil-derived DNA up to day-40 slots 1–4. Only in the day-40 samples, the background signals equaled
those from the inoculated soil. Quantification by scan- ning revealed initially strong signals in inoculated
soils day-0 and day-15 of about 10-fold background strength, after which signal intensity fell progressively
down to background level after 40 days. Comparison with the positive control strain A1501R genomic
DNA, slots 5 and 11 suggested that initially an esti- mated 10
7
copies of the target were present per gram of the inoculated soils, which declined to less than 10
6
over time. This roughly matched the data obtained by selective plating, and also supported the population
density estimated via PCR–DGGE Fig. 4; see below.
3.3. PCR–DGGE fingerprinting of bacterial communities in inoculated and control soil
Total community DNA was obtained over time from duplicate bulk and rhizosphere soil samples of
both inoculated and uninoculated soil microcosms. PCR-amplified community-level 16S rDNA frag-
ments were then generated. All DNA extracts yielded similar amounts of PCR products of expected size,
i.e. about 450 bp not shown. Results of DGGE pro- filing of these mixed amplicons showed that at each
time point, the fingerprints of replicate samples were indistinguishable from each other; one replicate per
sample is therefore shown in Fig. 4A and B. A dom- inant band, absent from the profiles of uninoculated
soil, was consistently visible in the profiles derived from the initial extracts of inoculated soils, i.e. up to
M. Lin et al. Applied Soil Ecology 15 2000 211–225 219
Fig. 4. PCR–DGGE fingerprinting of soil DNA obtained from uninoculated and inoculated soils: A bulk soils; B rice rhi-
zosphere soils. B in figure: 16S rDNA fragment amplified from strain A1501R using conserved primers; arrow indicates the
A1501R-specific band. Lanes 1 and 2: day-0 samples; Lanes 3 and 4: day-15 samples; Lanes 5 and 6: day-30 samples; Lanes 7
and 8: day-40 samples. Odd numbers: amplified with DNA from uninoculated soils; even numbers: amplified with DNA from inoc-
ulated soils. M: marker; products of, from top to bottom: Listeria innocua
ALM105, Rhizobium leguminosarum biovar trifolii R62, Arthrobacter
sp. A2, Burkholderia cepacia P2 faint band.
day-15 in bulk soil and up to day-30 in rhizosphere soil samples Fig. 4A and B. This band migrated
to the same position as the PCR product generated with strain A1501R. In later samples e.g. day-30
and day-40, these bands in the profiles of inoculated soils became weaker and of similar intensity as comi-
grating weak bands apparent in the profiles generated from uninoculated soils. These observations were
confirmed by using the strain A1501R specific V6 probe, which identified the A1501R-specific bands,
via strong signals, at the same time points indicated. The data suggested that the uninoculated soil samples
might contain relatively low numbers of bacteria es- timated, as numbers of target molecules, at ≤10
5
–10
6
cells per gram of soil with similar 16S ribosomal target sequences.
Fig. 4 further shows that at least six to seven dom- inant bands and about 30 weak bands were present in
almost all DGGE profiles. All dominant bands were very stable and similar between the profiles obtained
for control and inoculated samples. The remaining weak bands were more variable during incubation, as
compared to the major bands. Clustering of the bulk soil-derived profiles via the UPGMA Dice coeffi-
cient of similarity revealed an internally great resem- blance 90 between all profiles, and no evidence
for a trend towards an effect of inoculation Fig. 5A. The rhizosphere-derived profiles clustered together at
Fig. 5. Dendrograms showing clustering of PCR–DGGE generated community fingerprints using UPGMA: A fingerprints obtained
from bulk soil samples; B fingerprints obtained from rhizosphere soil samples. 1, 2: day-0; 3, 4: day-15; 5, 6: day-30; 7, 8: day-40.
Odd numbers: uninoculated soils; even numbers: inoculated soils.
220 M. Lin et al. Applied Soil Ecology 15 2000 211–225
about 80 similarity, and again no effect of inocu- lation was evident Fig. 5B. There was an effect of
incubation time i.e. root growth, as at about 92 of similarity, three clusters could be formed, i.e. the
day-40 samples, the day-30 samples and the day-0 plus day-15 samples Fig. 5B. All of the differences
occurred in the weak bands of the patterns.
3.4. Biolog GN community-level substrate utilization patterns
During 40 days, the substrate utilization patterns of the uninoculated as well as inoculated FSL soil
microcosms were examined using Biolog GN mi- croplates. Among the 95 substrates, 10 could not
be utilized at all by the microbial communities of the tested soil. These included erythritol, lactulose,
xylitol, a-hydroxybutyric acid, a-keto-butyric acid,
a -keto-valeric acid, sebacic acid, alaninamide, glycyl-
l-glutamic acid and threonine. These characteristics were, thus, similar between the bacterial communi-
ties of uninoculated and inoculated soil microcosms. On the other hand, utilization of four carbohydrate or
polymer substrates, i.e. glycogen, arabinose, gentio- biose and mannose, occurred consistently, albeit with
different rates, in all samples.
To assess the contribution of inoculation with A1501R to the substrate utilization patterns at the soil
microbial community level, the responses to 29 dis- criminative substrates Table 1 were analyzed; these
substrates were selected on the basis of substrate cat- egories Insam, 1997. Using this criterion, the effect
of inoculation on community-level substrate utiliza- tion was not at all clear. In bulk soil, overall there
were only up to five significant differences per time point between inoculated and control soils in the val-
ues obtained. However, the differences were erratic and a clear trend could not be detected. Moreover, the
patterns obtained with rhizosphere soil populations, sampled at day-23, were similar between inoculated
and uninoculated soils.
On day-30, 24 of the 29 substrates were utilized similarly between control and inoculated bulk soil
samples, whereas the utilization rates of the re- maining substrates were different Table 1. Six se-
lected substrates, i.e. dextrin, glycogen, citric acid, p
-hydroxyphenylacetic acid, lactic acid or asparagine, were interesting since, in spite of the similar color
Table 1 Response of bacterial communities obtained on day-30 from
uninoculated and inoculated soil microcosms to 29 selected sub- strates of the Biolog GN system
Substrate Class
Differential response U versus I or
remark
a
Dextrin Carbohydrate
SR; SS Glycogen
Carbohydrate IR; SS
Tween-40 Detergent
SR Tween-80
Detergent SR
Fructose Sugar
SR Gentiobiose
Sugar UI
Inositol Sugar
UI Sucrose
Sugar SR
Trehalose Sugar
SR Methyl pyruvate
Organic acid IR
cis -Aconitic acid
Organic acid SR
Citric acid Organic acid
SR; SS, prevalent in rice root exudates
Lin, unpublished p
-Hydroxy-phenyl acetic acid
Organic acid SR; SS
Lactic acid Organic acid
IR; SS, prevalent in rice root exudates
Lin, unpublished Malonic acid
Organic acid NR
Propionic acid Organic acid
UI Quinic acid
Organic acid SR
Saccharic acid Organic acid
IR Succinic acid
Organic acid IR, prevalent in
rice root exudates Lin, unpublished
l-alanyl glycine Amino acid
SR Asparagine
Amino acid SR; SS
Aspartic acid Amino acid
UI Glutamic acid
Amino acid UI
g -Aminobutyric acid
Amino acid IR
Inosine Pyrimidine
NR Thymidine
Pyrimidine IR
Phenylethyl amine Amine
SR Putrescine
Amine IR
Glycerol Alcohol
NR
a
For responses similar between uninoculated U and inocu- lated I soils, the strength of the color development is shown, as
follows: NR, no response 5 of maximal color development; IR, intermediate response 50; SR, strong response 50–
100. SS, substrate selected for community analyses by PCR–DGGE.
development rates observed, they might differentiate inoculated from uninoculated soils on the basis of a
differential response of the inoculant strain. These substrates were used for DGGE analyses of the un-
derlying communities, as outlined in Section 3.5.
M. Lin et al. Applied Soil Ecology 15 2000 211–225 221
3.5. PCR–DGGE community profiling applied to bacterial communities in selected Biolog wells
The DGGE profiles of soil microbial communities obtained on day-30 in the wells of the Biolog GN mi-
croplates containing dextrin, glycogen, citric acid, p- hydroxy phenylacetic acid, lactic acid and asparagine
were analyzed after incubation for 48 h Fig. 6. These six substrates belong to different main groups of
carbon substrates, i.e. carbohydrates, polymers, car- boxylic acid, amino acid and phenolic components.
All but p-hydroxyphenylacetic acid were utilized by strain A1501R.
First, during incubation, the numbers of total bac- teria in all wells increased from about 4×10
4
to over 10
8
CFU per milliliter Table 2. The density of strain A1501R was initially about 3×10
2
CFU per milliliter 0.75 of the total bacterial CFU. In the
lactic acid-containing well it increased to over 10
8
CFU per milliliter, whereas it remained lower around
Fig. 6. DGGE patterns obtained directly from uninoculated − and inoculated + soil, and from six wells of the Biolog GN system
inoculated with the microbial community from these soils. SS: DGGE fingerprints of the bacterial communities in uninoculated
and inoculated soils 30 days; 1–6: profiles of selected microbial communities in Biolog wells. 1, Dextrin; 2, glycogen; 3, citric
acid; 4, p-hydroxy-phenylacetic acid; 5, lactic acid; 6, asparagine and B, profile of strain A1501R.
10
5
CFU per milliliter in wells containing four other substrates dextrin, glycogen, citric acid, p-hydroxy
phenylacetic acid. In the asparagine-containing well, strain A1501R was not found. Hence, A1501R was
a dominant strain in the lactic acid-utilizing bacterial community, whereas it was a minor player in those
degrading the other substrates.
Fig. 6 provides, next to the soil-derived PCR–DGGE fingerprints, the fingerprints showing the effect of
soil inoculation on microbial communities with po- tential to oxidize the selected substrates. The DGGE
profiles of the bacterial communities selected with the six substrates were quite different from those
obtained directly with soil DNA in that a strongly re- duced number of bands was generally observed. For
three substrates, i.e. dextrin, glycogen and citric acid, the profiles were similar between uninoculated and
inoculated soils, with the exception of a consistent extra band from the inoculated soils which comi-
grated with the band generated with strain A1501. For p-hydroxyphenylacetic acid, this comigrating
band from inoculated soil was also observed in oth- erwise divergent profiles. Moreover, the lactic acid
well representing the uninoculated soil showed only two strong bands, whereas those from the inoculated
soils showed, next to other bands, one stronger band which comigrated with the A1501R-derived band.
Therefore, the presence of strain A1501R affected the composition of the bacterial communities capa-
ble of utilizing these five substrates, in particular lactic acid, since it made part of these functional
communities.
However, in spite of the fact that strain A1501R can utilize asparagine as the sole carbon source, its spe-
cific band was not observed in the DGGE profile of the asparagine well inoculated from A1501-containing
soil. This corroborated the fact that the A1501R CFU counts in this asparagine well were below the limit of
detection 10
2
CFU per milliliter, and suggested that indigenous bacterial strains that exhibit a higher ca-
pacity to utilize asparagine than strain A1501R might have outcompeted or inhibited this strain. Also, the
DGGE pattern of the asparagine-utilizing community in the well prepared with bacterial communities from
inoculated soils was quite different from that from uninoculated soil, which suggested that the effect of
inoculation with A1501R on the asparagine-utilizing bacterial community was indirect.
222 M. Lin et al. Applied Soil Ecology 15 2000 211–225
Table 2 Bacterial populations in six selected wells of Biolog GN plates inoculated with bacterial suspensions obtained from soil from microcosms
30 days after the introduction of inoculant strain A1501R
a
Bacterial numbers in wells of Biolog GN microplates CFU per milliliter containing the following substrates
Dextrin Glycogen
Citric acid P
-hydroxy-phenylacetic acid Lactic acid
Asparagine Total counts
4.4×10
8
9.9×10
8
4.7×10
8
8.7×10
8
6.6×10
8
5.5×10
8
A1501R counts 2.0×10
5
3.0×10
5
2.1×10
5
1.6×10
7
7.8×10
8
10
2
Percentage of A1501R in total 0.05
0.03 0.05
1.8 100
[0]
a
Bacterial density established before inoculation: total: 4×10
5
CFU per milliliter; A1501R: 3×10
2
CFU per milliliter.
4. Discussion
