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Applied and Environmental Microbiology, August 2001, p. 3418-3425, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3418-3425.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Impact of 2,4-Diacetylphloroglucinol-Producing Biocontrol Strain
Pseudomonas fluorescens F113 on Intraspecific
Diversity of Resident Culturable Fluorescent Pseudomonads Associated
with the Roots of Field-Grown Sugar Beet Seedlings
Yvan
Moënne-Loccoz,1,2
Hans-Volker
Tichy,3
Anne
O'Donnell,1
Reinhard
Simon,3 and
Fergal
O'Gara1,*
BIOMERIT Research Centre, Microbiology
Department, National University of Ireland, Cork,
Ireland1; UMR CNRS Ecologie Microbienne,
Université Claude Bernard (Lyon 1), 69622 Villeurbanne Cedex,
France2; and Abteilung Biologische
Sicherheit, TÜV Bau und Betrieb GmbH, D-79108 Freiburg,
Germany3
Received 21 November 2000/Accepted 8 May 2001
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ABSTRACT |
The impact of the 2,4-diacetylphloroglucinol-producing biocontrol
agent Pseudomonas fluorescens F113Rif on the diversity
of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was
evaluated. At 19 days after sowing, the seed inoculant F113Rif had
replaced some of the resident culturable fluorescent pseudomonads at
the rhizoplane but had no effect on the number of these bacteria in the
rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the
level of broad phylogenetic groups (by amplified ribosomal DNA
restriction analysis) and at the strain level (with random amplified
polymorphic DNA markers) as well as phenotypically (55 physiological
tests). The introduced pseudomonad induced a major shift in the
composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane
isolates capable of growing on three carbon substrates (erythritol,
adonitol, and L-tryptophan) not assimilated by the
inoculant was increased from less than 10% to more than 40%. However,
the pseudomonads selected did not display enhanced resistance to
2,4-diacetylphloroglucinol. The shift in the resident populations,
which was spatially limited to the surface of the root (i.e., the
rhizoplane), took place without affecting the relative proportions of
phylogenetic groups or the high level of strain diversity of the
resident culturable fluorescent Pseudomonas community.
These results suggest that the root-associated
Pseudomonas community of sugar beet seedlings is
resilient to the perturbation that may be caused by a taxonomically related inoculant.
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INTRODUCTION |
Biological control of diseases
and pests of crops using microbial inoculants is receiving increased
attention as an environmentally friendly alternative to the
use of chemical pesticides (6, 14, 45). For biocontrol
strains of fluorescent Pseudomonas spp., the
production of antimicrobial secondary metabolites often represents a
key factor in their ability to protect plant roots from fungal
soilborne diseases (12, 21, 40). One promising secondary
metabolite is 2,4-diacetylphloroglucinol (Phl), a polyketide synthesized by a diverse array of biocontrol pseudomonads (22, 45). Indeed, genetic enhancement of Phl-producing ability in pseudomonads can lead to higher Phl levels in the rhizosphere (2,
27) and increased plant protection against fungal root diseases
(27, 40, 41).
Efficient biocontrol requires that large numbers of microbial cells be
released into the environment, and issues relating to the biosafety of
this technology have been highlighted, regardless of whether wild-type
or genetically modified strains are considered (6, 9, 48).
Often, the impact of bacterial inoculants on nontarget populations has
been assessed quantitatively, i.e., with respect to the population
sizes of particular microbial groups defined on the basis of taxonomy
or physiological function (4, 11, 16, 33, 37, 47).
However, this approach is limited by the fact that important ecological
impacts may take place in terms of the composition of a particular
microbial group without modification of its population size.
Indirect evidence of perturbations caused by bacterial
inoculants on the indigenous bacterial community of the rhizosphere has
been obtained by studying community-level catabolic profiles on BIOLOG
plates, microbial enzymatic activities, profiles of fatty acid methyl
esters extracted from the rhizosphere, and/or the distribution of
r/K strategists on plates (10, 11, 34, 37, 46). The
impact of biocontrol pseudomonads on the diversity of the bacterial
community has been investigated at the level of the genus and/or
species (26, 37), and it can be expected that this type of
work will benefit from current developments in molecular,
culture-independent approaches (25, 38). However, lower
taxonomic levels (i.e., below the species level) have been neglected so
far. Because of niche overlap (33, 36), a
Pseudomonas inoculant will be expected to interact with
fellow fluorescent pseudomonads indigenous to the soil for colonization
of the rhizosphere. The community of resident culturable fluorescent
pseudomonads (RCFP) in the rhizosphere plays a key role in the
functioning of the ecosystem through its contribution to plant health,
nutrient cycling, and soil fertility (8). It is therefore
important to understand how Phl-producing Pseudomonas
biocontrol inoculants can influence this nontarget community.
The objective of the current work was to assess, under field
conditions, the impact of the Phl-producing biocontrol agent Pseudomonas fluorescens F113Rif on the community of RCFP
associated with the roots of sugar beet seedlings. This objective was
achieved by comparing the intraspecific diversities of rhizosphere and rhizoplane RCFP obtained from uninoculated sugar beet seedlings and
seedlings inoculated with strain F113Rif.
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MATERIALS AND METHODS |
Inoculation of seeds and field experiment.
P.
fluorescens F113, which was isolated from the roots of sugar beets
(42), can protect sugar beets from Pythium damping-off disease. Pythium spp. infect the plant shortly after sowing
(13). Strain F113Rif is a spontaneous rifampin-resistant
mutant of F113 that grows and produces Phl like the wild-type strain in
vitro (4) and whose disease-suppressive ability has been
demonstrated (13, 32).
Strain F113Rif was delivered at 6.0 log CFU per sugar beet seed
(cultivar Accord) using a biocontrol-compatible (32)
seed-pelleting formulation (30). The experiment was
carried out in 1994, near Bandon (County Cork, Ireland), at a site
where the ecological impact of F113Rif on ecosystem functioning has
been studied using a combination of different approaches (30, 31,
35). The field site, soil characteristics (brown podzolic soil),
and farming conditions have been described in detail elsewhere
(30). Damping-off disease of sugar beets often takes place
at this site, but there was no disease pressure in 1994 (probably for
climatic reasons), so that potential nontarget effects of the inoculant
could not be compensated for by its positive biocontrol effect. Four
plots (adjacent to plots I-1, III-1, V-1, and VII-1 defined by
Moënne-Loccoz et al. [30]) located along a 72-m
transect and comprising furrows of uninoculated sugar beet seeds and
furrows of F113Rif-inoculated seeds were studied. The distance between
the centers of two consecutive plots was 24 m.
Sampling and colony counts.
At 19 days after sowing, three
adjacent inoculated sugar beets (spaced 17 cm apart on the row), three
adjacent uninoculated plants, and a 100-g bulk soil sample (taken from
the surface soil horizon halfway between two uninoculated rows)
were collected at the center of each plot. Two neighboring rows
(distant by 56 cm) were used to sample the plants (one row with
inoculated sugar beets and the other with uninoculated sugar beets).
Plant shoots were 4 to 6 cm high, and roots (about 10 to 12 cm long)
were all located within the 22-cm-deep loamy surface soil horizon.
Each sample was processed individually as follows. Bulk soil (1 g) was
transferred into 10 ml of one-quarter-strength Ringer's
solution
(Oxoid, Hampshire, United Kingdom) in a McCartney bottle,
and the
bottles were vortexed for 5 min. Loosely adhering soil
was detached
from the roots by shaking and was discarded. The
soil remaining on the
roots (i.e., closely adhering soil) was
considered rhizosphere soil. A
diligent effort was made to remove
this soil adhering closely to the
roots by using a spatula and
then by dipping the root system for 2 s in 10 ml of one-quarter-strength
Ringer's solution in a McCartney
bottle. The rhizosphere soil
removed with the spatula was subsequently
added to those bottles,
which were vortexed for 5 min. The shoots were
excised. Each soil-free
root system was transferred to a new bottle
containing 10 ml of
one-quarter-strength Ringer's solution and was
extracted by vortexing
the bottle for 5 min. The extract was used to
recover rhizoplane
bacteria.
Each extract (from individual samples of bulk soil, rhizosphere soil,
and rhizoplane) was serially diluted in one-quarter-strength
Ringer's
solution and spread plated. Colony counts of F113Rif
were determined on
Luria-Bertani (LB) (
39) agar containing 100
µg of
rifampin/ml (i.e., Rif100) and the antifungal compound cycloheximide
(100 µg/ml). Colonies derived from inoculated plants and resistant
to
Rif100 displayed a random amplified polymorphic DNA (RAPD)
profile
(
29) identical to that of F113Rif (
30). A few
Rif100-resistant
colonies were obtained from bulk soil or uninoculated
plants,
but their RAPD profiles differed from that of F113Rif (data not
shown).
The total culturable fluorescent pseudomonads and the total
culturable aerobic bacteria were recovered on S1 agar (
17)
and
LB agar as described by Carroll et al. (
4) and
Moënne-Loccoz
et al. (
30), respectively. S1 agar is
a selective medium for
fluorescent pseudomonads (
17), and
colony counts on S1 agar
were shown to be in agreement with 16S
ribosomal DNA (rDNA) quantitative
direct PCR data for enumeration of
root-associated pseudomonads
(
20). In addition,
Pseudomonas diversity is higher on S1 agar
than on King's B
agar (
19), which is the medium traditionally
used to
recover fluorescent pseudomonads, and S1 has become the
medium of
choice for these bacteria (
19,
36,
47,
49).
All plates
were incubated at room temperature for 3 to 7 days
prior to counting of
colonies.
Isolation of resident fluorescent pseudomonads.
Colonies on
S1 agar were chosen at random, purified by subculturing, and replica
plated on LB Rif100 agar to distinguish resident bacteria from F113Rif
(and to determine the percentage of colonies on S1 agar that
corresponded to the inoculant). Colonies resistant to Rif100 were
discarded for all treatments, and a replica of the others was checked
for fluorescence under UV light. Thirty RCFP from bulk soil were
obtained from each of the four plots (for a total of 120 isolates).
Totals of 30 rhizoplane isolates and 30 rhizosphere isolates were
obtained from uninoculated sugar beets as well as from
F113Rif-inoculated sugar beets from each of the four plots. The 600 RCFP were stored at 
80°C in glycerol solutions and kept on LB agar
at 4°C for short-term maintenance. About 17% of the isolates were
lost during the study, and characterization was completed using the 498 remaining isolates.
Genotypic and phenotypic characterizations.
Strain F113Rif
and all 498 RCFP isolates were studied by amplified 16S rDNA
restriction analysis (ARDRA) and RAPD analysis. ARDRA was performed
using TaqI and AluI as described previously (50). Analysis of RAPD markers was done using primer DAF-4
(52). Banding patterns were generated using an automated
laser fluorescent sequencer (Amersham Pharmacia Biotech, Freiburg,
Germany) and compared with the use of WinCam2.2 software (Cybertech,
Berlin, Germany).
For phenotypic characterization, 55 physiological attributes (see
Tables
4 and
5) were studied by replica plating using
actively growing
colonies from LB plates. Gelatin liquefaction
was determined as
described previously (
15). Growth on single
carbon sources
was studied with a low-potassium minimal medium
(
3)
containing 0.05% yeast extract and 15 mM carbon source
and solidified
with purified agar (Oxoid). Growth of F113Rif and
the isolates did not
take place on this medium without a carbon
source. To study the growth
on seed exudates, sugar beet seeds
(250 g) were added to 500 ml of
sterile distilled water, and the
flasks were shaken at 100 rpm for
3 h. The solution was filtered
successively through a series of
five Millipore membranes 5, 3,
1.2, 0.45, and 0.20 µm in pore
diameter. The seed exudate solution
was mixed with a 3% agar solution
in a 1:1 ratio and poured into
plates. Growth in the presence of
antibiotics (see Table
5) was
assessed using LB plates amended with the
antibiotics. Synthetic
Phl was obtained from the Chemistry Department,
National University
of Ireland, Cork. Plates were scored (growth or no
growth) after
3 to 7 days of incubation at 28°C. The ability to
produce Phl
was investigated using the Phl-sensitive indicator
bacterium
Bacillus subtilis A1 (
14), and RCFP
that inhibited A1 growth were studied
further by high-pressure
liquid chromatography analysis (
43).
Indices of strain diversity.
Strains were defined based on
either phenotypic profiles (yielding phenotypically defined strains) or
RAPD profiles (yielding genotypically defined strains) as follows.
Isolates sharing the same phenotypic or RAPD profile were considered to
belong to the same phenotypically or genotypically defined strain,
respectively. The genotypic (RAPD) and phenotypic diversities of the
RCFP were evaluated with regard to the number of strains identified
(i.e., strain richness) using Shannon's H' index
(44) and the distribution of isolates among those strains
(i.e., strain evenness) using Shannon's E index
(44). Strain evenness was computed from H' and
the total number of strains (S) as follows:
E = H'/ln S.
Statistics.
Colony counts obtained from individual plants
were log transformed. The effect of inoculation on the numbers of RCFP
and resident culturable aerobic bacteria was assessed for the
rhizosphere (12 replications) and for the rhizoplane (12 replications).
This assessment was done by analyses of variance (P < 0.05), and Systat 5.05 was used (SPSS Science, Chicago, Ill.).
Three statistical approaches were used to study the composition of the
RCFP community at Bandon. In the first one, analyses
of variance were
carried out, followed (when appropriate) by Fisher's
least-significant-difference tests (
P < 0.05; Systat
5.05). Each
of the four plots was considered a replication (i.e.,
results
for isolates from each set of three adjacent plants were
pooled),
and arcsine-transformed values of the square root of
percentages
were used in the analyses. Two limitations need to be kept
in
mind with these types of analyses. First, there is always a small
number of isolates that die during a biodiversity study that focuses
on
environmental bacteria (
16); consequently, treatments do
not contain exactly the same numbers of isolates. In this work,
fluctuations in the final number of isolates from one treatment
to the
next were relatively modest (from 80 to 110 isolates per
treatment),
and statistical analyses gave the same results when
a total of 80 randomly chosen isolates were used for each treatment.
The second issue
corresponds to the normality of the data which,
considering the
experimental design, could not be established
formally in this
work.
Therefore, the statistical relationship between treatments was
confirmed using procedures of the general linearized model
(GLIM) as
described by Crawley (
7). In this situation, error
type is
binomial when the exact number of isolates in each treatment
is
considered. For each variable studied, the proportion of isolates
scoring positively was analyzed according to a first factor
corresponding
to the distance to the root (i.e., rhizosphere versus
rhizoplane)
and a second factor corresponding to inoculation (i.e.,
inoculation
with F113Rif versus no inoculation). The significance of
each
simple factor and the interaction between those factors were
studied
using
t tests with GLIM parameters
(
P < 0.05) as described previously
(
7).
This statistical approach is interesting when one is considering
the
differences in the number of isolates from one treatment to
the next
and the issue of normality, but the fifth treatment (i.e.,
bulk soil)
is excluded from the analyses. However, both statistical
approaches
gave similar results for the four root-associated treatments
in most
instances. Therefore, the statistical analyses presented
in this report
are those obtained with the first approach (
36),
which
were confirmed by GLIM
analysis.
The third statistical approach was based on
2
tests (
P < 0.05) and focused on resident isolates
obtained from the rhizoplane
of inoculated plants, with the objective
of comparing the subpopulation
of RCFP isolates that could assimilate
the substrates erythritol,
adonitol, and
L-tryptophan with the other isolates from the
same
treatment. Data expressed as the numbers of isolates were used
in
all
2 tests.
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RESULTS AND DISCUSSION |
The biocontrol inoculant had replaced some of the root-associated
RCFP at 19 days.
At 19 days after sowing, the biocontrol inoculant
P. fluorescens F113Rif was found at 5.61 ± 0.35 log
CFU/root system in the rhizosphere of sugar beet seedlings. The
presence of the inoculant in the rhizosphere had no effect on the
number of RCFP or that of the total culturable fluorescent pseudomonads
(Table 1), despite the fact that F113Rif
represented as much as 63% of the latter in the rhizosphere of
inoculated sugar beets. The inoculant was recovered at 5.29 ± 0.41 log CFU/root system at the rhizoplane, and the number of RCFP at
the rhizoplane of inoculated sugar beets was lower than that for
uninoculated plants (Table 1). This lower value was due to the presence
of F113Rif, as the numbers of total culturable fluorescent pseudomonads
at the rhizoplane did not differ statistically for inoculated and
uninoculated sugar beets. The inoculant represented 75% of the total
culturable fluorescent pseudomonads at the rhizoplane of inoculated
sugar beets.
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TABLE 1.
Effect of the biocontrol seed inoculant P. fluorescens F113Rif on populations of culturable fluorescent
pseudomonads and culturable aerobic bacteria associated with roots of
field-grown sugar beet seedlings at 19 days
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Inoculation of soil or seeds with pseudomonads for biocontrol purposes
implies the release of cells in large numbers (often
higher than the
number of RCFP), which can cause a transient increase
in the number of
total culturable fluorescent pseudomonads (
1,
49) and even
sometimes in that of total culturable aerobic bacteria
(
4,
49). At 19 days after sowing, the number of total culturable
fluorescent pseudomonads at the rhizoplane was not affected, but
that
of RCFP was reduced (Table
1), similar to the results of
other studies
(
10,
36,
47). This suggests that sampling
took place after
this transient situation and that the seed inoculant
interacted with
RCFP while colonizing the roots of sugar beet
seedlings.
The biocontrol inoculant had no impact on the structure of the
community of root-associated RCFP at 19 days.
A total of 498 RCFP
isolates were characterized. No colony was found on S1 agar when
uninoculated seeds were studied (as described by Moënne-Loccoz et
al. [32]) before sowing, which means that for the four
root-associated treatments, the RCFP sampled were unlikely to
have originated from populations of naturally occurring seed-borne
pseudomonads. Four phylogenetic groups were identified by ARDRA
for 496 of the 498 RCFP isolates (i.e., groups
AluI-2/TaqI-1, AluI-13/TaqI-1,
AluI-13/TaqI-7, and
AluI-13/TaqI-12) (Fig.
1). The percentage of resident isolates
within each of these four ARDRA groups was not influenced by the
presence of roots or inoculation with F113Rif (Table
2). Similarly, inoculation of a
pseudomonad did not alter the community structure of the culturable
aerobic bacteria colonizing cucumber roots (studied at the genus level) (26) or the 16S rDNA denaturing gradient gel
electrophoretic patterns of bacteria in the potato rhizosphere
(25). In summary, the biocontrol inoculant F113Rif had no
impact on the structure of the RCFP community associated with the roots
of sugar beet seedlings.
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FIG. 1.
Molecular analysis of P.
fluorescens F113Rif and the 498 isolates of RCFP by ARDRA. The
DNA fragments obtained by ARDRA were separated by electrophoresis
using an automated laser fluorescent sequencer. The resulting traces
were then incorporated into image analysis software (WinCam2.2) and
converted into the banding patterns shown. Four ARDRA groups were
identified for 496 of the 498 RCFP isolates when restriction analysis
of amplified 16S rDNA was done using AluI and
TaqI as follows. With AluI, the isolates
yielded profile AluI-2 or AluI-13 (A).
With TaqI, isolates with profile AluI-13
displayed profile TaqI-1, TaqI-7, or
TaqI-12, and those with profile AluI-2
yielded profile TaqI-1 (B). The biocontrol inoculant
F113Rif displayed profiles AluI-13 and
TaqI-1.
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TABLE 2.
Frequency of ARDRA groups
AluI-2/TaqI-1,
AluI-13/TaqI-1,
AluI-13/TaqI-7, and
AluI-13/TaqI-12 in RCFP following inoculation of
sugar beet seeds with the biocontrol agent P. fluorescens
F113Rif
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The biocontrol inoculant had no impact on strain distribution
patterns within the community of root-associated RCFP at 19 days.
The most frequent RAPD profile was shared by 27 of the 498 RCFP
isolates, which in turn displayed a total of 24 different phenotypic
profiles. One of the rhizoplane isolates displayed a RAPD profile
identical to that of F113Rif, but the isolate was phenotypically
different from the inoculant based on several properties in addition to
Rif100 sensitivity. A striking feature of the collection of 498 RCFP
isolates sampled in the experiment was its very high level of strain
diversity, regardless of whether strains were defined by molecular
(RAPD analysis) (Fig. 2) or phenotypic
(based on 55 independent tests) characterization. Indeed, RAPD and
phenotypic determinations identified as many as 310 and 442 different
strains, respectively, from the 498 isolates. Plasmids are infrequent
in fluorescent pseudomonads isolated from soil or roots
(23) and thus were unlikely to account for the high level
of strain diversity found in these isolates. Not surprisingly, only
small percentages (usually less than 10%) of the RAPD profiles and
phenotypic profiles observed with a given treatment also were found
with another treatment. In parallel, the level of strain diversity
(Shannon's H' index) was high and strain evenness
(Shannon's E index) was close to 1, regardless of
whether phenotypic or RAPD profiles were considered (Table
3). These results suggest that the
disturbance caused to soil RCFP by the plant and/or the inoculant was
small, if it existed at all.
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FIG. 2.
Diversity of RAPD profiles for 16 isolates randomly
chosen from the 498 RCFP isolates (lanes 3 to 23) and for strain
F113Rif (lane 1). PCR fragments generated by the RAPD technique were
separated by electrophoresis, and the photograph was taken after silver
staining of the gel. The size marker  X174-RF-DNA
(HaeIII digest; Amersham Pharmacia Biotech) is included
in lane 2 and shows bands (from top to bottom) of 1,358, 1,078, 872, 603, 310 to 271 (in fact, three bands too close to each other to be
distinguished), 234, 194, and 118 bp (a 72-bp band is too faint to be
seen). Isolates X109, X116, and X117 (lanes 14, 21, and 22, respectively) exhibited the same RAPD profile, as did isolates X113 and
X115 (lanes 18 and 20, respectively). The reproducibility of the RAPD
procedure is illustrated by duplicate assays of isolates X103 (in lanes
8 and 9) and X110 (in lanes 15 and 16).
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In summary, the strain diversity of RCFP at 19 days was high and did
not appear to have been influenced by the presence of
sugar beet roots
or inoculation with F113Rif. This finding contrasts
with the reduced
diversity levels observed for RCFP colonizing
tomato or flax in
mesocosms (
24), as well as for other resident
bacteria
(
Paenibacillus polymyxa) at the rhizoplane of wheat compared
with bulk soil in microcosms (
28), and suggests that the
community
of root-associated RCFP of field-grown sugar beet seedlings
may
be particularly resilient to ecological perturbation in terms
of
strain
diversity.
The biocontrol inoculant had a major impact on the composition of
root-associated RCFP at 19 days.
In the current work, specific
catabolic traits were used to compare the 498 isolates of RCFP, as was
done for resident culturable aerobic bacteria from soybeans inoculated
with Bacillus cereus UW85 (16). A large
proportion of the carbon substrates tested have been detected in the
exudates of plants (including sugar beets) but under laboratory
conditions (5, 8, 18, 51). The capacity to assimilate
D-xylose, a monomer of several plant cell
polymers that is present in sugar beet seed exudates (5), was less frequent in RCFP from bulk soil than in those from three of
the four treatments associated with roots (Table
4); this result indicates possible
selection by the roots of D-xylose-assimilating strains. D-Xylose was not assimilated by any RCFP
associated with flax roots, whereas this compound could be used by a
large percentage of RCFP isolated from tomato roots (23).
In the current work, however, the frequency of the other catabolic
traits in the resident isolates was not influenced by the presence of
roots, indicating that the perturbation caused to RCFP by the plant was
small. This may be a particular feature of young sugar beet plants
and/or of crop rotation systems, as suggested by the moderate (tomato) and strong (flax) perturbations caused to RCFP in greenhouse mesocosms under monoculture conditions (23, 24).
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TABLE 4.
Phenotypic properties of RCFP following inoculation of
seeds with the biocontrol agent P. fluorescens F113Rif
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In contrast, the introduced biocontrol agent had a major impact on
RCFP. This conclusion is indicated by the fact that the
percentages of
isolates assimilating trehalose, erythritol, adonitol,
or
L-tryptophan, which F113Rif cannot assimilate, were higher
at the rhizoplane of F113Rif-inoculated plants than with the other
four
treatments (Table
4). In parallel, the percentage of RCFP
from the
rhizoplane capable of growing on sugar beet seed exudates
was
statistically higher for inoculated plants than for uninoculated
sugar
beets. Whether these four compounds were actually present
in the
vicinity of sugar beet roots is unknown, but this result
suggests that
RCFP displaying catabolic abilities different from
those of F113Rif
(and thus more likely to secure carbon substrates
released by the root
and not used by the inoculant) were favored
over RCFP placed in more
direct competition with F113Rif for growth
substrates derived from the
plant. This notion is also suggested
by the observation that the
percentage of root-associated RCFP
isolates capable of assimilating
L-tryptophan was lower for cucumber
grown in soil
inoculated with the Phl-producing biocontrol strain
P. fluorescens CHA0 (which can assimilate this amino acid) than
for
cucumber grown in uninoculated soil (
36). In the
rhizosphere,
however, the percentage of RCFP capable of growing on
sugar beet
seed exudates was statistically lower for inoculated plants
than
for uninoculated sugar beets in the current work (Table
4), a
result which suggests movements of resident pseudomonads from
one root
compartment (rhizosphere) to the other (rhizoplane) for
inoculated
plants.
The major impact of the biocontrol inoculant on root-associated
RCFP at 19 days was unlikely to be mediated by Phl inhibition of the
RCFP.
Synthetic Phl is inhibitory at low levels to various
microorganisms in vitro (21, 42). Here, however, the
impact of F113Rif did not result from Phl-resistant RCFP enrichment, as
treatments had no influence on the percentages of RCFP capable of
growing in the presence of Phl at various concentrations (Table
5). This finding is consistent with the
fact that a Phl-negative mutant of strain F113 (lacking the ability to
suppress Pythium damping-off disease) (14) colonized the
roots of sugar beets in a manner similar to that of its Phl-positive
counterpart (4). In fact, a majority of isolates were
resistant to rather high levels of Phl, confirming previous results
(36). In contrast, the trifolitoxin-producing strain
Rhizobium etli CE3(pT2TFXK) [but not the
trifolitoxin-negative nearly isogenic mutant CE3(pT2TX3K)] had a
negative impact on taxonomically related bacteria indigenous to the
rhizosphere of field-grown beans, but these indigenous bacteria were
trifolitoxin sensitive (38).
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|
TABLE 5.
Resistance to Phl of RCFP following inoculation of seeds
with the Phl-producing biocontrol agent P. fluorescens
F113Rifa
|
|
In the current work, the resistance of RCFP to Phl was unlikely to have
resulted from previous exposure to Phl released by
pseudomonads
indigenous to the site, since none of the 498 RCFP
isolates studied
could produce Phl, as indicated by inhibition
tests of Phl-sensitive
indicator bacterium
B. subtilis A1 on plates
and
high-pressure liquid chromatography analysis. Determination
of the
patterns of resistance of RCFP to commercial antibiotics
has been
proposed as an efficient phenotypic approach to distinguish
between
different root-associated bacteria (
16). This was
confirmed
here, but the method failed to identify an effect of the
inoculant
on RCFP (Table
5). In summary, Phl inhibition of RCFP was not
an important factor in the ecological perturbation to the RCFP
community that followed the inoculation of Phl-producing strain
F113Rif.
Characterization of RCFP favored at the rhizoplane in the presence
of the biocontrol inoculant.
When we considered together the
phenotypic properties of the resident strains from the rhizoplane of
inoculated sugar beets, it appeared that the ability to assimilate
erythritol, adonitol, and L-tryptophan was shared by 42.9%
of the RCFP (i.e., 42 of 98 isolates) and that the latter could also
grow on trehalose and seed exudates (Table
6). This percentage of 42.9% was
statistically higher than that found with the other treatments (0% in
bulk soil and 7.6% or less with the three other root-associated
treatments). An ecological impact of this magnitude caused by a
Pseudomonas biocontrol inoculant on RCFP was not detected
before (36), perhaps for methodological reasons. The
impact of F113Rif was larger than that of Burkholderia
cepacia MCI 7 on resident Burkholderia populations associated with maize roots (33).
View this table:
[in this window]
[in a new window]
|
TABLE 6.
Characterization of the 42 RCFP that were capable of
assimilating the three compounds erythritol, adonitol, and
L-tryptophan and that were obtained from the
rhizoplane of sugar beets inoculated with the biocontrol agent P. fluorescens F113Rif
|
|
Considering the impact of F113Rif on the RCFP at the rhizoplane, it may
seem unexpected that a high level of strain diversity
was maintained
with this treatment (Table
3). In fact, RAPD and
phenotypic analyses
distinguished as many as 32 and 39 strains,
respectively, from the 42 isolates from the rhizoplane of inoculated
sugar beets with the ability
to assimilate the carbon compounds
erythritol, adonitol, and
L-tryptophan. The high number of phenotypic
profiles is
explained by the diversity of responses of the 42
isolates in the other
52 phenotypic tests (Table
6). In addition,
these isolates were
distributed over the four main ARDRA groups
(Table
6). Therefore, when
one is considering the meaning of
the shift caused by F113Rif at the
strain level, it appears that
(i) this shift took place in each
phylogenetic group and (ii)
in each of these groups several different
strains with particular
catabolic traits in common were selected, so
that the level of
strain diversity of the RCFP community at the
rhizoplane of F113Rif-inoculated
plants remained high (Table
3).
Ecological significance.
In this study, the interactions
between the Phl-producing inoculant P. fluorescens F113Rif
and RCFP appeared essentially to have involved phenomena other than
Phl-mediated antagonism (probably microbial competition), and these
interactions did not result in a modification of the structure or of
the high level of strain diversity of the RCFP community. Despite its
impact on RCFP, the inoculant had no effect on key aspects of ecosystem
functioning (30, 31, 35). Overall, the results indicate
that certain nontarget resident bacterial communities (here the
community of RCFP) may have the capacity to buffer the ecological
impact to which they are subjected following the introduction of
taxonomically related bacterial inoculants. Further work is needed to
assess whether these findings are specific to the pioneer community
colonizing seedlings or can be extended to the RCFP community
associated with roots of older plants. Nevertheless, they establish
novel baseline information for biosafety research and will be
important to consider in characterizing the fate of genetically
modified inoculants (e.g., Phl-overproducing Pseudomonas
strains displaying improved biocontrol activity) released into the soil environment.
| |
ACKNOWLEDGMENTS |
We thank P. Higgins and J. McCarthy (BIOMERIT Research Centre,
National University of Ireland, Cork) for technical assistance and F. Gourbière (UMR CNRS Ecologie Microbienne, Lyon 1, Villeurbanne) for help and discussions regarding statistics. Y.M.-L.
was a visiting professor at the BIOMERIT Research Centre during part of
this study.
This work was supported by grants from the Biotechnology
Programme (BIO2-CT93-0053 [IMPACT Project], BIO2-CT93-0196,
BIO4-CT96-0027 [IMPACT 2 Project], BIO4-CT96-0181,
BIO4-CT97-2227, and BIO4-CT98-0254) and the TMR Programme
(FMRX-CT96-0039) of the European Commission as well as by
grants awarded by Enterprise Ireland (SC/98/261 and SC/98/306).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: BIOMERIT
Research Centre, Microbiology Department, National University of
Ireland, Cork, Ireland. Phone: 353 21 4272097. Fax: 353 21 4275934. E-mail: f.ogara{at}ucc.ie.
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Applied and Environmental Microbiology, August 2001, p. 3418-3425, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3418-3425.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
