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Applied and Environmental Microbiology, June 2001, p. 2627-2635, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2627-2635.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Involvement of Nitrate Reductase and Pyoverdine in
Competitiveness of Pseudomonas fluorescens Strain C7R12
in Soil
Pascal
Mirleau,1
Laurent
Philippot,2
Thérèse
Corberand,1 and
Philippe
Lemanceau1,*
UMR INRA/Université de Bourgogne
BBCE-IPM1 and
MS-Geosol,2 CMSE-INRA, 21065 Dijon
Cedex, France
Received 1 December 2000/Accepted 14 March 2001
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ABSTRACT |
Involvement of nitrate reductase and pyoverdine in the
competitiveness of the biocontrol strain Pseudomonas
fluorescens C7R12 was determined, under gnotobiotic conditions,
in two soil compartments (bulk and rhizosphere soil), with the soil
being kept at two different values of matric potential (
1 and 10
kPa). Three mutants affected in the synthesis of either the nitrate
reductase (Nar
), the pyoverdine (Pvd), or
both (Nar Pvd) were used. The
Nar and Nar Pvd mutants were
obtained by site-directed mutagenesis of the wild-type strain and
of the Pvd mutant, respectively. The selective advantage
given by nitrate reductase and pyoverdine to the wild-type strain was
assessed by measuring the dynamic of each mutant-to-total-inoculant
(wild-type strain plus mutant) ratio. All three mutants showed a lower
competitiveness than the wild-type strain, indicating that both nitrate
reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest
competitiveness. Overall, the competitive advantages given to C7R12 by
nitrate reductase and pyoverdine were similar. However, the selective
advantage given by nitrate reductase was more strongly expressed under
conditions of lower aeration (1 kPa). In contrast, the selective
advantage given by nitrate reductase and pyoverdine did not differ in
bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in
the saprophytic ability of C7R12 in soil environments.
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INTRODUCTION |
Fluorescent pseudomonads can
suppress various soilborne diseases (40). Their efficacy
has been related both to their antagonistic activities and to their
rhizosphere competence (4, 6). Overall, biological control
of soilborne disease achieved by fluorescent pseudomonads is often
inconsistent (15, 40). This inconsistency has been
partially associated with inefficient root colonization by the
introduced bacteria (35). Indeed, a clear relationship has
been established between suppression of the wheat root disease take-all
and that of fusarium wilts by different strains of fluorescent pseudomonads and the densities of these bacteria in the rhizosphere of
the corresponding host plants (1, 33). In order to improve the efficacy and the consistency of the biological control, the use of
rhizosphere-competent strains is then required. However, since the
microbial inoculations would mainly be performed in soils before the
plant is grown, the strains should also be able to survive in the soil
and should then show a good saprophytic ability. To fulfil these
requirements, progress must be made in the knowledge of bacterial
traits promoting saprophytic ability under soil conditions.
Despite the abundance of iron in soils, the concentration of ferric
iron available to the soilborne microflora is very low (17). Since Fe3+ is an essential element for
most microorganisms, this ion is often a limiting factor for microbial
growth and activity in soil habitats (18). Most
microorganisms have developed an active strategy for iron acquisition
based on the use of siderophores and of the corresponding
ferrisiderophore membrane receptors (26). The major
siderophores of the fluorescent pseudomonads, called pyoverdines, show
a very high affinity for Fe3+ (23). Several
studies have stressed the role played by pyoverdine-mediated iron
competition in the microbial antagonism performed by biocontrol strains
against some pathogens (18). Other studies have underlined the involvement of pyoverdine-mediated iron uptake in the ecological fitness of different strains of fluorescent pseudomonads (20, 25,
32). Ferric iron is indeed known to play a major role in the
bacterial metabolism since it is an intermediate electron acceptor in
the respiratory chain. Some fluorescent pseudomonads are able to adapt
to limited oxygen conditions by using nitrogen oxides as alternative
electron acceptors (38), and respiratory nitrate and
nitrite reductase have been described to be implicated in the
competitiveness of model strains of fluorescent pseudomonad in soil
(8, 28).
The soil environment experiences major changes such as those resulting
from root growth and from rainfall and/or irrigation. The growth and
activity of the root system induce significant modifications in the
physicochemical and biological properties of the soil surrounding the
root; these modifications correspond to the so-called rhizosphere
effect. This effect is partly related to the higher concentration of
carbohydrates (electron donors) in the rhizosphere than in the bulk
soil, due to rhizodeposition (22). This results in higher
oxygen consumption due to microbial respiration in rhizosphere compared
to bulk medium as reported by Højberg and Sørensen (9).
This may account for the fact that the frequency of populations of
fluorescent pseudomonads able to reduce nitrates in the rhizosphere is
higher than that in bulk soil (2). Aeration of the soil is
also affected by the hydrous pattern. Rainfall and/or irrigation leads
to an increase of the soil porosity filled with water and to a
reduction of the aeration, as demonstrated using an oxygen-sensing
reporter strain of Pseudomonas fluorescens
(10). This reduced aeration is expected to decrease the
concentration of ferric iron (17), and roots were shown to
influence the biological availability of ferric iron (19).
Under soil conditions where the oxygen status and the ferric iron
availability are subject to variations, fluorescent pseudomonads able
to synthesize both nitrate reductase and pyoverdine should then have a
competitive advantage.
The aim of our study was to assess the involvement of pyoverdine
and nitrate reductase in the competitiveness of a biocontrol strain of
P. fluorescens. This comparison was based on the use of
isogenic mutants unable to synthesize pyoverdine, nitrate reductase, or
both. The relative importance of nitrate reductase and of pyoverdine in
the competitiveness of the wild-type strain was evaluated by comparing
the dynamics of each mutant-to-total-inoculant (wild-type strain plus
mutant) ratio. This strategy is commonly considered the most accurate
in evaluating the role of a specific genotype and/or phenotype in the
competence of a bacterial strain (8, 28, 36, 39). The
impact of soil conditions, expected to influence the oxygen status and
the ferric iron availability, was assessed by performing the
competitiveness experiments in a given soil, cultivated or not with
tomato and kept at two different matric potential values (1 versus
10 kPa). The soil was sterilized prior to inoculation and the
competitiveness experiments were then conducted under gnotobiotic
conditions in order to make a more straightforward demonstration and to
avoid any possible interference with the native microflora. Previous
studies have indeed shown that the competitive advantage of different
wild-type strains over their mutants was more clearly expressed under
gnotobiotic than under nongnotobiotic conditions (25, 28).
Therefore, the experiments presented here deal with intraspecific
competition between the wild-type strain and the defective mutants.
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MATERIALS AND METHODS |
Organisms and growth conditions.
P. fluorescens
strain C7R12 is a spontaneous mutant of strain C7 resistant to rifampin
(100 mg liter1) (7). The wild-type strain C7
was previously isolated from the rhizosphere of flax cultivated in the
Châteaurenard, France, soil that suppresses fusarium wilts
(16). The strain C7R12 was shown (i) to improve the
suppression of fusarium wilts achieved by nonpathogenic Fusarium
oxysporum (14) and (ii) to be rhizosphere competent
(7). Pyoverdine-deficient mutant PL1 was previously obtained from strain C7R12 by Tn5 insertion into a
pyoverdine synthetase gene (25). Pseudomonad strains were
grown in KB (12) or Luria broth (LB) medium
(24). The antibiotics incorporated into these media were
gentamicin (10 µg/ml), tetracycline (10 µg/ml), ampicillin (10 µg/ml), kanamycin (50 µg/ml), and rifampin (100 µg/ml).
Escherichia coli host strains for plasmids were grown in LB
medium at 37°C.
Construction of Nar mutants of P. fluorescens C7R12.
Using primers narG-TET
(5'-ACG-TTG-CCA-AGG-ACT-ATG-AC-3') and narG-END
(5'-CGG-TGA-TGG-TGC-GCC-ATG-GG-3'), a 1.6-kb fragment of
the narG gene of P. fluorescens C7R12 was
amplified (for 3 min at 95°C; followed by 35 cycles at 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min; and then 5 min at 72°C)
and cloned into the pT7 plasmid according to the manufacturer's
procedures (Novagen-Merck, Nogent-sur-Marne, France). To obtain the
Nar mutants, the wild-type chromosomal copy of
narG of P. fluorescens C7R12 and PL1 was replaced
after homologous recombination by a copy of the deleted gene with an
insertion of the apra3 gentamicin resistance gene (Fig.
1). DNA restriction, agarose gel
electrophoresis, ligation, and transformation were carried out by
standard methods (31). The different steps describing the
construction of the cosmid vector pL3NR carrying a copy of the deleted
narG gene with an insertion of the apra3
gentamicin resistance gene are presented in Fig. 1. Briefly the
apra3 gene encoding resistance to gentamicin from plasmid
pHP45
(34) was ligated into pT7NR. The
narG::apra3 construct was then
excised from pT7NR and cloned into the pLAFR3 cosmid (37),
yielding pL3NR. Electrotransformation of P. fluorescens C7R12 and PL1 with plasmid pL3NR was achieved in a Bio-Rad pulser apparatus (with settings of 2.5 kV, 25 µF, and 200 for 4 to 6 ms). Transconjugants of C7R12 and of PL1 were selected on LB medium
supplemented with gentamicin and tetracycline. Recombinants showing
double crossing-over were identified after several rounds of growth on
LB medium containing gentamicin at 4°C to cure the pL3NR and scoring
for Tcs Gmr colonies. Two Nar
mutants, namely, NR2 and PL1NR6 of C7R12 and PL1, respectively, were
chosen. Southern blot analysis of chromosomal DNA of P. fluorescens C7R12 and of the NR2 and PL1NR6 mutants was performed
to check the replacement of the wild-type chromosomal copy of the
narG gene with the copy with the deletion and containing the
apra3 gentamicin resistance gene.
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FIG. 1.
Construction of a cosmid vector containing the deleted
narG gene with an insertion consisting of a gentamicin
resistance (Gmr) gene. A 1.8-kb BamHI fragment
containing the apra3 gene from pHP45 was ligated into the
NarI sites of pT7NR after filling in to generate blunt ends.
The resulting deleted narG gene with an insertion of the
apra3 gentamicin resistance gene was then cut from the
pT7NRgm plasmid with SmaI-XbaI and cloned into
the EcoRI site of pLAFR3 (37).
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Growth characteristics of C7R12, NR2, and PL1NR6 in liquid
medium.
For preparation of inocula, strains were cultured in 10 ml
of LB medium. After 24 h of incubation, bacterial cells were
collected by centrifugation at 8,000 × g for 20 min. The cells were resuspended in LB medium to obtain an absorbance at
600 nm of 0.1.
For anaerobic treatments, 150-ml plasma flasks containing 50 ml of LB
medium, supplemented with 10 mM KNO
3 or 10 mM
KNO
2,
were made anaerobic by evacuation and flushing three
times with
helium and then were aseptically inoculated through the
rubber
stopper with each of the different microbial inocula to
obtain
a final absorbance of 0.01. The denitrification rates by
P. fluorescens C7R12 in succinate medium
supplemented with 10 mM KNO
3 in the
presence and in the
absence of Fe
3+ (50 µM) were compared. For these
experiments, 10% acetylene was
introduced in the anaerobic plasma
flasks to avoid any reduction
of N
2O to N
2.
Nitrous oxide concentration was determined by using
an MTI high-speed
microgas chromatograph equipped with a catharometer
detector (SRA
Instruments).
For aerobic treatments, Erlenmeyer flasks containing 50 ml of LB medium
were aseptically inoculated with each of the different
microbial
inocula to obtain a final absorbance of 0.01.
All the flasks were incubated at 25°C on an orbital shaker. Each
treatment was tested in triplicate. Bacterial growth was
periodically
monitored by measuring the absorbance at 600 nm with
a Shimadzu UV-160
spectrophotometer (Roucaire, Velizy-Villacoublay,
France).
Soil and plant experiments.
The experiments were performed
in the calcic silt-clay soil from Châteaurenard. This soil is
known for its natural suppressiveness to fusarium wilts, and its main
characteristics have been previously described (13). The
soil was air dried and sieved (for particles <2 mm in diameter).
Population dynamics of the wild-type strain C7R12 and of the PL1, NR2,
and PL1NR6 mutants were compared in bulk soil and in the rhizosphere of
tomato grown in the Châteaurenard soil, which was kept at two
matric potential values (1 and 10 kPa). These values
correspond to a proportion of the porosity filled with water equal to
51 and 35%, respectively (16). The experiments were
performed under gnotobiotic conditions.
Briefly, 31 g of soil was introduced into containers (30 ml),
corresponding to an apparent density of 1.06. These containers
were
sterilized by gamma radiation (40 kGy; Conservatome, Dagneux,
France).
Some containers were kept uncultivated, and others were
cultivated with
tomato (
Lycopersicon esculentum Mill. cv. H63-5).
Tomato
seeds were sterilized in a 1.25% (vol/vol) solution of
NaOCl for 20 min, washed three times with sterile distilled water,
and placed on a
sterile filter at 25°C for 48 h. Five tomato seedlings
were
transferred per container. The cultivated and uncultivated
containers
were placed in a flow cabinet used as a growth chamber
on a cycle of
16 h of light (25°C) and 8 h of darkness (22°C).
Each
mutant was introduced in combination with the wild-type strain
(1:1) to
obtain a bacterial density of 10
5 CFU/g of dry
soil.
The extraction of the bacteria for enumeration was performed as
follows. Approximately 0.5 g of dry soil was collected (i)
from each
uncultivated container (bulk soil) and (ii) from the
soil remaining at
the root surface of three root systems, after
a gentle brushing, in
each cultivated container (rhizosphere soil).
Each soil sample from the
two compartments (bulk and rhizosphere
soil) was suspended in 10 ml of
sterile distilled water for 60
s with a Vortex shaker. The soil
suspensions were diluted in sterile
distilled water and plated (three
plates per suspension) on KB
supplemented with the antibiotics
corresponding to the resistance
phenotypes of the different strains.
Bacterial enumeration was
performed 0, 10, and 20 days after
inoculation on five independent
replicates (containers) per
experimental treatment and date. The
bacterial densities were expressed
as CFU per gram of dry
soil.
Statistical analyses.
Since populations of bacteria
approximate a log normal distribution (21), values were
log transformed before analysis. Competitiveness of each mutant in the
presence of the wild-type strain was represented as the
mutant-to-total-inoculant (wild type plus mutant) CFU ratio. These
ratios were angular transformed before statistical analysis. Transformed values of microbial enumeration and of
mutant-to-total-inoculant ratio were submitted to repeated-measure
analysis using Statview software (1996 release; Abacus Concepts, Inc.,
Berkeley, Calif.). The significant threshold value was fixed at
P = 0.05.
Nucleotide sequence accession number.
The sequence of the
1.6-kb narG fragment has been deposited in the GenBank
database under accession number AF 197465.
| |
RESULTS AND DISCUSSION |
Construction of the Nar mutants of P. fluorescens C7R12.
The two Nar mutants, NR2
and PL1NR6, were constructed in the present study by disrupting the
narG gene in the wild-type strain (C7R12) and in the
Pvd mutant PL1, respectively. The deduced amino acid
sequence of the 1.6-kb amplified fragment shows 83% identity to the
corresponding NarG subunit from P. fluorescens YT101
(29). Southern blot analysis on chromosomal DNA digested
with XbaI from C7R12, NR2, and PLINR6 confirmed the allelic
exchange of the narG gene in the NR2 and PL1NR6 mutants as
described in Fig. 2.
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FIG. 2.
Deletion-insertion of the narG gene coding
for the catalytic subunit of the membrane-bound nitrate reductase of
the wild-type strain C7R12 (WT) and of the PL1 mutant. (A) The 1.6-kb
fragment of the narG gene (probe 1), the deleted fragment of
the narG gene (probe 2), and an internal fragment of
apra3 gene (probe 3) were used as probes; their sizes and
localizations are indicated. (B) Southern analysis of
XbaI-digested chromosomal DNA from the wild-type strain and
NR2 mutant with probes 1, 2, and 3, labeled with digoxigenin-11-dUTP
(Boehringer, Mannheim, Germany). As identical hybridization profiles
were obtained with both NR2 and PL1NR6, only those of NR2 are
presented. Probe 1 lanes show the presence of a higher band for the NR2
mutant than for C7R12 resulting from the 425-bp NarI
deletion and newly introduced 1.8-kb apra3 gene in the
narG gene. Probe 2 lanes show the presence of a
hybridization signal only for the wild-type strain C7R12, indicating
replacement of the narG gene with the deleted one in the NR2
mutant. Probe 3 lanes show the presence of a band similar in size to
the one obtained with probe 1 only in the NR2 mutant, indicating the
presence of apra3 in the narG deleted gene.
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Growth characteristics of the Nar mutants of P. fluorescens C7R12.
Growth characteristics of the
Nar mutants and wild-type strain under conditions of
aerobiosis and anaerobiosis with nitrate or nitrite as the electron
acceptor were compared.
Since similar growth kinetics were recorded with the wild-type strain
and the Nar
mutants under aerobic conditions, the mutants
appeared to be
not affected in their ability to use oxygen as the sole
electron
acceptor (Fig.
3A). As expected
the Nar
mutants were unable to use nitrate as the sole
electron acceptor
to sustain growth, while the wild-type strain reached
an absorbance
of 0.6 after 12 h (Fig.
3B). Altogether, these data
validate the
use of the Nar
mutants obtained for further
ecological studies, in the same
way that the use of the
Pvd
mutant PL1 was previously validated
(
25).
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FIG. 3.
Growth kinetics of C7R12 ( ), NR2 ( ), and PL1NR6
(×) under aerobic conditions in LB medium (A) and under anaerobic
conditions in LB medium supplemented either with KNO3 (20 mM) (B) or with KNO2 (10 mM) (C). Error bars, standard
deviations.
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With nitrite as the sole electron acceptor, the wild-type strain and
NR2 mutant had similar growth rates, reaching an absorbance
of 0.3 after 20 h (Fig.
3C). Unexpectedly, the mutant PL1NR6,
affected in
the synthesis of both pyoverdine and nitrate reductase,
showed a lower
growth rate than that of the wild-type strain and
of the
Nar
mutant NR2 when cultivated with nitrite as the sole
electron
acceptor (Fig.
3C). A reduced growth of the Pvd
mutant PL1 was also recorded under these experimental conditions
(data
not shown). Altogether, these data suggest a possible interaction
between the pyoverdine-mediated iron uptake and the nitrite reduction.
Expression of respiratory enzymes containing heme molecules, such
as
the denitrifying nitrate reductase, was previously showed to
be iron
dependent (
5). The heme
d1 was also
described in the
nitrite reductase encoded by the
nirS gene
(
41), and thus, the
expression of this denitrifying enzyme
would also be expected
to be iron dependent. The presence of the
nirS gene, recently
described in
P. fluorescens
C7R12 (EMBL accession number
AF197467)
(
30), is then in
favor of our hypothesis on the possible relation
between the
pyoverdine-mediated iron uptake and the nitrite reduction
in C7R12.
This hypothesis is further supported by the lower denitrification
rate
by
P. fluorescens C7R12 recorded under iron-limiting
conditions
as indicated by the level of N
2O production by
C7R12, which was
4.3 times lower in the absence than in the presence of
ferric
iron after 29 h of growth in succinate
medium.
Compared competitiveness of Pvd mutant and
Nar mutants.
Data for all environmental conditions
(matric potential and soil compartments) were combined for each mutant
(Fig. 4). All three mutants showed a
reduced competitiveness compared to the wild-type strain as
indicated by the decrease of the different mutant-to-total-inoculant
ratios. These data indicate that nitrate reductase and pyoverdine
are involved in the fitness of the wild-type strain P. fluorescens C7R12. This conclusion is in agreement with previous
studies showing the involvement of nitrate reductase and of pyoverdine
in the competitiveness of P. fluorescens YT101 and P. fluorescens C7R12, respectively (8, 25).
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FIG. 4.
Dynamics of the PL1 ( ), NR2 (), and PL1NR6 (×)
mutant-to-total-inoculant ratios, with all experiments conditions (soil
compartments and values of matric potential) being combined.
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Combination of all data from the different environmental conditions
further showed that overall the competitive advantages
given by nitrate
reductase and by pyoverdine are similar, as indicated
by the lack of
interaction between the mutant-to-total-inoculant
ratio and time
(
P = 0.11) (Fig.
4). As expected, the PL1NR6 curve
differed significantly (
P < 0.05) from those of PL1
and NR2, indicating
that the double mutation of genes encoding nitrate
reductase and
pyoverdine synthesis resulted in a lower competitiveness
of the
double mutant compared to the single mutants NR2 and
PL1.
Nitrate reductase and pyoverdine were shown to give a competitive
advantage over the wild-type strain, although the intensity
of this
advantage varied according to the environmental conditions
and to the
mutants as described
below.
Influence of matric potential on competitiveness of the
Pvd mutant and Nar mutants.
Figure 5A
to C show, at matric potentials of 1
and 10 kPa, the population dynamic of the combinations of the
wild-type strain C7R12 together with the mutant PL1, NR2, or PL1NR6,
respectively. For all microbial combinations, the total bacterial
density (wild-type strain plus mutant), initially at 105 to
106 CFU · g of dry soil1, increased to
roughly 108 to 109 CFU · g of dry
soil1 10 days after inoculation and remained constant at
day 20. The densities of microbial combinations, including mutants PL1
and PL1NR6, were nonsignificantly different (P = 0.1
and P = 0.9, respectively) at both values of matric
potential (Fig. 5A and C), while a significant difference (P < 0.05) was recorded for the combination including NR2 (Fig. 5B).
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FIG. 5.
Dynamics of the combinations of the wild-type strain
C7R12 with the mutant PL1 (A), NR2 (B), or PL1NR6 (C) and of the ratios
of the mutants PL1 (D), NR2 (E), and PL1NR6 (F) to total inoculant in
soil kept at 1 kPa (open symbols) and 10 kPa (closed symbols).
Error bars, standard deviations.
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For all microbial combinations, the mutant-to-total-inoculant ratio
decreased with time (Fig.
5D to F), indicating a reduced
competitiveness of the mutants compared to the wild-type strain.
However, the influence of the matric potential varied according
to the
mutants. For the combination including the Pvd
mutant
PL1, the decrease of the mutant-to-total-inoculant ratio
did not differ
significantly (
P = 0.07) at either matric potential
(Fig.
5D). In contrast, this ratio decreased significantly
(
P < 0.05) more at
1 than at
10 kPa for the
combinations including
the Nar
mutants NR2 and PL1NR6
(Fig.
5E and F). Altogether, these data
indicate that the
competitiveness of the Pvd
mutant PL1 did not differ with
regard to the matric potential
and that the one of the
Nar
mutants NR2 and PL1NR6 was significantly lower at a
potential
of
1 kPa than at a potential of
10 kPa. These
observations confirm
our initial hypothesis suggesting that the
selective advantage
given to the wild-type strain C7R12 by the nitrate
reductase would
be expressed under conditions of lower aeration.
Several studies
have stressed the impact of matric potential on
soilborne fluorescent
pseudomonads (
3,
10,
11,
27).
However, to our knowledge,
the present study is the first to
demonstrate the implication
of an enzyme in the bacterial adaptation to
variations of matric
potential.
Influence of plant roots on competitiveness of Pvd
mutant and Nar mutants.
Figure 6A to
C show, in bulk and rhizosphere soil, the
population dynamic of the combinations of the wild-type strain C7R12 together with the mutants PL1, NR2, and PL1NR6, respectively. For all
microbial combinations, the total bacterial density (wild-type strain
plus mutant), initially at 105 to 106 CFU
· g of dry soil1, increased to 108 to
109 CFU · g of dry soil1 10 days after
inoculation and remained constant at day 20. Total bacterial densities
were always significantly higher (P < 0.05) in the
rhizosphere than in bulk soil (Fig. 6A to C). These data show that a
rhizosphere effect was expressed towards the strains introduced even if
this expression was not as high as the one recorded under
nongnotobiotic conditions as previously described (25).
The observation of a rhizosphere effect under the present experimental
conditions warrants the comparison of the competitiveness of the
different mutants made in soil and rhizosphere when combined with the
wild-type strain.
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FIG. 6.
Dynamics of the combinations of the wild-type strain
C7R12 with the mutant PL1 (A), NR2 (B), or PL1NR6 (C) and of the ratios
of the mutants PL1 (D), NR2 (E), and PL1NR6 (F) to total inoculant in
bulk soil (open symbols) and rhizosphere soil (closed symbols). Error
bars, standard deviations.
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For all microbial combinations, the mutant-to-total-inoculant ratios
decreased with time (Fig.
6D to F), indicating a reduced
competitiveness of the mutants compared to the wild-type strain.
In
contrast, the competitiveness of the mutants did not differ
significantly in the bulk and rhizosphere soil, as shown by the
lack of
significant differences in the decrease of the
mutant-to-total-inoculant
ratio (
P = 0.23,
P = 0.82, and
P = 0.48, for PL1, NR2, and PL1NR6,
respectively) in these two environments (Fig.
6D to F). Altogether,
these data indicate that nitrate reductase and pyoverdine were
implicated in both soil and rhizosphere competence, and then more
generally in the saprophytic competence of the wild-type strain.
This
conclusion supports the data of Mirleau et al. (
25)
showing
that the higher competitiveness of the wild-type C7R12 over the
Pvd
mutant PL1 was expressed both in bulk and rhizosphere
soil.
Conclusion.
In this study, nitrate reductase and pyoverdine
were shown to be involved in the intraspecific competitiveness of the
biocontrol agent P. fluorescens C7R12 under soil conditions.
The competitive advantage given to the wild-type strain by nitrate
reductase and pyoverdine over the defective mutants was expressed not
only in the rhizosphere but also in bulk soil, indicating that these
two bacterial traits are implicated in the bacterial saprophytic
competence in soil environments. The selective advantage given to C7R12
by the nitrate reductase was more strongly expressed under conditions of lower aeration, in such a way that the ability of P. fluorescens C7R12 to switch from one metabolic pathway
(respiratory chain) to the other (nitrogen respiration) would account
for its ability to remain competent under various soil environment
conditions. Further research should now be performed to determine if
the bacterial traits shown in the present study to be involved in the
intraspecific competitiveness of the biocontrol agent P. fluorescens C7R12 also play a role in this agent's
interspecific competitive ability in the presence of indigenous microflora.
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ACKNOWLEDGMENTS |
We are grateful to C. Steinberg for performing the statistical
analyses and for stimulating discussions, to P. Potier and R. Lensi for
their advice, to J. Raaijmakers for critical reading of the manuscript,
and to J. Thomas and J. Rupe for correcting the English text.
This work was partly supported by the Conseil Régional de Bourgogne.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: UMR
BBCE-IPM, CMSE-INRA, 17 rue Sully, BP 86510, 21065 Dijon
Cedex, France. Phone: 33 3 80 69 30 56. Fax: 33 3 80 69 32 26. E-mail:
Philippe.Lemanceau{at}dijon.inra.fr.
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Applied and Environmental Microbiology, June 2001, p. 2627-2635, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2627-2635.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
