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Applied and Environmental Microbiology, September 2000, p. 4012-4016, Vol. 66, No. 9
Laboratoire d'Ecologie Microbienne, UMR-CNRS
5557, Université Claude-Bernard Lyon I, 69622 Villeurbanne
Cedex, France
Received 28 February 2000/Accepted 7 July 2000
Selection of the denitrifying community by plant roots (i.e.,
increase in the denitrifier/total heterotroph ratio in the rhizosphere) has been reported by several authors. However, very few studies to
evaluate the role of the denitrifying function itself in the selection
of microorganisms in the rhizosphere have been performed. In the
present study, we compared the rhizosphere survival of the denitrifying
Pseudomonas fluorescens YT101 strain with that of its
isogenic mutant deficient in the ability to synthesize the respiratory
nitrate reductase, coinoculated in nonplanted or planted soil. We
demonstrated that under nonlimiting nitrate conditions, the
denitrifying wild-type strain had an advantage in the ability to
colonize the rhizosphere of maize. Investigations of the effect of the
inoculum characteristics (density of the total inoculum and relative
proportions of mutant and wild-type strains) on the outcome of the
selection demonstrated that the selective effect of the plant was
expressed only during the phase of bacterial multiplication and that
the intensity of selection was dependent on the magnitude of this
phase. Moreover, application of the de Wit replacement series technique
to our results suggests that the advantage of the wild-type strain was
maximal when the ratio between the two strains in the inoculum was
close to 1:1. This work constitutes the first direct demonstration that
the presence of a functional structural gene encoding the
respiratory nitrate reductase confers higher rhizosphere competence to
a microorganism.
Denitrification is considered to be
an important soil process, since it influences (i) the functioning of
ecosystems by controlling the global soil N budget and the balance
between the mineral nitrogen forms (and, consequently, the nitrogen
nutrition of plants) and (ii) the quality of the atmosphere by
producing nitrogen oxides and, especially, N2O, which is
involved in the terrestrial greenhouse effect (10, 16) and
affects the chemistry of O3 in the upper troposphere and
lower stratosphere (9). Most of the numerous studies on this
process have dealt with how biotic and/or abiotic parameters regulate
the denitrifying activity. However, the denitrifying activity is an
integrative measurement depending, among other variables, on the
density and the distribution of denitrifiers. The distribution of
denitrifiers in physicochemically heterogeneous environments basically
results from (i) multiplication of denitrifiers The purposes of the present work were (i) to evaluate the role of
nitrate reduction in the ability of a Pseudomonas
fluorescens strain to colonize roots of maize grown in a
nonsterilized soil and (ii) to evaluate the effects of the inoculum
characteristics (i.e., density of the total inoculum and relative
proportions of mutant and wild-type [WT] strains) on the outcome of
the selection exerted by the plant.
Soil.
The soil used was a permanent pasture silt loam from
the region of Lyon, France, air dried and sieved to a particle size of less than 2 mm. The properties of the soil were as follows: clay, 31.4%; loam, 36.4%; sand, 32.2%; pH 7.5; organic C, 2.69%; total N,
0.35%; water holding capacity, 0.4 g g Organisms and growth conditions.
Pseudomonas
fluorescens YT101 (WT) is a natural, rifampin-resistant clone of
strain AK-15 isolated from a loam soil from the Kellog Biological
Station, Kalmazoo County, Mich. (24). Strain LP59JG
(Nar
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of Respiratory Nitrate Reductase in Ability of
Pseudomonas fluorescens YT101 To Colonize the
Rhizosphere of Maize
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
most being
heterotrophswhich essentially depends on the availability of
assimilable organic substrates and (ii) selection of denitrifiers within the heterotrophic microflora (i.e., increase in the
denitrifier/total heterotroph ratio). With regard to the first aspect,
several authors reported an increase in the density of denitrifiers in
the rhizosphere and attributed this observation to the carbon compounds
exudated by roots (14, 17, 19, 20). Few authors have
demonstrated that the denitrifier/total heterotrophic microflora ratio
may be modified by plants (3, 4, 11). However, these works did not demonstrate that the ability to dissimilate nitrate or nitrite
is actually responsible for the selection of the dissimilating community in the rhizosphere. Indeed, other characteristics associated with denitrification could be responsible for this selection. The best
way to evaluate the role of denitrification in the adaptative and/or
competitive advantage for root colonization is the use of mutants
affected in the ability to perform the various steps of the
denitrifying pathway. Using this approach, Philippot et al.
(15) demonstrated that inactivation (by Tn5
insertion) of the structural gene encoding the cd1-type nitrite
reductase decreased the ability of a Pseudomonas fluorescens
strain to colonize the rhizosphere. Recent acquisition of a respiratory
nitrate reductase mutant by allelic exchange of the narG
gene in the same strain (8) allowed for an extension of
these studies to the first step of the denitrifying pathway (the most
energetic one).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1.
mutant) was obtained from strain YT101 by allelic
exchange of a gentamicin resistance gene in the narG gene
encoding the catalytic subunit of the respiratory nitrate reductase
(8). The isogenic character of the mutant LP59JG was
confirmed by molecular, biochemical, and immunological assays.
Moreover, the similar growth rates of the WT strain and the
Nar mutant under aerobic conditions (with oxygen as the
sole electron acceptor) and the total absence of growth of the mutant
under anaerobic conditions with nitrate as the sole electron acceptor (8) validate the use of the biological models for the
present study.
1) for the
WT strain and rifampin (50 µg ml1) plus gentamicin (50 µg ml1) for the Nar mutant.
Plant experiments.
The effect of plant roots was assayed by
using microcolumn systems previously described by Steinberg et al.
(18) and Philippot et al. (15). Briefly, 10 g (nonplanted treatment) or 8 g (planted treatment) of nonsterile
soil was introduced into 10-ml syringes (microcolumn) fitted with a
cotton wick. Each microcolumn was introduced into a test tube
containing a KNO3 solution (10 mM) in distilled water. The
cotton wick allowed provision of sufficient water and nitrate to
maintain constant soil moisture and nonlimiting nitrate conditions in
the two treatments (nonplanted and planted soil) during the entire
experiment. This experimental design does not allow adjustment of soil
moisture to different values but does allow its maintenance at around
90% of the water-holding capacity for both treatments. For planted
soil, maize seeds were allowed to germinate for 24 h on moistened
filter papers at 28°C in the dark before being placed on the soil.
Then, an additional 2 g of soil was added to the microcolumns in
order to cover the seeds. For preparation of inocula, strains were
cultured separately in 40 ml of LB medium. After 24 h of growth,
bacterial cells were collected by centrifugation at 5,500 × g for 15 min. The two strains were mixed in different
relative proportions (50, 20, or 80% of Nar) in
appropriate quantities of KNO3 solution (10 mM) to achieve total (WT + Nar) densities of 103,
104, or 107 cells g of dry soil1
in the microcolumns. Then, 4 ml of each of the WT + Nar mutant mixtures was inoculated into the microcolumns.
The proportion of 50% Nar mutants was associated with
the three cell densities, while the proportions of 20 and 80%
Nar mutants were associated only with 104
cells g of dry soil1.
2 s1) and 9 h of dark; temperature
and humidity, 25°C and 70 to 80% during the day and 19°C and 90%
during the night, respectively.
At set times, three planted and three nonplanted microcolumns were used
for bacterial enumeration, water content determination, denitrifying
activity measurements, and NO3 and
NO2 concentration measurements.
NO3 measurements were performed in order to
maintain concentrations between 20 and 100 µg of
NO3-N g of dry soil1 by
adjusting the NO3 concentration in the test
tubes containing the remaining microcolumns. During all experiments,
NO2 accumulation was never observed in soil.
Denitrifying activity measurements. At 3, 7, and 10 days, three noninoculated nonplanted and three noninoculated planted microcolumns were used to compare the denitrifying activities in the presence and absence of the plant. The cylinders of soil were extracted from each microcolumn and transferred (after cutting the aerial part of the plant for the planted systems) into 150-ml plasma flasks. The flasks were then sealed with rubber stoppers. In each flask, 15 ml of atmosphere was replaced by 15 ml of C2H2 in order to ensure N2O reductase inhibition. After 3 days of incubation at 25°C, gas samples were analyzed for N2O with a gas chromatograph equipped with an electron capture detector.
Enumeration.
To investigate the dynamics of the total
(WT + Nar) population and the evolution of the
proportion of Nar mutants in the total population,
enumeration was made at 0, 3, 7, 10, and 14 days. Bacteria were
extracted by blending whole samples (soil or soil plus roots) for 1.5 min in 100 ml of sterile NaCl solution (8 g liter1) with
a Waring blender. Appropriate dilutions of the soil suspensions were
spread on LB agar supplemented with rifampin (50 µg
ml1) for enumeration of the total (WT + Nar) inoculant. The colonies were then transferred by
velvet replication on LB agar supplemented with rifampin (50 µg
ml1) plus gentamicin (50 µg ml1) for
enumeration of Nar mutants. Cycloheximide (200 µg
ml1) was added to LB agar to prevent fungal growth.
Between 30 and 300 CFU of bacteria per plate were counted after 24 h of incubation at 28°C. For each enumeration, three replicates of
the appropriate dilution were analyzed. The use of the velvet replica
technique allows accurate statistical analysis of the observed
proportions. Background counts of Lyon soil on LB agar with 50 µg of
rifampin ml1 indicated a naturally rifampin-resistant
population lower than 102 CFU g1. It was
previously verified that no loss of the rifampin or gentamicin resistance marker occurred by comparison of counts on selective LB agar
plates (rifampin or rifampin plus gentamicin) and nonselective LB agar
plates after 2 weeks of incubation of sterile soil inoculated with both
strains (15).
Statistical treatment of data. For population values, the homogeneity of nine estimations (3 microcosms × 3 replicates) was tested by one-way analysis of variance at each time for each treatment. Since no significant difference between these estimates was observed, means and their confidence intervals were calculated from the nine values (3 microcosms × 3 replicates). Means in planted and nonplanted soil were compared at each time by using the Student t test at the 5% significance level.
For proportion values, the homogeneity of the nine estimations (3 microcosms × 3 replicates) was tested by using the
2 test at each time for each treatment. Since no
significant difference between these estimates was observed,
proportions and their confidence intervals were calculated with pooled
samples. Proportions in planted and nonplanted soil were compared at
each time by using the 2 test for the pooled samples.
The results were summarized and discussed by the de Wit replacement
series technique (6): the plot of the proportion of mutants
at the end of the experiment measured against the proportion of mutants
at the beginning of the experiment (i.e., in the inoculum).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Comparison of the denitrifying activities of planted and nonplanted
soil.
The values of denitrifying activity were 0.27 (standard
deviation [SD], 0.36) and 2.73 (SD, 0.96) µg of N2O-N g
of dry soil1 at 3 days, 0.42 (SD, 0.29) and 2.85 (SD,
0.29) µg of N2O-N g of dry soil1 at 7 days,
and 0.13 (SD, 0.14) and 5.97 (SD, 1.01) µg of N2O-N g of
dry soil1 at 10 days for nonplanted and planted soil,
respectively. This shows that, during our experiments, the potential
for denitrification (measured under aerobiosis and nonlimiting nitrate
conditions) was at least 7 times higher in planted soil than in
nonplanted soil.
Evolution of the total (WT + Nar) inoculated
P. fluorescens population in planted and nonplanted
soil.
Figure 1A, B, D, and E show
that when the inoculum densities were 103 and
104 cells of g dry soil1, the dynamics of the
total (WT + Nar) population consisted of an increase
until day 7 followed by a stabilization from day 7 to day 14. From the
beginning of the experiment to day 3, no significant difference was
observed between the planted and nonplanted treatments. Then, the
dynamics systematically and significantly differed between the two
treatments, and the levels of stabilization were about 106
and 107 cells g of dry soil1 in nonplanted
and planted soil, respectively. When the initial inoculum was
107 cells g of dry soil1 (Fig. 1C), the total
population remained at the initial value until the end of the
experiment, and no significant difference between planted and
nonplanted soil was observed during the whole period dynamics (14 days).
|
Evolution of the proportion of Nar mutants in the
total (WT + Nar) population in planted and
nonplanted soil: influence of the inoculum characteristics.
Figure
1F, G, H, I, and J show that in all nonplanted microcolumns, the
proportion of Nar mutants in the total (WT + Nar) population remained constant and equal to the
proportion of the inoculum during the entire experiment: about 50% in
Fig. 1F, G, and H; about 20% in I; and about 80% in J.
1 (Fig. 1F, G, I, and J), the
proportion of Nar mutants in the total (WT + Nar) population was significantly lower in planted soil
than in nonplanted soil after day 3 (except in Fig. 1J, where the
values were not significantly different at day 3). Then, at day 7, the
proportion of Nar mutants in the planted soil remained
constant until the end of experiment at the following densities: about
20% when the density of the total inoculum was 103 cells g
of dry soil1 (with 50% of Nar mutants)
(Fig. 1F); about 25% when the density of the total inoculum was
104 cells g of dry soil1 (with 50%
Nar mutants) (Fig. 1G); about 5% when the density of the
total inoculum was 104 cells g of dry soil1
(with 20% Nar mutants) (Fig. 1I); and about 60%
when the density of the total inoculum was 104 cells g of
dry soil1 (with 80% Nar mutants) (Fig.
1J).
A radically different trend was observed when the density of the total
(WT + Nar) inoculum was 107 cells g of
dry soil1 (with 50% Nar mutants) (Fig.
1H). In this case, no significant difference in the evolution of the
proportion of Nar mutants was observed between the
planted and nonplanted treatments (the proportion of Nar
mutants remained roughly constant and equal to the proportion of the
inoculum during the entire experiment for planted and nonplanted soil).
de Wit plot.
The experimental design could be viewed as a de
Wit replacement series (6). Figure
2 shows a graphical summary obtained by
plotting the proportion of Nar mutants at the end of the
experiment against the proportion of Nar mutants in the
inoculum. In nonplanted soil, the observed relation was close to the
null hypothesis of the de Wit representation, i.e., a similar
proportion of Nar mutants at the end of the experiment
and in the inoculum (intracompetition = intercompetition). A
slight divergence from the null hypothesis was observed only when the
initial ratio between the two strains was 1:1. In planted soil, the
observed relation shows that intercompetition was higher than
intracompetition: the WT strain had the advantage for all the tested
proportions of Nar mutants in the total inoculum, but its
advantage was higher (maximal divergence from the null hypothesis) when
the initial ratio between the two strains was 1:1.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The major result of this study was to demonstrate that the
presence of a functional structural gene encoding the dissimilative nitrate reductase confers to the P. fluorescens YT101 strain
an advantage in its ability to colonize the rhizosphere of maize. This
result was obtained by comparing the evolution of the proportion of an
isogenic Nar mutant in a total (Nar mutant
plus the corresponding WT) population in the presence and absence of
the selective factor (the plant in this study). Such an approach is
generally considered to be the most accurate to demonstrate the role of
a given microbial function (i.e., the selective value of the
corresponding genes) on the environmental competence of a
microorganism. Using the same P. fluorescens strain, Philippot et al. (15) reported that the presence of plant
roots is able to lower the survival ability of a Tn5 mutant
affected in the nirS gene (encoding the cd1-type nitrite
reductase) compared to that of the WT denitrifying strain. They
concluded that the presence of a functional nirS gene
conferred a selective advantage in the rhizosphere of maize. The
results of the present study improve our understanding of the
importance of denitrifying ability for competition of microorganisms in
the rhizosphere by (i) demonstrating the role of the narG
gene (encoding the catalytic subunit of the respiratory nitrate
reductase) in the rhizosphere competence of the studied strain and (ii)
investigating to what extent the outcome of the selection exerted by
the plant depends on the population dynamics of the studied strain.
Measurements of denitrifying activity demonstrated that conditions in
the planted systems were significantly more conducive for
denitrification than conditions in the nonplanted ones. This was an
absolute prerequisite to ensure that the experimental systems used were
satisfactorily adapted to reach our objectives.
Influence of plants and of the density of the total (WT + Nar) inoculum on the dynamics of the total P. fluorescens population.
When the densities of total (WT + Nar) P. fluorescens inocula were
103 and 104 cells g of dry soil1,
an increase followed by a stabilization of the population was observed
(Fig. 1A, B, D, and E). Under these conditions, the level of
stabilization in the nonplanted soil was 106 cells g of dry
soil1. For the same strain and the same soil but under
gnotobiotic conditions (i.e., the conditions defining the carrying
capacity stricto sensu of the soil for the strain used), the level of
stabilization observed by Philippot et al. (15) was about
109 cells g of dry soil1 (i.e., 3 orders of
magnitude higher). This means that the biotic components of the soil
(through competition and/or predation processes) drastically decreased
its ability to receive the introduced P. fluorescens strain
but nevertheless allowed the strain to multiply and maintain itself in
the soil during at least the 2 weeks of the experiments. This suggests
that irrespective of its denitrifying abilities, P. fluorescens YT101 shows high adaptative and competitive potentialities in the studied soil and thus can be considered as a
denitrifier that exhibits good competitive abilities as an aerobic
heterotroph (12, 13). The presence of a plant improved these
potentialities, as shown by the level of stabilization, which was about
1 order of magnitude higher in planted than in nonplanted microcolumns.
Thus, approximately 106 and 107 cells g of dry
soil1 might be considered to be the carrying capacities
of the studied strain in the nonplanted and planted soil, respectively
(lato sensu, i.e., under conditions in which both physicochemical and biotic components can interfere with the survival of the introduced strain). One may assume that the introduced strain must systematically stabilize at these densities, whatever the density of inoculum. Indeed,
several studies demonstrated that the carrying capacity of a given soil
for a given strain can be considered as a constant and that the
dynamics of the strain decrease or increase to reach the carrying
capacity (2, 5, 21). As expected, the population in the
planted soil remained constant until the end of the experiment, when
the density of the WT + Nar P. fluorescens inoculum was 107 cells g of dry
soil1, corresponding to the carrying capacity of the
planted soil (Fig. 1C). However, the population of the nonplanted soil
did not decrease to the carrying capacity (106 cells g of
dry soil1) but remained constant and equal to the
inoculum density. A possible explanation is that the limited duration
of the experiment (14 days) would not allow us to observe the decrease
in P. fluorescens density to the carrying capacity, which
may be a slow process, especially in silt loam soils (22).
Influence of the density of the total (WT + Nar)
inoculum on the outcome of the selection exerted by the plant.
Another major result of our work was to demonstrate that the selective
effect of the plant was expressed only during the phase of cell
multiplication (when such phase occurred) of the introduced P. fluorescens population and that the intensity of the selection is
dependent on the magnitude of the phase of multiplication. In order to
base our interpretations on treatments differing in one single
parameter, only the experiments involving the same proportion (50%) of
mutants in the inoculum are discussed (Fig. 1F, G, and H).
P. fluorescens inocula were 103 and 104 cells
g of dry soil1, the proportion of Nar
mutants in the total population decreased in the planted soils until
day 7 (Fig. 1F and G), i.e., during the phase of cell multiplication. From day 7 to the end of the experiment, while the WT + Nar population was stabilized at the value of the
carrying capacity (Fig. 1A and B), the proportion of Nar
mutants remained constant. Moreover, the differences observed in the
evolution of the proportions of Nar mutants between
planted and nonplanted soil (Fig. 1F and G) were higher when the total
population increase was about 4 orders of magnitude (103 to
107 cells g of dry soil1 [Fig. 1A]) than
when it was only about 3 orders of magnitude (104 to
107 cells g of dry soil1 [Fig. 1B]). The
absolute requirement of a cell multiplication phase to induce a plant
selection of the WT strain was clearly confirmed by the fact that, when
no cell multiplication phase occurred (inoculum density of
107 cells g of dry soil1 [Fig. 1C]), no
selection was observed (Fig. 1H).
Influence of the proportion of mutants in the WT + Nar inoculum on the outcome of the selection exerted by
the plant.
The advantage of the WT P. fluorescens
strain in the rhizosphere was always observed, whatever the proportions
of mutants in the inoculum, unless a cell multiplication phase was not
occurring. In order to base our interpretations on treatments differing
in one single parameter, only the experiments involving the same density of total (WT + Nar) inoculum
(104 cells g of dry soil1) are discussed
(Fig. 1G, I, and J). The general trend observed in the evolution of the
proportion of Nar mutants in the planted microcolumns was
similar for the different tested proportions of mutants in the inoculum
(50, 20, and 80%): a decrease until day 7 (corresponding to the cell
multiplication phase) followed by a stabilization until the end of the
experiment. In order to assess the effect of the proportion of mutants
in the inoculum on the intensity of the selection, we used the de Wit
replacement series technique as a representation of the competition between the two strains. This technique (initially devoted to plant
ecology [7] and further applied to the analysis of
fungal and bacterial competition [1, 23]) confirms
that (i) the soil conditions existing in the nonplanted treatments did
not induce a marked discrimination between the two strains, and (ii) in
the planted treatments, the WT strain had an advantage for all the
tested proportions of Nar mutants in the inoculum (Fig.
2). These observations are in agreement with denitrifying activity
measurements, which showed that, under the experimental conditions used
in this study (especially soil moisture), the planted systems were
significantly more conducive for denitrification than the nonplanted
ones. Since the maximal distance from the curve representing the null
hypothesis was obtained for an initial ratio of 1:1, application of
this technique also suggests that the intensity of the selection
exerted by roots was minimized when the initial ratio diverged from
1:1. The fact that a slight advantage of the WT strain was also
observed in the case of an initial ratio of 1:1 in the nonplanted soil
(where conditions allowed a small but detectable amount of denitrifying activity) supports the predominance of the 1:1 condition in
discriminating between the strains.
) in the
inoculum may result from this complex network of interactions.
Several studies of the indigenous soil microflora have demonstrated
that denitrifying bacteria predominantly occur near or inside the roots
(3, 4, 11) and proposed that the function of denitrification
itself may constitute a significant advantage for root colonization.
This assumption has now clearly been proved for one P. fluorescens strain (either for the role of nitrite reductase
[15] or for the role of nitrate reductase [this
study]), but generalization to other denitrifiers or a fortiori to the whole denitrifying community in a given soil remains to be investigated.
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ACKNOWLEDGMENTS |
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We are very grateful to Agnès Richaume for helpful comments on the manuscript. We also thank Nadia Salin and Amandine Tabouret for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire d'Ecologie Microbienne, UMR-CNRS 5557, Université Claude-Bernard Lyon I, 43, Bvd. du 11 Novembre 1918, Bât. 741, 69622 Villeurbanne Cedex, France. Phone: (33) 4 72 43 13 79. Fax: (33) 4 72 43 12 23. E-mail: lensi{at}cismsun.univ-lyon1.fr.
Present address: Laboratoire de Microbiologie des Sols, INRA, 21034 Dijon Cédex, France.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 2000, p. 4012-4016, Vol. 66, No. 9
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