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Applied and Environmental Microbiology, October 2000, p. 4193-4199, Vol. 66, No. 10
Cardiff School of Biosciences, Cardiff
University, Cardiff CF10 3TL,1 and
N. E. R. C. Institute of Virology and
Environmental Microbiology, Oxford OX1 3SR,2
United Kingdom
Received 23 February 2000/Accepted 19 July 2000
We describe two prolonged bacteriophage blooms within sugar beet
rhizospheres ensuing from an artificial increase in numbers of an
indigenous soil bacterium. Further, we provide evidence of in situ
competition between these phages. This is the first in situ
demonstration of such microbial interactions in soil. To achieve this,
sugar beet seeds were inoculated with Serratia liquefaciens
CP6RS or its lysogen, CP6RS-ly- Bacteria are ubiquitous in the
environment, with a global estimate of 4 × 1030 to
6 × 1030 cells (26). With this ubiquity
comes an importance to the biosphere that is well recognized; thus, any
process that substantially affects natural bacterial communities will
also be significant. One such process may be predation by
bacteriophages (phages). It is thought that predatory phages could
control the numbers of bacteria and facilitate gene transfer between
bacteria by transduction (5, 6, 14). Certainly phages are as
common as bacteria. In addition, estimates of phage abundance in
aquatic habitats suggest their numbers are 10 times greater than those
of bacteria (5). Extrapolating this estimate to the
biosphere at large would make phages the most abundant organisms on earth.
Clearly then, phages have a potentially significant global impact. But
is this potential realized? This is a difficult question to answer, as
the natural population ecology of phages has been little studied. Most
knowledge derives from investigations using chemostats, mainly because
phage-bacterium interactions serve as a useful paradigm of
predator-prey interactions generally, and chemostats afford the
opportunity to test the validity of mathematical models (see reference
16 for a review). However, chemostat conditions are
far removed from the complexity of nature. A few studies have attempted
to follow long-term phage population changes in situ, but these
concentrated on aquatic habitats, considering only gross overall
changes in bacterial and phage populations (6, 24). In
addition, some microcosm studies have considered specific
bacterium-phage systems over short time scales (7, 9, 10,
20-22).
Our present study is the first to describe interactions between
competing phages within a natural habitat over a prolonged (i.e.,
6-month) time scale. The data we present provide compelling evidence of
competition between two indigenous predatory phages for the same prey
bacterium within a natural environment that is consistent with
established interspecific-competition theory (19).
Bacteria used in this study.
The bacteria used in this study
(Table 1) were derived from
Serratia liquefaciens CP6, previously isolated from a sugar
beet grown at our field site (3). For the present study, a
spontaneous spectinomycin- and nalidixic acid-resistant mutant of
S. liquefaciens CP6 was isolated and called CP6SpN. In
addition, a lysogen of S. liquefaciens CP6RS was isolated
(4) from a CP6RS culture inoculated with the temperate
Siphoviridae phage
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Seasonal Population Dynamics and Interactions of
Competing Bacteriophages and Their Host in the Rhizosphere
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1. These were sown, along with
uninoculated seeds, in 36 field plots arranged in a randomized Latin
square. The plots were then sampled regularly over 194 days, and the
plants were assayed for the released bacteria and any infectious
phages. Both the lysogen and nonlysogen forms of CP6RS survived equally
well in situ, contradicting earlier work suggesting lysogens have a
competitive disadvantage in nature. A Podoviridae phage,
identified as CP6-4, flourished on the nonlysogen-inoculated plants
in contrast to those plants inoculated with the lysogen. Conversely,
the Siphoviridae phage CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some CP6-1 phage up to day 137, yet hardly any CP6-4 phages were found, and this was consistent with
previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be
explained by application of optimal foraging theory to phage ecology.
This is the first time that such in situ evidence has been provided in
support of this theoretical model.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
CP6-1, and this lysogen was named
CP6RS-ly-1. These isogenic forms of the wild-type CP6 grew equally
well in soil in the laboratory. All bacteria were maintained on
nutrient agar (CM3; Oxoid) at 4°C, with stocks kept at 
80°C in
50% glycerol.
TABLE 1.
S. liquefaciens strains used in this study
CP6-1 and CP6-4 are double-stranded-DNA-tailed phages
belonging to the families Siphoviridae and
Podoviridae, respectively (4). They both infect
S. liquefaciens CP6 and were previously isolated from our
field site (3).
The field site.
Fieldwork took place during 1997 and 1998 at
a site on Oxford University Farm, Wytham, Oxford, United Kingdom. The
site was originally pastureland, and sugar beets had been grown there
since 1990. The soil is an Evesham series heavy clay soil with 53%
clay, 25% sand, 22% silt (pH 7.7), and 8.5% organic content. As in
previous years, the soil was fertilized with an NPK (13:13:20)
fertilizer (dosage, 0.15 kg m
2) and treated with
herbicide (Roundup; 2 kg ha1) and insecticide (Gamma-col;
ICI; 1.4 liter ha1) prior to sowing (3).
First field experiment, 1997. On 16 May 1997 (day zero), five soil samples were randomly collected from a 2.25- by 5.1-m plot within the field site. Next, around 500 sugar beet seeds (EB3 pellets; Germains UK Ltd., Kings Lynn, United Kingdom) were soaked in sterile quarter-strength Ringer's solution (QSR) (BR52; Oxoid) for 5 min and sown at 15-cm intervals within the plot.
Homogenates were prepared from the soil samples by mixing 1 g of soil with 20 ml of QSR and thoroughly homogenizing the resulting suspension by adding sterile 5-mm-diameter glass beads, vortex mixing the suspension for 1 min, and then shaking it on an orbital shaker for 10 min. The homogenates were screened for phages antagonistic towards CP6 by the overlay agar technique (2). The base medium was nutrient agar, while the overlay agar was made from nutrient broth (CM1; Oxoid) (13 g liter1) and bacteriological agar (L11;
Oxoid) (6.5 g liter1). The homogenates (1 ml) were
centrifuged for 5 min at 14,000 × g, and 100 µl of
the resulting supernatant was used to inoculate the overlay agar (2.5 ml) with an equal volume of CP6 culture.
The plates were incubated overnight at a plaque size-optimizing
temperature of 15°C, and any resulting plaques were counted. Phages
from these plaques were identified using the methodologies detailed in
our previous studies (3, 4). That is, the plaques were
initially classified according to appearance (the two most dominant
phages at the field site have very distinct plaque morphologies [4]). The classifications were then tested by
producing phage lysates from the plaques and assaying them for the
ability to lyse the CP6 lysogens listed in Table 1 (i.e., superimmunity testing [4]). As a final confirmatory step, DNA was
extracted from representative lysates (3) and cut with
EcoRI (Promega) as described by the manufacturer. Digests
were run on 0.7% agarose gels at 0.32 V cm2, along with
HindIII-cut lambda DNA (D-9780; Sigma), and the
resulting restriction profiles were compared with the expected banding
patterns (4). When no phages were detected, the homogenates
were enriched with nutrient broth, spiked with an overnight culture of
CP6, and reassayed after overnight incubation at 20°C to optimize
phage proliferation.
Soil bacteria were counted by plating the homogenates on tryptone
soy broth agar (TSBA) (17), which, when enumerated, gave an
estimate of total viable heterotrophic bacterial numbers, and on
Pseudomonas selection isolation agar (PSIA) (15)
to determine total pseudomonad counts. TSBA plates were incubated at
15°C for 7 days before enumeration, while PSIA plates were incubated
at 30°C for 48 h.
On days 25, 52, 82, 123, 160, 209, and 286 after being sown, 10 sugar
beet plants were collected at random from the field site and weighed.
After as much loose soil as possible was dislodged, the rhizosphere
from each plant was sampled by scraping the surface of each root with a
sterile scalpel and collecting the resulting thin layer of soil in a
sterile 50-ml centrifuge tube. Homogenates were then prepared and
assayed for phages and bacteria, as described for the soil samples.
Additionally, the homogenates were enumerated on a Serratia
selective medium (SSM) that we had designed for estimating total
CP6-like bacterial counts (i.e., D3 Erwinia medium [13] modified by eliminating arabinose and increasing
the sucrose to 40 g liter1). Prior tests with
CP6-spiked soil samples had shown that, when incubated for 48 h at
30°C, this medium selected against most non-Serratia
bacteria while CP6 appeared as small (~1-mm-diameter) orange colonies
with yellow borders that were clearly discernible against the dark-blue
background of the surrounding agar. The identities of randomly selected
CP6-like isolates were confirmed by checking their sensitivities to our
CP6 phage collection (3) and their API 20E strip
(bioMérieux sa) profiles.
Second field experiment, 1998.
On 22 May 1998 (day zero), in
a separate region of the field site, 3.9 by 4.8 m was partitioned
off and divided into 36 plots in a six-by-six matrix, with a 30-cm-wide
border separating each plot from its neighbor. Soil was collected from
each plot. The plots were then randomly assigned to one of five sugar
beet seed treatments (Fig. 1). The seeds
were treated as follows: for treatment 1, the seeds were uninoculated
as in 1997; for treatment 2, the seeds were inoculated with CP6SpN; for
treatment 3, the seeds were inoculated with CP6SpN and CP6RS; for
treatment 4, the seeds were inoculated with CP6RS-ly-1; for
treatment 5, the seeds were inoculated with CP6SpN and CP6RS-ly-1.
The purpose of these inoculations was to release differently marked
lysogen (CP6RS-ly-1) and nonlysogen (CP6SpN) forms of S. liquefaciens to investigate cross infection of the temperate phage
CP6-1 in situ and to see whether transduction might occur. CP6RS was
released as a nonlysogen control of CP6RS-ly-1. The inoculations
were achieved by soaking the seeds for 5 min in the appropriate
bacterial suspensions prepared with QSR (3). Estimates of
inoculum density were obtained by drop plate counting (12)
on nutrient agar with the appropriate selective agents. Inocula were
also checked for phages by similarly drop plating serial dilutions of
samples filtered through 0.2-µm-pore-size membranes onto
CP6-inoculated overlay plates.
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1 (to select for bacteria with the CP6RS
phenotype), and SSM supplemented with spectinomycin at 100 µg
ml1 (to select for the CP6SpN phenotype). Plates were
incubated as described above.
On days 19, 47, 68, 96, 137, and 194 after being sown, one plant from
each of the 36 plots was randomly selected and weighed. The rhizosphere
of each plant collected was then sampled and assayed for phages and
bacteria as for the soil samples. Putative S. liquefaciens CP6RS or S. liquefaciens CP6SpN colonies were confirmed by
growing them on nutrient agar supplemented with rifampin (100 µg
ml1) or nalidixic acid (200 µg ml1),
respectively, which selected for their second phenotypic markers. Additionally, selected isolates were assayed for sensitivity towards phages CP6-1 to CP6-6 (4). This confirmed whether CP6
bacterial strains (i.e., CP6RS and CP6SpN) had been reisolated and
identified any CP6-1 lysogens (i.e., CP6RS-ly-1) by their
sensitivities to all phages except CP6-1. Lysogeny was confirmed by
stabbing colonies onto CP6-inoculated overlay lawns to detect zones of lysis after incubation.
Statistics. Calculations were done using the MINITAB version 11 computer package (Minitab Inc., University Park, Pa.). Sugar beet weights and bacterial counts were compared statistically using analysis of variance, after log10 (x + 1) transformation, with group means compared by calculating the minimum significant difference at a P value of 0.05, according to the Tukey-Kramer method (8). Phage counts were compared using the mood-median test, and linear regression lines were compared by analysis of covariance (8). Contour plots showing in situ phage distributions were generated using MINITAB.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The 1997 field experiment.
Prior to sowing, the mean
total-viable bacteria count per gram of soil was 2.5 × 107 CFU, as estimated on TSBA medium. A mean of 2.6 × 106 CFU of pseudomonads g1 was also
calculated from PSIA plates. No phages were detected from any of the
five soil homogenates directly after preparation (limit of detection,
200 PFU per g of soil). Only after the homogenates had been enriched
with host bacteria were phage detected in one of the soil samples, and
these were all identified as CP6-1.
1). Small significant (i.e., P < 0.05)
variations in numbers of both CP6-like bacteria and pseudomonads were
seen (Fig. 2A), but these did not coincide with significant changes in
the populations of their phages.
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CP6-1 (Fig. 2C) and, to a lesser
extent, CP6-4, a Podoviridae phage frequently isolated
from our site in 1996 (3). A few CP6-3
(Myoviridae) phage (3) and several previously
unisolated phages were also present.
The 1998 field experiment.
Prior to sowing, there was no
significant difference among the treatment plots in terms of total
viable bacterial counts (P = 0.897) or total CP6-like
bacteria (P = 0.879). The mean total viable count was
3.3 × 107 CFU g1 of soil, while for
CP6-like bacteria it was 8.6 × 103 CFU
g1. As in 1997, no CP6-antagonistic phages were isolated
from freshly prepared soil homogenates (limit of detection, 100 PFU
g1). Only after the samples had been enriched were 3 of
the 36 samples shown to harbor phages for this bacterium. Specifically,
soil from plot A6 was shown to contain an unidentified CP6-antagonistic phage and plot B4 soil carried CP6-4, while soil from plot E4 harbored phage CP6-3.
1 prior to seed inoculation. Cultures of the lysogen
CP6RS-ly-1 contained 103 PFU ml1 of phage
CP6-1 due to spontaneous lysis. No free phages were detected from
either the CP6SpN or CP6RS cultures.
The sugar beets reached maximum weight by around day 137. The resulting
mean weight, 275.4 g, was much less than in the previous year, probably
due to the combined effects of later sowing and a particularly dry
summer. In addition, analysis of variance showed that the overall mean
total viable bacterial count recorded from the rhizosphere samples of
these 1998 plants (9.3 × 107 CFU g1)
was significantly lower (P < 0.05) than that
determined from the 1997 plants (1.3 × 108 CFU
g1).
Both S. liquefaciens CP6RS and S. liquefaciens
CP6RS-ly-1 inocula survived well after release (Fig.
3B and C). S. liquefaciens CP6SpN, however, was never detected on any plants sampled, showing that
this bacterium had not survived in situ. Moreover, a comparison of
sugar beet weights and bacterium and phage counts between treatments revealed that the S. liquefaciens CP6SpN inoculation had no
detectable effect on the experiment (all P > 0.05).
Consequently, for the purposes of subsequent analysis, treatments 1 and
2 were judged to be the same (i.e., "uninoculated" controls), and
their results were combined (Fig. 3A, D, and G). Treatments 4 and 5 were also determined to be equivalent (i.e., a CP6RS-ly-1 release),
and their data sets were merged (Fig. 3C, F, and I). Treatment 3 was effectively a release of CP6RS alone (Fig. 3B, E, and H).
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1). Subsequently, fewer control
plants carried released bacteria, until by day 194, only one plant
harbored these organisms. When bacteria were detected, the abundances
were around 102 CFU g1. In total, 14 out of
the 108 control plants collected over the experiment had detectable
quantities of released bacteria.
Overall, five different types of S. liquefaciens-infecting phages were identified from sugar beet
samples, and these corresponded to phages CP6-1 to CP6-5
(3, 4). The vast majority isolated (84.4%) were either
CP6-1 or CP6-4.
(i) Phage CP6-1.
Relatively small numbers of CP6-1 phage
were isolated from uninoculated plants (Fig. 3D and
4A), and they were only very apparent
when the homogenates were enriched (Fig. 3D and 4B). No CP6-1 phage
were isolated from plants inoculated with bacterium CP6RS (Fig. 3E and
4A) unless their homogenates were also enriched, whereupon plants from
two plots (B3 and E4) were shown to harbor small amounts of CP6-1 on
days 68 and 137 (Fig. 3E and 4B).
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1 repeatedly produced plants harboring high densities of
CP6-1 from fresh homogenates (Fig. 3F and 4A), and high titers (up
to 1.6 × 104 PFU g1) were regularly
recorded. The only exception was plot A4, and even then, when enriched,
homogenates from this plot occasionally elicited phages (Fig. 4B). The
CP6-1 titers decreased with time, and this decline mirrored the
observed drop in CP6RS-ly-1 numbers on the same plants (Fig. 3C).
From days 19 to 96 inclusive, the mean phage and lysogen counts
appeared to decrease at the same rate, and a comparison of the slopes
confirmed this, with no significant difference in gradient detected
(P = 0.972).
In all, CP6-1 counts were significantly greater (P < 0.001) in lysogen-inoculated plots (mean, 2.7 × 103 PFU g1) than in both uninoculated (mean,
5.8 × 101 PFU g1) and
nonlysogen-inoculated plots, where CP6-1 were below the limit of
detection unless enriched.
(ii) Phage CP6-4.
Phage CP6-4 was rarely isolated from
untreated plants (Fig. 3G and 4C) even after nutrient enrichment (Fig.
3G and 4D). However, for inoculated plants the patterns of significant
difference between treatments were different for CP6-4 and CP6-1.
That is, phage CP6-4 titers were significantly higher in
nonlysogen-treated plots (mean, 9.1 × 102 PFU
g1) than in either the uninoculated (mean, 3.6 × 102 PFU g1; P < 0.001) or
the lysogen-inoculated (mean, 3.5 × 102 PFU
g1; P = 0.009) plots. There was no
difference between uninoculated and lysogen-inoculated plots
(P = 0.110). Phage CP6-4 was not inoculated into the
site, and so it was not as abundant or as widely distributed as
CP6-1 (Fig. 4). More of the nonlysogen-inoculated plants (44.1%)
carried CP6-4 than either the lysogen-inoculated plants (19.7%) or
uninoculated plants (12.0%). Furthermore, 83.3% of all
nonlysogen-inoculated plots, 66.7% of all lysogen-inoculated plots,
and 55.5% of uninoculated plots harbored plants with detectable CP6-4 at some point during the experiment (Fig. 4D).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This is the first in situ study to unambiguously show that an
increase in numbers of an indigenous soil bacterium can lead to an
equally substantial rise in a naturally occurring bacteriophage (CP6-4). This work is unique, because it took place in a completely natural environment with native bacteria and phages and occurred over a
long, ecologically relevant time scale. In no other natural habitat has
this been achieved. Previous equivalent terrestrial studies have all
employed microcosms, over far shorter time scales, and often with very
simplified microbial communities (7, 9, 21, 22). A few
aquatic studies have been undertaken in situ. However, these followed
gross changes in total bacterial and virus populations (6,
24). Interactions between individual bacterial and phage species
in water have only been reported from microcosms (10, 20).
What makes our results so remarkable is that another S. liquefaciens phage, CP6-1, failed to benefit from the release
of S. liquefaciens CP6RS. Contrast the almost complete
absence of this phage within CP6RS-inoculated plots (Fig. 3E) with its
repeated occurrence elsewhere (Fig. 3D and F). This statistically
significant difference leads us to conclude that phages CP6-1 and
CP6-4 competed with each other in situ and that the different state of health of the released CP6RS, relative to wild-type CP6 indigenous within the soil, predisposed it to successful predation by CP6-4 in
preference to CP6-1.
We assert these two conclusions for the following reasons. We already
have strong evidence of temporal succession, and hence competition,
between CP6-1 and CP6-4 occurring in situ in 1996 (3).
During that field experiment, we observed an explosion in CP6-1
numbers between days 48 and 99. This situation continued until day 156, when a dramatic decline in abundance occurred; thereafter numbers
remained low until the end of the experiment. Concurrent with this
decline was an even more substantial increase in the numbers of phage
CP6-4, which until that point had been almost completely absent.
Our subsequent research (4) confirmed CP6-1 and CP6-4
to be very different. (i) We found no DNA homology between the two
phages. (ii) CP6-1 was shown to be a Siphoviridae phage, while CP6-4 was a member of the family Podoviridae. (iii)
CP6-1 was temperate for CP6, while CP6-4 was entirely virulent.
(iv) The latent period for CP6-1 was almost three times that of
CP6-4, while its burst size was over five times greater. The last
attributes are particularly pertinent to this discussion, as they have
been identified as possible phage survival strategies (1, 23, 25).
For example, Stewart and Levin (23) theorized that virulence
would be favored as a survival mechanism over lysogeny in those environments where there are high numbers of a physiologically "suitable" host available. According to their theory, CP6-4
would predominate over CP6-1 at our field site when such host cells became abundant. CP6RS may have been this physiologically suitable host. Besides being abundant as a consequence of our release, CP6RS
would have been physiologically different from contemporaneous indigenous CP6.
The scenario we outline is also consistent with the work of Abedon
(1) and Wang et al. (25), who applied optimal
foraging theory to phage ecology. From their theoretical models, they
concluded that phages with short latent periods and small burst sizes
(like CP6-4) would outcompete phages with longer latent periods and larger burst sizes (like CP6-1) when the numbers of physiologically suitable host bacteria are high (as for CP6RS). Taken together, all
these factors provide strong evidence of competition occurring between
phages in situ.
We did not add CP6-4 phage to our site, so the bloom we triggered
derived entirely from naturally present virions. Phage CP6-1 was
also native; however, in this experiment its numbers were only
increased substantially by a lysogen release. This inoculation, coincident with the CP6RS release, generated large numbers of CP6-1
phage in all but one of the lysogen-inoculated plots. In these plots,
CP6-1 was up to 1,000-fold more numerous than in uninoculated plots.
This and the pattern of significant differences in observed phage
titers showed that these large titers came from the inoculated lysogen.
Several points arise from this concurrent release of lysogen and
nonlysogen. First, it is clear that the proximity of all the plots to
one another led to some movement of phage and released bacteria between
plots, with small numbers of released bacteria repeatedly occurring in
untreated controls (Fig. 3A) and the apparent spread of CP6-1 from
lysogen-inoculated plots to neighboring plots (Fig. 4B). Yet, in spite
of these factors favoring CP6-1, it was CP6-4 that entirely
dominated the nonlysogen plots, emphasizing its competitive advantage
over CP6-1 for CP6RS.
Second, CP6-4 did not do so well in the lysogen-inoculated plots. It
is unclear why this should be, as in the laboratory the lysogen was
CP6-4 sensitive. Perhaps the lysogen had a level of resistance to
CP6-4 that was only discernible under the nonoptimal growth
conditions experienced in situ.
Third, this is the first study to simultaneously release lysogen and nonlysogen forms of the same bacterium into a natural environment and, in doing so, to demonstrate that a bacterium "burdened" with prophage DNA can survive as well as its wild type. This contradicts earlier microcosm studies (11, 18) that found lysogens surviving less well than nonlysogens.
Fourth, our study also illustrates what can happen to an environment
into which a lysogen is released artificially. Not only is the
microbial community altered by the new bacterium, but phages released
from that lysogen also have the potential to affect indigenous bacteria
and facilitate gene transfer through transduction. CP6-1, for
example, is a transducing phage (4).
A final important point to be drawn from our study is the unique
observation that the temporal dynamics of specific phage populations in
soil are repeated over successive years. Specifically, our results show
that, over three consecutive years, CP6-1 predominated at the
beginning of the growing season (3) (Fig. 2C and 3D). In
contrast, CP6-4 was never abundant at that time. If it did bloom, it
did so some time after the sugar beets had fully matured and CP6-1
numbers had begun to fall (3). Thus, we conclude that the
seasonality described in this paper highlights the potential predictability of bacterium-phage interactions in soil.
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ACKNOWLEDGMENT |
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This work was supported by the Ministry of Agriculture Fisheries and Food (UK) grant RG0112.
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FOOTNOTES |
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* Corresponding author. Mailing address: Cardiff School of Biosciences, Cardiff University, P.O. Box 915, Cardiff CF10 3TL, United Kingdom. Phone: 44 (0)2920 876002. Fax: 44 (0)2920 874305. E-mail: ashelford{at}cardiff.ac.uk.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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