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Applied and Environmental Microbiology, June 2001, p. 2469-2475, Vol. 67, No. 6
Biotechnology Research Institute, National Research Council
Canada, Montreal, Quebec,1 and Fisheries and
Oceans Canada, Dartmouth, Nova Scotia,4
Canada, and School of Civil
Engineering2 and School of
Agronomy,3 Purdue University, West LaFayette,
Indiana
Received 2 November 2000/Accepted 18 March 2001
Plant-bacterial combinations can increase contaminant degradation
in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The
purpose of this study was to determine if plants had the ability to
selectively enhance the prevalence of endophytes containing pollutant
catabolic genes in unrelated environments contaminated with different
pollutants. At petroleum hydrocarbon contaminated sites, two genes
encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were
two and four times more prevalent in bacteria extracted from the root
interior (endophytic) than from the bulk soil and sediment,
respectively. In field sites contaminated with nitroaromatics, two
genes encoding nitrotoluene degradation, 2-nitrotoluene reductase
(ntdAa) and nitrotoluene monooxygenase (ntnM),
were 7 to 14 times more prevalent in endophytic bacteria. The addition
of petroleum to sediment doubled the prevalence of
ndoB-positive endophytes in Scirpus pungens,
indicating that the numbers of endophytes containing catabolic
genotypes were dependent on the presence and concentration of
contaminants. Similarly, the numbers of alkB- or
ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not
with the numbers of alkB- or ndoB-positive
bacteria in the bulk soil. Our results indicate that the enrichment of
catabolic genotypes in the root interior is both plant and contaminant dependent.
The potential to use plants to
remediate polluted soils has recently attracted considerable interest
(19, 28, 29, 34). Plants can stimulate contaminant
disappearance by accumulation and transformation (30), by
extracellular transformation (13, 37) and by stimulating
microbial degradative activity in the rhizosphere (3, 35).
Several authors have investigated the role of microorganisms in
phytoremediation, and they have found that certain plant-bacterial
associations can increase degradation (1, 8, 11, 33). This
suggests that, under certain circumstances, such as in microbially
inoculated plants, microorganisms play an important role in
phytoremediation systems, but it is not clear what role they play in
phytoremediation systems having only indigenous microbial populations.
Plants routinely encounter allelopathic compounds, many of which are
analogous to organic contaminants (9), and thus there may
be a plant response that stimulates microbial defenses against a soil
toxicant or toxin (48). If this is true, plants could recruit bacteria that contain genotypes specific for toxicant degradation into the rhizosphere and root interior, and this selection should be contaminant specific. These bacteria would presumably protect
the plant from the phytotoxic effects of the contaminants. In a similar
manner, plants growing in herbicide-contaminated soils have high
herbicide mineralization activity in their rhizospheres (2). Inoculating herbicide-degrading bacteria into the
rhizosphere also protects plants from the phytotoxic effects of the
herbicides (17, 32).
The selective pressure of plants on bacterial populations has its
maximum effect near the root surface or in the root interior (16,
21, 23). For example, the maximum impact of altering plant
genotypes was observed in the root interior of canola with substantially less impact observed on the rhizosphere microbial community (36, 40). Endophytic bacteria enhance the
ability of plants to resist pathogens (49), herbivores
(10), and other plants (44). Thus, if
bacteria do play a role in the plant's ability to tolerate
contaminants, we hypothesized that this effect would occur in the
endophytic zone.
In this study, gene probes for alkane monooxygenase (alkB)
(47), naphthalene dioxygenase (ndoB)
(41), and catechol-2,3-dioxygenase (xylE)
(24) were used to assess the prevalence of bacteria
involved in petroleum hydrocarbon degradation. Gene probes developed
for the ntdAa and the ntnM genes
(38) were used to test for the prevalence of bacteria
involved in nitrotoluene metabolism. The ntdAa gene of
Pseudomonas sp. strain JS42 encodes the reductase component
of the three-component dioxygenase system that converts 2-nitrotoluene
to 3-methylcatechol (27). The ntnM gene from Pseudomonas putida encodes the hydroxylase component of
nitrotoluene monooxygenase (18).
The purpose of this study was to assess the influence of plants on
contaminant-degrading bacteria in upland terrestrial or freshwater
intertidal environments. The effect of different contaminants, petroleum hydrocarbons, or nitroaromatics was also assessed. A wide
range of environments and contaminants were purposely selected to
investigate if plant selection of catabolic genotypes occurred as a
general phenomenon or rather as a special case of a particular plant-soil-contaminant combination.
Terrestrial petroleum site.
The terrestrial petroleum
contaminated study site was located at the Port Hueneme, Calif.,
Department of Defense National Test Site. Each plot was approximately
30 by 50 m and was contaminated intermittently over a 20-year period
with 1.5 g of total petroleum hydrocarbons per kg of soil
originating from diesel fuel and heavy oil. Heavy metal concentrations
were not significantly above background levels. There were three
treatments with four replicate plots each of mixture 1, consisting of
Bromus hordeaceous, Festuca arundinacea, Trifolium fragiferum, T. hirtum, and Vulpia microstachys; mixture 2, consisting
of B. carinatus, Elymus glaucus, F. ruba, Hordeum californicum,
Leymus triticoides, and Nassella pulchra; and an unvegetated plot. These plant mixtures were selected for their suitability to the northern California climate. Mixture 1 contained two
clovers, which may increase the nitrogen supply to the soil organisms,
whereas mixture 2 only contained native grasses. Plants were seeded in
August 1997. Both plant mixtures were sampled at the petroleum site in
September, January, and April 1998 and in August 1999 for a total of 16 replicates. The culturable and nonculturable community was analyzed for
the F. arundinacea and T. fragiferum treatments
and for a composite sample of mixture 2. The soil had a pH of 7.2 and
an organic matter content of 2.1% and was composed of 59% sand, 26%
silt, and 15% clay. The site was irrigated as needed and fertilized
with 11.3 kg of urea and 11.3 kg of diammonium phosphate every other month.
Terrestrial nitroaromatic site.
The nitroaromatic site is a
former 2,4,6-trinitrotoluene (TNT) manufacturing facility near
Montreal, Quebec, Canada. Soil from which plants were sampled was
contaminated with approximately 390 mg of TNT, 12 mg of
2,4-dinitrotoluene, 12 mg of 2,6-dinitrotoluene, 0.38 mg of
2-nitrotoluene, and 1.39 mg of 1,3,5-trinitrobenzene per kg. Production
on site was terminated in 1972 and has not been in use since this time.
The site was naturally vegetated by a variety of indigenous grasses
since production stopped. The soil was a clay-loam with a pH ranging
between 6.8 and 7.8. In June, July, and August 1998, the nitroaromatic
site was sampled at three replicate locations, with three replicates
per site for a total of 27 replicates. This site was not irrigated or
fertilized during this study.
Freshwater intertidal petroleum site.
Eight replicate plots
(5 by 4 m) were constructed in an intertidal region on the St.
Lawrence River, Quebec, Canada. Four plots were contaminated at 0.6 liters/m2 or 12 g kg of sediment Growth chamber study.
Control garden soil was a sandy loam
consisting of 70% sand, 7% clay, and 23% silt with a pH of 7.9, water-holding capacity of 76%, total organic C of 11.2%, and Kjeldahl
N of 0.38% (14). The garden soil was mixed with soil
obtained from an active wood treatment facility contaminated with 43 g
of C10 to C50 aliphatic hydrocarbons kg of
soil Catabolic gene probe analysis of the cultured and noncultured
communities.
Bacteria were extracted from the root interior,
rhizosphere, and bulk soil as previously described (36)
with the exception that bacteria were resuspended in 0.1% (wt/vol)
tetrasodium pyrophosphate (pH 7.0). Aliquots (0.1-ml) of serial
dilutions in tetrasodium pyrophosphate (10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2469-2475.2001
Selection of Specific Endophytic Bacterial
Genotypes by Plants in Response to Soil Contamination
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 with
weathered light crude oil in June 1999. Oil was raked into the top 5 cm
of the sediment during low tide. Nitrogen and phosphorus as nutrients
[NH4NO3 + Ca(H2PO4)2 · H2O] were added initially at a rate of 1,000 g of N and
300 g of P per plot. After 4 weeks, all fertilized plots received
weekly applications of fertilizers. Bulk soil and endophytic samples
were collected from the on-site natural vegetation (Scirpus
pungens) 6 and 8 weeks after contamination for a total of eight
replicates per treatment.
1 and 11 g of polycyclic aromatic hydrocarbons kg of
soil1 at five different concentrations (0, 5, 10, 20, and
40% [wt/wt]). The soil from the wood treatment facility had aged,
and there was intermittent fresh contamination as a result of spills
that had occurred during treatment operations. Soil from the
nitroaromatic site was mixed with uncontaminated garden soil for a
final concentration of nitroaromatics of 340 mg of TNT, 0.4 mg of
2,4-dinitrotoluene, and 0.3 mg of 1,3,5-trinitrobenzene per kg. The
contaminated soil was placed in 5-cm pots, F. arundinacea
was planted, and pots were placed in a greenhouse with an average
daytime temperature of 18°C, a night-time temperature of 5°C, a
relative humidity of 40%, and a light intensity of 400 µmol
s1 m2. Plants were thinned to five per pot
14 days after planting and harvested 6 weeks after planting.
2,
103, and 104) were spread plated onto
triplicate plates containing 250 mg of yeast extract (Becton Dickinson,
Cockeysville, Md.), 250 mg of tryptone (Difco Laboratories, Detroit,
Mich.), 250 mg of soluble starch (Anachemia, Montreal, Quebec, Canada),
and 15 g of granulated agar (Becton Dickinson) per liter of tap water.
Bacterial colonies were counted and lifted onto nylon membranes after
incubation at room temperature (21 to 24°C) for 2 weeks. Cells
adhering to nylon membranes were lysed, and the DNA was denatured,
fixed, and cross-linked to the membrane and hybridized to the
alkB (17), ndoB (41),
ntdAa (27), ntnM (18),
or xylE (24) gene probes as previously
described (12).
Statistics and data transformation.
Typically, the average
bacterial population density in the root interior is between
103 and 105 CFU g of root1
(16) compared to population densities in soil of
107 and 108 CFU g of soil1.
Therefore, in order to compare populations between habitats, genotype
prevalence is commonly expressed as the percent composition of the
community present in the habitat of interest (21), which in this case is the root interior, rhizosphere, and bulk soil. The
percent composition data were arcsine transformed to approximate the
normal distribution, and a Skewness and Kurtosis analysis of the data
indicated that the data were normally distributed (43).
Canonical correlation and regression analysis were performed using
Systat 10.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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At the terrestrial petroleum hydrocarbon contaminated site, the
alkB genotype was 10 times more prevalent in the culturable endophytic microbial community (4.3% of 21,182 colonies probed from
288 plates) compared to the bulk soil microbial community (0.42% of
25,700 colonies probed from 96 plates) (Fig.
1). Similarly, alkB was more
prevalent (P < 0.05; Student's t test) in
the total community DNA extracted from the rhizosphere, 5.6%, compared
to that from the bulk soil, 3.9% (data not shown). In contrast, there were fewer ndoB-positive bacteria present in the root
interior (0.13%) than in the bulk soil (4.8%), and no significant
difference was seen in ndoB prevalence in the total
community DNA of the rhizosphere (5.1%) compared to the bulk soil
(4.7%). The xylE results from the culturable community were
similar to ndoB, with more xylE-positive bacteria
detected in the bulk soil (3.2%) than in the root interior (0.23%).
The gene associated with mononitroaromatic metabolism,
ntdAa, was not detected at the petroleum-contaminated site.
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At the nitroaromatic site, ntdAa-positive bacteria were more
prevalent in the root interior (4% of 14,000 colonies probed or
6.2 × 104 positive CFU g of root1) than
in the bulk soil (0.6% of 21,000 colonies probed or 1.7 × 106 positive CFU g of soil1) (Fig. 1).
Similarly, the ntdAa gene was six times more prevalent in
the total community DNA extracted from the rhizosphere, 0.24%, compared to the bulk soil, 0.04% (data not shown). The ntnM
genotype was also more prevalent inside the root, with
ntnM-positive bacteria 14-fold more prevalent in the root
interior (0.35%) than in the bulk soil (0.03%). There were no
endophytic bacteria positive for alkB or ndoB,
and very few alkB (0.01%)- or ndoB
(0.03%)-positive bacteria in the bulk soil microbial communities of
the nitroaromatic site.
The increase in catabolic genotypes in the rhizosphere and root
interior was dependent on the catabolic genotype being investigated as
well as the plant treatment (Table 1).
All plant treatments had an increased prevalence of
alkB-positive bacteria in their root interior with little
difference seen between plant treatments. However, in the rhizosphere
of Rose Clover or mixture 2, alkB bacteria were more
prevalent compared to the bulk soil and levels were greater than
observed in the rhizosphere of the Tall Fescue. In contrast,
ndoB prevalence displayed an opposite trend. The prevalence
of ndoB-positive endophytes was lower compared to the rhizosphere in all three plant treatments, and ndoB-positive
bacteria in the root interior of Rose Clover and mixture 2 were
significantly less prevalent compared to Tall Fescue. Only Tall Fescue
increased the prevalence of ndoB-positive bacteria in its
rhizosphere compared to bulk soil. Bacteria positive for
xylE were not enhanced in the rhizosphere or root interior
by any plant treatments.
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The enrichment of catabolic gene-containing bacteria in the root
interior of S. pungens at the freshwater intertidal site was
dependent on the presence of contaminants. When plants were exposed to
crude oil, the numbers of ndoB- or xylE-positive
bacterial endophytes increased (P < 0.05) by an order of
magnitude (Fig. 2). There was not a
corresponding increase in ndoB-positive organisms in the
bulk sediment. Total endophytic or soil heterotrophs were not
significantly affected by hydrocarbon contamination with little difference observed between treatments. The numbers of
xylE-positive bacteria in the bulk sediment increased
significantly, which may explain the corresponding increase in
xylE probe-positive organisms in the root interior.
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Interestingly, no alkB-positive endophytes were found, and the presence of oil did not increase the prevalence of alkB-positive bacteria in the bulk sediment. This may be the result of using an alkB DNA probe derived from the terrestrial bacterium P. oleovorans (47) on bacteria extracted from a freshwater sediment environment. Smits and colleagues (42) demonstrated a wide divergence in DNA sequences of alkane monooxygenase genes among microorganisms.
In the growth chamber experiment, the enrichment of alkB- or
ndoB-positive endophytic bacteria in F. arundinacea was correlated to the concentration of contaminant in
the soil. The numbers of endophytic bacteria that were positive for
alkB or ndoB were proportional (r2 = 0.988, P < 0.001 for
alkB; r2 = 0.967, P < 0.001 for ndoB) to the soil creosote concentration and
not to the prevalence of alkB- or ndoB-positive
bacteria in the rhizosphere (Fig. 3). In
contrast, the prevalence of rhizosphere bacteria positive for
alkB or ndoB was not correlated
(r2 = 0.111, P = 0.308 for
alkB; r2 = 0.189, P = 0.259
for ndoB) with increasing soil creosote concentrations. The
numbers of xylE-positive bacteria increased in the
rhizosphere (r2 = 0.673, P = 0.052),
as well as in the root interior (r2 = 0.891, P < 0.001). The numbers of endophytic (ca. 4 × 106 CFU g of root1) or rhizosphere (ca.
3 × 109 CFU g of soil1) heterotrophs
did not differ with increasing creosote concentrations (data not
shown). The prevalence of the ndoB genes in the culturable community of the root interior increased 20 times from 0.01% in plants
grown with no creosote to 0.2% of the total heterotrophic community.
|
) or
endophytes (
) present in F. arundinacea in response to
increasing creosote concentrations in soil. F. arundinacea
was planted in garden soil mixed with five concentrations (0, 5, 10, 20, and 40%) of soil heavily contaminated with creosote (43 g
C10 to C50 aliphatic hydrocarbons and 11 g
of polycyclic aromatic hydrocarbons kg of soilSimilar trends were seen in the total community DNA extracted from the
root system (Fig. 4), with
alkB or ndoB increasing in prevalence as the
creosote concentrations in soil increased. For example, the
concentration of alkB or ndoB genomic equivalents in the total community DNA extracted from the roots of plants grown in
the 40% creosote soil increased by 25 or 10 times, respectively, over
plants grown in control soil in a concentration-dependent manner
(r2 = 0.994, P < 0.001 for
alkB; r2 = 0.998, P < 0.001 for ndoB). No bacteria positive for the
ntdAa genotype were found in the creosote-contaminated soils
(data not shown).
|
When F. arundinacea was grown in the presence of
nitroaromatics, there was no enrichment for ntdAa-positive
endophytes, with 0.3% (1,660 colonies probed or 1.3 × 105 positive CFU g of soil1) of the bulk soil
population positive for ntdAa and only 0.05% (1,224 colonies probed or 8.7 × 101 CFU g of
root1) of the endophyte population (data not shown). The
numbers of alkB- or ndoB-positive endophytes did
not increase in the presence of nitroaromatics in bulk soil. In
contrast, the numbers of xylE-positive endophytic bacteria
increased from 339 CFU g of root1 in control plants to
1,040 CFU g of root1 in plants grown in the presence of nitroaromatics.
The results of colony lifts obtained using the ndoB, alkB,
and ntdAa gene probes for both soil and root interior
samples were compared with values obtained by dot blot analysis of the
total community DNA. Catabolic gene probe results obtained with colony lifts were well correlated (r2 = 0.875, P < 0.001, n = 44) with results obtained from dot blots (Fig.
5), and a strong linear relationship was
observed between the two parameters.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our results indicate that certain bacterial catabolic genotypes (alkB, ndoB, ntdAa, and ntnM) were enriched in the interior of plant roots in response to pollution in a contaminant-dependent manner. In contrast, xylE increased in response to all classes of contaminants. Enrichment of specific catabolic genotypes was also dependent on plant species in combination with environmental parameters. For example, S. pungens exposed to oil enriched ndoB but not alkB, whereas the phytoremediation mixture enriched alkB but not ndoB. Similarly, F. arundinacea exposed to nitroaromatics did not increase the prevalence of ntdAa in the root interior, but the grass community at a nitroaromatic-contaminated site had increased prevalence of ntdAa in the root interior. An increase in contaminant-degrading bacteria in the rhizosphere of plants exposed to petroleum hydrocarbons was observed previously (26). Our results extend this observation to other contaminants and indicate that the enrichment is dependent on the type and amount of contaminant present in soil. At terrestrial upland sites, endophytic bacteria only contained the petroleum hydrocarbon-degrading genes, alkB or ndoB, when petroleum hydrocarbons were present in the surrounding soil and/or sediment. Similarly, genes encoding nitrotoluene degradation were only identified when nitrotoluenes were present in the environment. Furthermore, the numbers of endophytic catabolic genotypes were not related to the presence of bacteria containing catabolic genotypes in the surrounding soil but rather with contaminant concentrations in soil.
The increase in endophytic catabolic genotypes may be a plant-dependent or -independent process. The plant may be selectively enriching catabolic genotypes in its root interior via some as-yet-unknown plant process. Roots are known to exert a selective influence on the root-associated bacterial communities (15, 20, 25, 50), and the influence of roots is at least partly plant specific (36). Alternatively, the numbers of endophytic catabolic genotypes may increase due to contaminant flux through the root system. Both explanations account for the contaminant-dependent increase in endophytic catabolic genotypes. It is likely that the enrichment is plant mediated because this explanation accounts for the differences observed between plant species and the dependence of catabolic gene prevalence in the root interior on contaminant levels. For example, alkB-positive endophytes of F. arundinacea increased in proportion to the amount of creosote present in soil, but when this grass was exposed to nitroaromatics no enrichment was detected. In contrast, grasses revegetating a nitroaromatic contaminated terrestrial site contained significant levels of nitroaromatics in their root interior. Thus, if F. arundinacea did not influence the enrichment process, its roots would need to be permeable to alkane hydrocarbons but not nitrotoluenes. Typically, nitrotoluenes are readily absorbed by grasses, so this is probably not the case (30, 31, 45).
The enrichment of contaminant-degrading bacteria in the root interior
is at least partially plant species specific. F. arundinacea did not promote an increase in ntdAa-positive endophytic
bacteria despite being exposed to nitroaromatic concentrations
comparable to that seen at a contaminated field site. For example,
there were 6.2 × 104 ntdAa-positive
endophytes g of root1 in plants growing at the
contaminated field site but only 8.7 × 101
ntdAa endophyte CFU g of root1 in F. arundinacea grown in the growth chamber experiment. This plant
species is not exceptionally tolerant of nitroaromatics and is used for
the phytoremediation of petroleum hydrocarbon-contaminated sites rather
than nitroaromatic contaminated sites (38). Similarly, at
the petroleum-contaminated site, F. arundinacea had a
significantly greater prevalence of ndoB in its root
interior compared to T. fragiferum, whereas T. fragiferum had more alkB endophytes than F. arundinacea. In addition to the effects of specific plants, environment plays an important role in altering endophytic community composition. S. pungens, grown inside an intertidal zone,
promoted ndoB prevalence inside the root, whereas grass
mixtures grown in a terrestrial upland site promoted alkB.
It is difficult to distinguish plant effects from those of the
environment. The composition of the endophytic microflora is known to
be dependent on both plant and soil type (15, 20, 25, 50).
There is likely a similar interaction between contaminant, soil, and
plant species that determines the level and identity of catabolic genes enriched.
Phytoremediation systems that use microbial inoculants are typically more effective than noninoculated plant systems (2, 8, 11, 33). This work examined one of the mechanisms, i.e., root interior colonization, that may be occurring to explain the increased success of plant-bacterial associations in degrading contaminants. In a similar manner, plant growth-promoting activity has been linked to colonization of the root interior (4, 46). Previous research has correlated the success of a plant-bacterial association to remediate nitroaromatics, in which the ntdAa genotype prevalence in the rhizosphere was promoted (38), but gene prevalence in endophytes was not investigated.
Our results indicated that culture-independent methods of detecting catabolic gene prevalence were more sensitive compared to culture-dependent methods. Gene probes have consistently been shown to be an effective method of monitoring gene prevalence in the environment (6) but do not estimate the in situ activity of these genes. Further, gene probes may underestimate actual prevalence of contaminant-degrading bacteria. Gene probes such as ndoB detect less than 50% of bacteria capable of polycyclic aromatic hydrocarbon metabolism (22). Similarly, sequence divergence between petroleum hydrocarbon genes (7, 42) implies that catabolic gene probes will underestimate the prevalence of bacteria capable of contaminant degradation. More work is required to assess gene probe efficacy in different environmental compartments.
The results from this study indicate that the enrichment of catabolic genotypes in the root interior occurs in different plants in a variety of environments and in response to different contaminants. The enrichment is dependent on plant species and the contaminant in which the plant is growing. The process by which this occurs is not yet known and is the subject of current investigations. The flexible and specific nature of the selective pressure observed here suggests that the mechanism by which plants control the composition of the endophytic microbial community is much more dynamic and responsive than previously believed. The impact of this dynamic response on plant survival at contaminated sites has yet to be determined.
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FOOTNOTES |
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* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada. Phone: 514-496-6182. Fax: 514-496-6265. E-mail: charles.greer{at}nrc.ca.
Present address: Department of Biology, University of Ottawa,
Ottawa, Canada.
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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, June 2001, p. 2469-2475, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2469-2475.2001
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