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Applied and Environmental Microbiology, December 2000, p. 5410-5418, Vol. 66, No. 12
Crop and Soil Sciences, Michigan State
University, East Lansing, Michigan 488241;
Department of Molecular and Cell Biology, University of
Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25
2ZD, United Kingdom2; and W. K. Kellogg
Biological Station, Department of Botany and Plant Pathology,
Michigan State University, Hickory Corners, Michigan
490603
Received 5 April 2000/Accepted 14 September 2000
The aim of this study was to determine the effects of different
agricultural treatments and plant communities on the diversity of
ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing,
was used to analyze 16S rRNA genes of ammonia oxidizers belonging to
the Autotrophic ammonia-oxidizing
bacteria carry out the first and rate-limiting step of nitrification,
namely, the oxidation of ammonia to nitrite. Ammonia oxidation often
involves the direct evolution of the greenhouse gas N2O to
the atmosphere (7) and indirectly leads to additional losses
of N through denitrification of nitrate. The
NO3 Understanding the effects of agricultural practices on the structure
and function of microbial communities, in particular, the
ammonia-oxidizing bacteria, may aid in the development of lower-input
sustainable systems. Evaluation of the early system effects of
management, for example, tillage, N inputs, and crop rotation, on
parameters such as total microbial populations, bacterial/fungal ratios, and overall microbial activity is difficult. Naeem et al.
(27), using experimental model systems, showed that soil community respiration and plant productivity were higher in more diverse plant communities. Organically based agricultural systems that
include multiple crops often have higher yields under stress conditions, such as drought, than do fertilizer-based one- or two-crop
systems (32). The functional composition and functional diversity of plant communities have been shown to be the principal factors controlling productivity and plant nitrogen uptake (15, 44). Findings such as these suggest that management practices that affect plant diversity and composition can have a profound effect
on ecosystem processes. Assessment of the impact of changes in plant
communities on soil community structure is made difficult by the high
level of diversity of total bacterial communities in terrestrial
environments (11, 24, 28, 29) but may be facilitated by
investigation of specific groups of organisms. The oxidation of ammonia
to nitrite therefore holds great promise as an indicator process in N
cycling studies and in the study of soil microbial diversity relative
to ecosystem disturbance.
Autotrophic ammonia oxidizers belong to two phylogenetic groups, one
within the Studies using molecular tools to characterize ammonia oxidizer
communities in soils at the Long Term Ecological Research (LTER) experiment at the W. K. Kellogg Biological Station (KBS), Michigan State University, have demonstrated a reduced diversity of
ammonia-oxidizing bacteria in cultivated soils. Cluster 3 Nitrosospira sp. 16S ribosomal DNA (rDNA) sequences were
found in cultivated soils but not in noncultivated soils from the same
area (5). Analysis of above-ground plant diversity in
successional treatments demonstrated the replacement of initially
dominant annual species by biennials and herbaceous perennials within 4 years (16). Although nitrogen addition significantly increased above-ground plant biomass, it had no significant effect on
plant species diversity. Annual tillage of the nonseeded land produced
low-diversity annual grassland. These results raise the question of
which parameters, fertilization, tillage, or plant community, drive
plant and microbial diversity shifts within these soils.
The objective of this study was to assess the relationship between the
diversity of ammonia oxidizer populations and to assess differences in
plant productivity and diversity brought about by different fertilizer
N and tillage regimens. The LTER plots in southwestern Michigan enabled
simultaneous measurement of the effects of tillage, fertilizer, and
plant type on potential nitrification, nitrifier numbers, and diversity
of ammonia oxidizers in cultivated and noncultivated soils.
Soil sampling.
Soil samples were collected in October 1996 and March 1997 from the LTER experiment at the KBS in southwestern
Michigan. The site was established in 1988 from a field that had been
under cultivation for over 100 years. Replicate plots of 0.9 hectare had six replicate plots of seven management treatments
(http://www.lter.kbs.msu.edu). Molecular characterization of ammonia
oxidizer communities was carried out with samples collected from
cultivated and successional treatment plots. In addition, soil was
investigated from a nearby native deciduous forest (NDF).
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effects of Agronomic Treatments on Structure and
Function of Ammonia-Oxidizing Communities
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
subgroup of the division Proteobacteria by use of
DNA extracted from cultivated, successional, and native deciduous
forest soils. Community profiles of the different soil types were
compared with nitrification rates and most-probable-number (MPN)
counts. Despite significant variation in measured nitrification rates
among communities, there were no differences in the DGGE banding
profiles of DNAs extracted from these soils. DGGE profiles of DNA
extracted from samples of MPN incubations, cultivated at a range of
ammonia concentrations, showed the presence of bands not amplified from
directly extracted DNA. Nitrosomonas-like bands were seen
in the MPN DNA but were not detected in the DNA extracted directly from
soils. These bands were detected in some samples taken from MPN
incubations carried out with medium containing 1,000 µg of
NH4+-N ml
1, to the exclusion of
bands detected in the native DNA. Cell concentrations of ammonia
oxidizers determined by MPN counts were between 10- and 100-fold lower
than those determined by competitive PCR (cPCR). Although no
differences were seen in ammonia oxidizer MPN counts from the different
soil treatments, cPCR revealed higher numbers in fertilized soils. The
use of a combination of traditional and molecular methods to
investigate the activities and compositions of ammonia oxidizers in
soil demonstrates differences in fine-scale compositions among
treatments that may be associated with changes in population size and function.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
produced during nitrification is also a
major cause of water pollution (45). Inhibition of ammonia
oxidation occurs in many ecosystems (41), and the ability to
achieve inhibition in agricultural systems could result in large
financial savings in fertilizer costs while preventing much
environmental pollution.
subdivision of the division Proteobacteria (-proteobacteria) and one within the -proteobacteria.
Representatives from the former have been isolated only from marine and
brackish waters (47), whereas all soil ammonia oxidizers
enriched or isolated to date belong to the -proteobacteria (40,
46). Phylogenetic analysis of 16S rRNA genes amplified from
extracted environmental DNA by PCR with primers selective for the
-proteobacterial ammonia oxidizers has indicated the existence of at
least seven distinctive clusters, four belonging to the genus
Nitrosospira and three belonging to the genus
Nitrosomonas (40). The distribution of clone
sequences among these clusters is related to the environments from
which they were obtained (25, 34, 39, 40).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1
for corn and 84 kg of N ha1 for wheat). Treatment 2 was
like treatment 1, but a no-till practice was in place. Two perennial
treatments were also sampled. Treatment 5 was a long-term perennial
crop of Populus trees established in 1989. Successional
grasslands (treatment 7) had been left to revert to native flora
following establishment of the LTER plots (16). Within these
treatments were microplots (5 by 5 m), established in 1989, amended or not amended with fertilizer. In addition, within treatment 7 there were microplots that had either tillage or no tillage (Table
1). This design enabled investigation of the effects of both tillage and fertilization in agricultural and
successional treatments.
TABLE 1.
Management histories and fertilization applications for
the soils from the LTER plots at the KBSa
20°C, and the remaining soil was stored
at 4°C for further analysis. Sterilized soil for competitive PCR
(cPCR) calibrations was prepared by autoclaving three times at 121°C
for 15 min each time.
Potential nitrification.
Potential nitrification was
determined by incubation at 25°C of 10 g of soil in a 250-ml
Erlenmeyer flask containing 200 ml of phosphate buffer (1 mM; pH 7.2)
and 1.5 mM (NH4)2SO4
(13). After 0, 2, 4, 12, 22, and 24 h, soil particles
were removed from 10-ml samples by centrifugation in a Sorvall CE25
centrifuge at 6,000 rpm for 10 min. The supernatant was decanted into
glass scintillation vials and stored frozen at 20°C for nutrient
analysis. Nitrate and ammonia concentrations were determined with a
Lachet automated nutrient analyzer. Nitrification rates were determined from the linear regression of nitrite and nitrate concentrations versus time.
MPN counts.
Most-probable-number (MPN) counts of ammonia
oxidizers were determined with microtiter plates (36) using
twofold dilution series and modified Skinner-Walker (38)
growth medium (35) containing 5, 50, or 1,000 µg of
NH4+-N ml1, giving a wide range
of substrate concentrations to enumerate ammonia oxidizers with
different substrate requirements. The microtiter plates were placed on
top of a pad of water-saturated tissue, wrapped in plastic wrap to
avoid evaporation, and incubated for 4 weeks in the dark at room
temperature. Growth was assessed by color change from pink to yellow
due to acid production. Ammonia oxidation was confirmed by measurement
of nitrate and nitrite concentrations by the addition of diphenylamine
reagent (0.2 g in 100 ml of concentrated sulfuric acid). MPN values
were calculated using the tables of Rowe et al. (36). After
MPN values were calculated, the contents of wells from the column with
the highest dilution that showed growth in all eight replicates were
harvested. Cells and soil in each sample were harvested by
centrifugation in a microcentrifuge at 14,000 × g for 10 min. The supernatant was discarded, and the pellet was frozen at
20°C for DNA extraction and PCR amplification.
Direct microscopic counts. Soil bacteria were stained with 5(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) (Sigma Chemical Co., St. Louis, Mo.) (2 mg in 10 ml of phosphate-buffered saline, consisting of 0.05 M Na2HPO4 in 0.85% [wt/vol] NaCl [pH 9.0]) for 30 min (31). Random images, obtained by epifluorescence microscopy with a charge-coupled device camera (Princeton Instruments, Trenton, N.J.), were transferred to a Power Macintosh 7100/66 computer for analysis with Adobe PhotoShop.
DNA extraction.
DNA was extracted from 5 g of soil
(48), and humic contaminants were removed by gel
electrophoresis in a 1% (wt/vol) low-melting-point agarose gel (Gibco
BRL, Gaithersburg, Md.) (40 V for 4 h). The DNA was excised from
the gel, and the agarose was digested by -agarose treatment
(Boehringer Mannheim Corp., Indianapolis, Ind.) and concentrated
through a Microcon 100 column (Amicon Inc., Beverly, Mass.). DNA
quantity and purity were determined by measuring the absorbances at 260 and 280 nm on a model 8452A spectrophotometer (Hewlett Packard Co.,
Sunnyvale, Calif.).
PCR amplification.
PCR was carried out in a total volume of
50 µl in 0.3-ml Eppendorf tubes on a GeneAmp PCR system 9600 (Perkin-Elmer, Foster City, Calif.). Reactions were carried out in a
solution containing PCR buffer (Perkin-Elmer), 25 mM each
deoxynucleoside triphosphate, 20 pmol of each primer, 1.5 mM
MgCl2, 400 ng of bovine serum albumin (20), and
1 U of Taq DNA polymerase (Perkin-Elmer). A nested PCR was
carried out for all samples. Initial amplification was carried out
using primers selective for -proteobacterial ammonia oxidizers (AMO
primers), AMOf and AMOr (26). Secondary amplification was carried out using ammonia oxidizer-specific CTO primers
(19), which amplify a 426-bp fragment, including a 30-bp
GC-rich domain, for denaturing gradient gel electrophoresis (DGGE)
analysis. Conditions for each round of PCR with both AMO and CTO
primers were initial denaturation at 95°C for 5 min; 94°C for
40 s, 55°C for 30 s, and 72°C for 2 min for 30 cycles;
and 72°C for 5 min. PCR products were resolved by electrophoresis of
5 µl of the reaction mixture in a 1 or 1.5% (wt/vol) agarose minigel
in Tris-acetate-EDTA (TAE) buffer for AMO or CTO primers, respectively.
DGGE.
Following the nested PCR, CTO products were resolved
on double-density DGGE gels (8) using a D-gene system
(Bio-Rad Laboratories, Hertfordshire, United Kingdom). Polyacrylamide
gels (6 to 12% polyacrylamide; 1.5 mm thick; TAE; 37:1
acrylamide-bisacrylamide; 35 to 50% denaturant; 20 by 20 cm) were
poured using a gradient maker (Bio-Rad). A 5-ml stacking gel (8%
acrylamide, 0% denaturant) was added to the top of the denaturing gel,
and a 25-well comb was inserted, allowing between 5 and 15 µl of PCR
product to be loaded onto each gel. Control clusters from the database
of Stephen et al. (40) were included. The gels were run for
5.5 h at 200 V and 65°C. Migration patterns were visualized by
staining with 1 mg of ethidium bromide ml1 in TAE for 15 min followed by rinsing for 10 min in TAE or by silver staining.
Electroblotting of DGGE gels. Ethidium bromide-stained gels were electroblotted onto Hybond N+ nylon membranes (Amersham International plc, Little Chalfont, Buckinghamshire, United Kingdom) using an electroblotter (HEP-1; Owl Scientific, Woburn, Mass.). Gels were trimmed to size and electroblotted for 1.5 h at 200 mA with TAE buffer according to the membrane manufacturer's instructions. Efficiency of transfer was checked by restaining the gels with ethidium bromide. Membranes were stored dry at 4°C prior to oligonucleotide probing.
Oligonucleotide probing.
Membranes were probed with a
selection of the ammonia oxidizer-specific probes of Stephen et al.
(39). Probes -AO233, Nsp436, and Nmo254, which recognize
all ammonia oxidizer, all Nitrosospira, and all
Nitrosomonas sequences, respectively, were used in
conjunction with the cluster-specific probes (NspCL2_458, NspCL3_454,
and NspCL4_446) for Nitrosospira clusters 2, 3, and 4, respectively (39). Each probe (20 pmol) was end labeled
using T4 polynucleotide kinase (Promega) and 20 µCi of
[-32P]ATP (3,000 Ci mmol1; Amersham) in
a 10-µl final volume.
DGGE band sequencing.
The middle portion of each selected
DGGE band was excised for sequence analysis and placed in a 500-µl
Eppendorf tube. The acrylamide was crushed using a sterile pipette tip,
10 µl of sterile MilliQ water was added to each tube, and the sample
was incubated at 4°C overnight. Acrylamide was removed by
centrifugation at 13,000 × g for 5 min, and PCR was
carried out using the CTO primers as described previously. Products
were cleaned and concentrated with Microcon 100 filter units (Amicon
Inc., Bedford, Mass.) by rinsing several times with sterile MilliQ
water. Products were quantified and checked for purity on 1% (wt/vol)
agarose gels prepared in TAE buffer using a mass ladder (Life
Technologies, Paisley, United Kingdom). Sequence analysis was carried
out on both strands using the CTO forward primer (without the GC clamp) and the 537r internal 16S rDNA sequencing primer (10) with
an automated sequencer. Sequence data were assembled and checked by
using the Chromas 1.42 program (C. McCarthy, Griffith University, Brisbane, Queensland, Australia) before analysis using the Genetic Database Environment running in ARB. Phylogenetic analysis was carried
out by aligning the partial 16S rDNA sequences from clones and the
sequences of ammonia oxidizers and other -proteobacteria contained
in the ribosomal database project (22). Trees were generated
from a 276-bp region of the 5' region of the 16S rDNA using the
Jukes-Cantor (18) correction and neighbor joining (37) with PHYLIP version 3.1 software (12) in ARB.
cPCR.
cPCR was carried out on all of the soils using the
COMP1 internal standard and conditions reported by Phillips et al.
(33). The calibration curve was prepared by the addition of
107 Nitrosomonas europaea cells to 5 g of
gamma-irradiated soil, and the DNA was extracted as described above.
The calibration series was prepared from a 10-fold dilution series of
DNA amplified with 7 pg of COMP1. Products were run on 2% (wt/vol)
agarose gels in TAE buffer at 40 mV for 45 min. The gels then were
stained in 1 mg of ethidium bromide solution ml1 for 30 min at room temperature and destained in TAE for 10 min. UV gel images
were captured by using the Imager System Amphigene, Illkirch, France)
and were quantified by densitometry using Molecular Analyst software
(Bio-Rad).
Nucleotide sequence accession numbers. All sequences were deposited in GenBank under accession numbers AF157707 to AF157741.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Potential nitrification.
Potential nitrification rates were
determined in October 1996 for all plots in treatments 1, 2, and 7. There was a marked tillage effect in cultivated plots, with potential
nitrification values in treatment 2 (no tilling) almost twice those in
treatment 1 (P = 0.03) (Fig.
1). However, in successional grasslands
(treatment 7), there was no significant difference between tilled and
nontilled plots. Fertilization did not affect the potential
nitrification activities of cultivated soils (P = 0.73)
or successional grasslands (P = 0.15) (Fig. 1). Samples
from poplar plots (treatment 5) and NDF were taken in November 1996. Potential activities in these sites were lower than those in cultivated
and successional soils; the lowest rate, 2.5 nM g of
soil1 h1, was found in the poplar plots.
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Ammonia oxidizer population size.
Cell concentrations of
ammonia oxidizers from these different communities were estimated by
two methods, conventional MPN counts and cPCR. MPN analysis was carried
out at three ammonia concentrations to allow quantification of groups
of ammonia oxidizers with different sensitivities to ammonia. In
cultivated soils (treatments 1 and 2), MPN counts determined with
medium containing the highest ammonium concentration, 1,000 µg of
NH4+-N ml1, were between 1 and 2 orders of magnitude lower than those determined with 5 µg of
NH4+-N ml1, while counts
determined with 50 µg of NH4+-N
ml1 were intermediate (Fig.
2). MPN counts from successional
grasslands (treatment 7) showed similar patterns, with the lowest
numbers from counts determined with medium containing the highest
ammonium concentration, although the differences between counts
determined with 5 and 50 µg of NH4+-N
ml1 were less marked. The effect of ammonium
concentration was greatest in NDF, which showed counts of 4 cells g of
soil1 when the highest ammonium concentration was used
and 21 × 104 and 20 × 104 cells g
of soil1 when intermediate and low concentrations were
used, respectively. Both tillage and fertilization increased MPN counts
in successional soils (P = 0.0175 and P = 0.062, respectively). In contrast to the results obtained for the
other experimental plots, the lowest counts were obtained from medium
containing 5 µg of NH4+-N ml1
for the poplar plots (treatment 5). There were no significant differences in MPN counts from cultivated and native soils at an
intermediate ammonia concentration (P = 0.431), but
counts obtained with the highest and lowest concentrations indicated higher numbers in cultivated soils (P = 0.065 and
P = 0.0005, respectively).
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1. The
numbers of ammonia oxidizers could not be determined for NDF soils, as
nested PCR was required for the amplification of ammonia oxidizer DNA,
probably due to the higher organic content in these soils. In all other
situations, except for treatment 5, cPCR counts were significantly
higher than MPN counts with medium containing 5 µg of
NH4+-N ml1. Differences ranged
from 0.3 to 2.5 orders of magnitude. For treatment 5, a single MPN
count, determined with an intermediate ammonium concentration, was
higher than the equivalent cPCR count. Both MPN and cPCR counts
increased with tillage and fertilization in successional soils. In
cultivated soils, cPCR counts, in contrast to MPN counts, increased
significantly with fertilization but were not affected by tillage. The
numbers of ammonia oxidizers determined by cPCR ranged from 1.5 × 104 to 1.1 × 107 cells g of
soil1; in comparison, total bacterial cell numbers ranged
from 5.8 × 109 to 8.5 × 109 cells g
of soil1. Thus, ammonia oxidizers likely constitute a
maximum of approximately 0.01% the total bacterial population in these soils.
DGGE analysis and oligonucleotide probing.
The composition of
ammonia oxidizer communities from the sites tested here was
characterized by DGGE analysis of ammonia oxidizer 16S rDNA partial
sequences amplified from extracted DNA using a nested PCR approach.
Banding patterns of ethidium bromide-stained DGGE gels were similar for
all plots, regardless of plant community or fertilization or tillage
treatments (Fig. 3). Banding patterns were reproducible when gels were run on several occasions and from
different sets of PCRs. The DGGE patterns from all sites included a
group of slowly migrating bands (band C) that appeared to comigrate
with the cluster 2 and cluster 3 Nitrosospira controls and a
group of faster migrating bands (band E) that comigrated with the
controls for cluster 4 Nitrosospira. Between two and three
bands appeared in each group, due to small variations in the denaturing
gradients. DGGE banding patterns of PCR products from these soils did
not include any indication of representatives of
Nitrosomonas.
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-proteobacterial ammonia
oxidizer 16S rDNA partial sequences amplified from DNA extracted from
the highest dilutions showing positive results for MPN counts. PCR
amplification and DGGE analysis were not successful for all MPN samples
obtained. The inability to obtain PCR products was not related to soil
treatments and might have resulted from difficulties in removing
sufficient material from the wells of microtiter plates, particularly
where evaporation was significant. Although no sequence representative
of the Nitrosomonas clade was detected in the DNA extracted
directly from soil samples, banding patterns typical of
Nitrosomonas were detected in DGGE gels of DNA amplified
from the MPN samples after incubation for 1 month. Representative DGGE banding profiles from MPN samples of cultivated and successional soils
with 5, 50, and 1,000 µg of NH4+-N
ml1 are illustrated in Fig.
5. In many samples, banding patterns were
similar to those obtained from DNA extracted directly from the soil,
but in several samples, a band typical of Nitrosomonas was
observed (for example, Fig. 5, lanes 2, 3, 5, 9, and 11). This result
was particularly evident for samples from cultures obtained with medium
containing 1,000 µg of NH4+-N
ml1, where a Nitrosomonas band frequently
appeared to the exclusion of the Nitrosospira bands (Fig. 5,
lanes 14, 15, 16, and 17).
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Sequence analysis.
The presence in soil and MPN cultures of
particular clusters of -proteobacterial ammonia-oxidizing bacteria
was confirmed by sequencing of bands excised randomly from DGGE gels.
Phylogenetic analysis (Fig. 6 and
7) showed
that all of the bands fell within the known -proteobacterial ammonia
oxidizer groupings described by Stephen et al. (40). All
bands sequenced that were representatives of the
Nitrosospira grouping belonged to cluster 3. However, bands excised from unusual banding profiles obtained from MPN cultures of
samples from the NDF and from poplar plots (treatment 5) were representative of cluster 4 Nitrosospira. The
Nitrosomonas bands detected in the MPN samples were closely
related to N. europaea. One sequence from the MPN
samples was found to lie within cluster 6 and was closely related to
another sequence, KZOO_D27, that was also isolated from this location
(5). Within the cluster 3 grouping, sequences from the NDF
samples clustered together, suggesting that there might be a treatment
effect within cluster 3. Some sequences from the slowly (band C) and
quickly (band E) migrating bands were different by only 1 bp over the
290 bp used for phylogenetic analysis; however, despite this fact, they
consistently migrated at different rates in gels. This mismatch was in
the middle of the sequence, but single mismatches within the primer region due to an ambiguous base led to closely migrating bands (19).
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tillage
(T), no tillage (NT), fertilization (F), and no fertilization (NF)
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Abundance estimates. This study used conventional and molecular techniques to assess the relationship among the abundance, activity, and diversity of ammonia oxidizer populations in soils. The communities reflected treatments ranging from intensive cultivation to NDF. Estimated concentrations of ammonia-oxidizing bacteria were dependent on the enumeration method and protocol. With the exception of the poplar plot, the use of higher concentrations of ammonia in the growth media significantly reduced MPN counts. Similar results have been reported by other workers (3, 41) and may result from growth inhibition of the ammonia oxidizers at high ammonia concentrations (41, 42). Ammonia oxidizer cell concentrations obtained by cPCR were 10- to 1,000-fold higher than MPN counts at these sites, with the exception of the poplar soil. The anomalous results found for this treatment may have been due to differences in the cover crop and potential consequent changes in the activities of different groups of ammonia oxidizers in this soil. DeGrange and Bardin (9) also found that the numbers of bacteria calculated by MPN-PCR counts were 100 times higher than those calculated by traditional MPN counts in a sandy calcareous soil, whereas the difference was only 10-fold in a sandy loam soil.
Detection limits for cPCR methods were observed to be between 10 and 1,000 times lower than those for standard dilution plating methods when a genetically modified strain of the fungus Trichoderma virens in soil was investigated (2). The differences might also reflect limitations of laboratory growth media and incubation conditions, which do not support the growth of all culturable organisms within natural populations and which will not detect nonculturable cells. Populations with lag periods longer than the incubation period also will not be detected, and Matulewich et al. (23) found increasing MPN counts of nitrifying bacteria even after incubation for 90 days. Belser and Schmidt (3) showed that the use of different media for MPN enumeration of ammonia oxidizers produced different results in an actively nitrifying soil. They also found dominance by Nitrosomonas in media inoculated with lower sample dilutions and by Nitrosospira at higher dilutions. In our study, comparison of 16S rDNA partial sequences amplified from DNA extracted directly from the soil and from positive MPN cultures indicated a similar shift in composition. Samples from the MPN cultures were dominated by sequences representative of Nitrosomonas, which were not detected in soil DNA extracts, while Nitrosospira-like sequences, which dominated in soil DNA extracts, were less frequent in MPN cultures and sometimes were not detected. Selection for Nitrosomonas was greatest in MPN counts when 1,000 µg of NH4+-N ml1 was used. Hiorns et al. (14) have detected
Nitrosomonas DNA in lake water and sediment enrichments but
not in extracted DNA, supporting the belief that
Nitrosomonas-like organisms are better adapted to growth on
laboratory media (3).
Compositional differences in ammonia oxidizers.
Despite the
significant differences in potential nitrification rates among these
communities, ammonia oxidizers were found to constitute a relatively
small proportion of the total bacterial population detected by
microscopic DTAF staining. MPN estimates were 6 to 8 orders of
magnitude lower than total cell counts. cPCR may provide a more
accurate estimate of total cell counts; in this study, cPCR
indicated that -proteobacterial ammonia oxidizers constituted a
maximum of 0.01% of the total population. This low relative abundance
in soil may explain the lack of detection of ammonia oxidizer sequences
in clone libraries generated by amplification of 16S rDNA using
eubacterial primers (17, 21, 25, 28). Borneman et al.
(4) found that the majority of the -proteobacterial clones from a Wisconsin soil showed 80% homology to the ammonia oxidizers. Our data indicate that the characterization of several thousand eubacterial clones would be necessary for the detection of
ammonia oxidizers, even in agricultural soils, and that detection by DGGE analysis of eubacterial PCR products would be unlikely.
-proteobacterial ammonia oxidizers. Soils from all sites were
dominated by members of Nitrosospira cluster 3, which are
commonly found in soil (5, 40) and which contain the
majority of cultured representatives of the genus Nitrosospira. Bruns et al. (5) did not detect
Nitrosospira cluster 3 in native and unfertilized soils, but
we found no effects of fertilization or cultivation on community
structure, and Nitrosospira cluster 3 dominated in all soils
sampled. Sequence analysis of DGGE bands indicated that for different
soils, there was a clustering of sequences within cluster 3. This
result was particularly evident for deciduous forest soils, although
the conclusions drawn must be considered tentative given the small
number of sequences analyzed. The stability of other components of the
microbial community in these soils has been reported by Buckley et al.
(6), who found no differences in Crenarchaeota
sequences in cultivated and native soils. However, significant
differences were seen in a comparison of two Norwegian agricultural
soils for total bacterial diversity (29).
Relating structure and function.
There are several
explanations for the lack of correlation between -proteobacterial
ammonia oxidizer population structure and nitrification rates. The
treatments imposed, i.e., tillage and fertilizer, may not drive ammonia
oxidizer community structure, which may be more dependent on soil
properties, which were initially the same for all treatments. The
already established populations survived in systems that lowered the
available NH4+ substrate levels, and substrate
additions would be required to bring in new populations. On a
phylogenetic level, it has been suggested that two sequences showing up
to a 0.3% difference in sequence homology in the 16S rDNA gene could
represent two species with different ecological functions
(43). Pankhurst et al. (30) suggested that there
does not need to be great taxonomic diversity for there to be
functional diversity in soils. In this study, differences seen in the
sequences of cluster 3 may mean that, although the organisms are very
closely related phylogenetically, they are in fact physiologically
different, leading to the differences in the nitrification rates
observed between treatments.
-proteobacterial
ammonia oxidizers but, in combination with CTO primers, amplify all
known sequences representative of this group. Although primer
bias cannot be dismissed, similar findings have been reported with either set of primers for amplification of 16S rDNA sequences from
the same soils and marine sediments (25, 39, 40). The possibility that ammonia oxidizers in natural communities have sequences that are not amplified by these primers cannot be excluded.
This study has demonstrated that the structures of -proteobacterial
ammonia oxidizer populations were quite similar in soils collected from
a wide range of communities under different soil cultivation
conditions, which resulted in significant changes in potential rates of
nitrification and in the sizes of ammonia oxidizer populations.
Community structure was assessed at the level of precision provided by
analysis of clusters characterized by 16S rDNA sequences and indicated
dominance by Nitrosospira cluster 3. Further studies are
required to determine whether subtle changes occur within this cluster
or whether stability under a variety of environmental conditions is due
to physiological and functional diversity within the populations.
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ACKNOWLEDGMENTS |
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This project was funded by NSF grants to the Center for Microbial Ecology (DEB912006) and to KBS LTER (DEB8702332).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Phone: 44 1224 273148. Fax: 44 1224 273144. E-mail: j.prosser{at}abdn.ac.uk.
Present address: NCIMB Ltd., Aberdeen AB24 3RY, United Kingdom.
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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, December 2000, p. 5410-5418, Vol. 66, No. 12
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