George E. Brown Jr. Salinity Laboratory, USDA
Agricultural Research Service, Riverside, California
92507,1 and Department of
Environmental Sciences, University of California, Riverside, California
925212
Agricultural soils are typically fumigated to provide effective
control of nematodes, soilborne pathogens, and weeds in preparation for
planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel
electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments
amplified directly from soil DNA) approaches. Changes in soil
microbial community structure were examined in a microcosm experiment
following the application of methyl bromide (MeBr), methyl
isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin.
Variations among Biolog fingerprints showed that the effect of MeBr on
heterotrophic microbial activities was most severe in the first week
and that thereafter the effects of MeBr and the other fumigants were
expressed at much lower levels. The results of PLFA analysis
demonstrated a community shift in all treatments to a community
dominated by gram-positive bacterial biomass. Different 16S rDNA
profiles from fumigated soils were quantified by analyzing the DGGE
band patterns. The Shannon-Weaver index of diversity,
H, was calculated for each fumigated soil sample. High
diversity indices were maintained between the control soil and the
fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H
increased to 0.73 in the MeBr-treated samples. Sequence analysis of
clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were
dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much
higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects
of fumigants on soil community composition and structure. Our results
indicate that MeBr has the greatest impact on soil microbial
communities and that 1,3-D has the least impact.
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INTRODUCTION |
Application of methyl bromide (MeBr)
to agricultural soils before planting of high-value cash crops has been
the mainstay for the control of nematodes, soilborne pathogens, and
weeds for many years in warm regions of the United States. The
chemistry and air pollution potential of this fumigant have been
documented (2, 7, 8). It has also been shown that MeBr may
have the ability to destroy stratospheric ozone (25, 43),
and a ban on its production in and importation into the United States is scheduled to be fully implemented by 2005 (35).
1,3-Dichloropropene (1,3-D), methyl isothiocyanate (MITC), and
chloropicrin (CP) have been proposed as most likely to be chemical
alternatives to MeBr. While most of these fumigants are known to have
broad biocidal activity (1), their effects on soil
microbial communities are largely unknown. Recently, the effect of MITC
(the toxic degradation product of metam sodium) on soil microbial
community structure and function was studied by the use of traditional
heterotrophic activity measures, catabolic potential, and biochemical
assay (18). Those authors showed that abundances of
indicator fatty acids for bacteria after 5 weeks of incubation were
correlated to MITC doses but that after 18 weeks very few were related
to MITC dose. MITC was also observed to reduce populations of
culturable organisms dramatically in the Biolog assay.
Garland and Mills (11) adapted the Biolog redox technology
based on community-level carbon source utilization patterns to characterize and classify microbial communities from environmental (soil, aquatic, and rhizosphere) samples. In a comparative study of
rhizosphere bacterial communities and hydrocarbon-polluted environments, Garland and Mills (12) and Wünsche et
al. (41) showed that substrate utilization patterns can be
used as an indicator of community structure and function. Although the
Biolog assay has been proposed as a measure of functional diversity
(44), assay responses are attributed mainly to a small
subset of heterotrophic bacteria in the soil.
White and Findlay (37) developed a community-level
approach to characterize microbial community structure by evaluating shifts in phospholipid fatty acids (PFLA) from environmental samples. Different groups of bacteria are characterized by specific PLFA profiles; therefore, a change in the phospholipid pattern in soil would
indicate a change in the bacterial composition of that soil. This
concept has resulted in the identification and quantification of viable
biomass and community structure in sediments (3, 27, 29)
and in agricultural soils (45, 46).
Analysis of cloned ribosomal gene sequences retrieved from the
environment can provide detailed information about the community composition and the structural diversity of the environment without bias. To monitor the structural diversity of microbial communities, denaturing gradient gel electrophoresis (DGGE) was introduced by Muyzer
et al. (21). This technique is based on the separation of
ribosomal gene sequences directly amplified from community DNA by using
conserved primers on a denaturing gel according to their melting
properties. This allows direct comparison of many samples with
different treatments (19). Our objectives were to monitor
the biocidal effects of these compounds and compare their
ecotoxicological effects on a soil microbial community in response to
the application of fumigants.
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MATERIALS AND METHODS |
Soil type and experimental design.
Soil samples (Arlington
sandy loam) were taken from the top 15 cm in a fallow field at the
University of California Riverside Agricultural Experiment Station.
Soil samples were collected with a 10-cm-diameter stainless-steel auger
that was washed with diluted methanol between samplings to avoid lipid
contamination. There has been no history of fumigant treatment on this
plot. The soil had a pH of 7.2 and an organic carbon content of 0.92%.
Moist field soil was passed through a 4-mm sieve, and the water content of the original soil was determined and readjusted to 12%. Soil samples were stored at room temperature for 48 h before being used
in the experiment.
Soil samples were placed in microcosms containing about 1.5 kg (dry
weight) of soil. The experimental design consisted of four fumigants at
three different concentrations and a control in three replicate
microcosms (Table 1). Fumigants were
added as freshly prepared aqueous solutions. Table 1 shows the test ranges used in the different microcosms. Microcosms were sealed for
24 h after fumigant application and were vented continuously through a small opening on the cover. Samples for community analysis were taken at 1, 8, and 12 weeks after fumigant treatment.
Substrate utilization patterns of microbial populations
determined using the Biolog system.
Biolog GN (gram-negative)
microtiter plates were used to analyze functional diversity through the
substrate utilization patterns shown by soil microorganisms. The Biolog
GN plates contained 95 separate carbon sources and a blank well with no
substrate. In order to obtain substrate utilization patterns of whole
soil communities, the cell suspensions were prepared by extracting the
soils with phosphate buffer solution, serially diluting, and adding a
150-µl suspension of a 10
3 dilution to the
Biolog plates with an eight-channel repeating pipette. Plates were
inoculated with three replicates of soil extract and incubated at
25°C, and absorbance data were recorded at 595 nm at 0, 24, 48, 72, and 96 h with a Bio-Rad (Richmond, Calif.) Plate Reader. The
patterns of sole carbon source utilization in Biolog plates were
expressed as an index of color development in each of the wells as
reported by Wünsche et al. (41) and modified by
Ibekwe and Kennedy (16). The index was obtained by
subtracting the color response reading in the spectrophotometer for
each of the 95 wells from that for the control well and then dividing
by the reading for the control well according to the formula
WE = (WA 
W0/W0)100,
where WA is the absorbance in each well
from well A2 to well H12
and W0 is the absorbance in the blank
well (no color development). A WE value of
greater than 100 was regarded as indicating a positive reaction
(evidence of substrate utilization), and a
WE value of less than 100 was regarded as
indicating a negative reaction. The variables were coded as binary
values (1 for a positive reaction and 0 for a negative reaction). This
approach was used to eliminate the problem of inoculum cell density and
produced a weighted data set that could be used in principal-component
analysis (PCA). PCA was used to reduce the number of variables (95 variables) to the number of principal components (PCs) that explain
80% or more of the variance (six PCs in this study). Since we used the
correlation matrix to compute variables (substrates), all substrates
with eigenvalues of greater than 1 were used in our analysis. We
computed the correlation between the PCs and the treatments to examine
the effects of substrates.
Phospholipid extraction and separation.
Triplicate soil
samples (5 g from each microcosm) were extracted by using the modified
method of Bligh and Dyer (38) as described by Petersen and
Klug (24). The total lipid extract was fractionated into
glycolipids, neutral lipids, and polar lipids (13, 19).
The polar lipid fraction was transesterified with mild alkali to
recover the PLFA as methyl esters in hexane. The PLFA were separated,
quantified, and identified by gas chromatography-flame ionization
detection (19). Samples were run for 38 min, which is long
enough for fatty acids with up to 28 carbons to elute from the column.
The system consisted of a gas chromatograph (HP6980; Hewlett-Packard,
Wilmington, Del.) with a flame ionization detector and HP3365
ChemStation software.
Fatty acid nomenclature.
The suffixes c for cis
and t for trans refer to geometric isomers. The prefixes i,
a, and me refer to isomethyl, anteisomethyl, and mid-chain
methyl branching, respectively, with cyclopropyl rings indicated by cy
(17).
DNA extraction, PCR primers, and DGGE analysis.
Total
bacterial community DNA was extracted to assess the effects of
fumigants on bacterial community diversity. DNA was extracted by
placing 500 mg of soil in FastPrep tubes (Bio 101, Vista, Calif.) containing lysing matrix and shaken for 30 s. Isolation of total DNA was accomplished with a FastPrep DNA isolation kit according to the
protocols of the manufacturer (Bio 101).
PCR was performed using 20 to 80 ng of template DNA with primers
PRBA338f and PRUN518r, located at the V3 region of the 16S rRNA genes
of bacterioplankton (23). PRBA338f consists of a region
that is conserved among the domain Bacteria, and PRUN518r is
located at a universal conserved region. PCR mixtures contained Ready-To-Go PCR beads from Amersham-Pharmacia Biotech (Piscataway, N.J.), 10 pmol of each primer, 4 µg of bovine serum albumin, template DNA, and sterile distilled water in a final volume of 25 µl. PCR conditions were 92°C for 2 min, followed by 30 cycles of 92°C for 1 min, 55°C for 30 s, and 72°C for 1 min and a single final extension at 72°C for 6 min.
DGGE was performed with 8% (wt/vol) acrylamide gels containing a
linear chemical gradient ranging from 30 to 70% denaturant, with 100%
defined as 7 M urea and 40% formamide. Gels were run for 3 h at
200 V with a Dcode Universal Mutation System (Bio-Rad). DNA was
visualized after ethidium bromide staining by UV transillumination and
photographed with a Polaroid camera. Major bands were excised for
identification of bacterial species. Bands were placed into sterilized
vials with 20 µl of sterilized distilled water and stored overnight
at 4°C to allow the DNA to passively diffuse out of the gel strips.
Ten microliters of eluted ribosomal DNA (rDNA) was used as the DNA
template with eubacterial primers. The sizes of the PCR products were
checked on a 1.5% agarose gel, and the DNA was cloned into a pGEM-T
Easy vector (Promega, Madison, Wis.) and transformed into
Escherichia coli JM109. Isolation of plasmids from E. coli was performed using standard protocols from the Qiagen
(Valencia, Calif.) plasmid minikit. The purified plasmids were
sequenced with the ABI PRISM Dye Terminator Cycle Sequencing Kit with
AmpliTaq DNA polymerase, FS (Perkin-Elmer).
Statistical analysis of PLFA profiles and DGGE bands.
Data
analysis of PLFA profiles was performed using SAS (30).
Analyses of variances, means, and standard deviations for the individual fatty acids in triplicate-sample PLFA profiles were determined to compare the moles percent of each PLFA in each sample. Correspondence analysis was used to compare PLFA profiles among sampling times. The mean moles percent data were presented as cluster
analysis or a two-dimensional plot for better understanding of
relationships. Minitab statistical software (version 13) was used to
cluster observations into groups.
DNA fingerprints obtained from the 16S rDNA banding patterns on the
DGGE gels were photographed and digitized using ImageMaster Labscan
(Amersham-Pharmacia Biotech, Uppsala, Sweden). The lanes were
normalized to contain the same amount of total signal after background
subtraction. The gel images were straightened and aligned using
ImageMaster 1D Elite 3.01 (Amersham-Pharmacia Biotech, Uppsala, Sweden)
and analyzed to give a densitometric curve for each gel. Band positions
were converted to Rf values between 0 and
1, and profile similarity was calculated by determining Dice's
coefficient for the total number of lane patterns. Dendrograms were
constructed by using the unweighted pair group method with mathematical
averages (UPGMA). Dice's similarity coefficients were generated,
converted into x-y line plots, and transferred to Excel
files. Community similarities based on peak areas from the Excel files
for the different bacterial groups (16S rDNA bands) were analyzed by
correspondence analysis (CANACO 4.0; Microcomputer Power, Ithaca, N.Y.)
to generate an ordination diagram as described by Yang and Crowley
(42). Dice's similarity coefficients generated from the
three sampling points were integrated and analyzed using ImageMaster 1D
database 2.01 (Amersham-Pharmacia Biotech, Uppsala, Sweden). The data
obtained were used for the construction of a library to determine the
best-fit profile and to integrate the area under each peak for every
gel and for the construction of a dendrogram between treatments. For analysis of diversity, each band was presumed to represent the ability
of that bacterial species to be amplified. The Shannon-Weaver index of
diversity (H) was used to compare changes in diversity of
microbial communities within the four treatments at each time (31) by using the function H = (Pi log
Pi), where Pi = ni/N, ni is the peak height, and N is
the sum of all peak heights in the curve.
Phylogenetic analysis.
Sequence identification was performed
by using the BLAST database (National Center for Biotechnology
Information [www.ncbi.nlm.nih.gov]) and the Sequence
Match Facility of the Ribosomal Database Project (www.cme.msu.edu/RDP).
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RESULTS AND DISCUSSION |
Analysis of heterotrophic microbial communities by using
Biolog GN microplates.
The results of the PCA performed on the
mean values of the Biolog GN fingerprints of the different soil
microbial communities obtained after 72 h of incubation are shown
in Fig. 1. The functional abilities of
the heterotrophic soil microbial communities were altered by the
application of the fumigants, especially MeBr, during the first week of
the experiment. The PCA plot (Fig. 1) for the four microbial
communities and the control showed that the first component accounted
for 28% of the variance, while the second component accounted
for 16% and the third component accounted for 14%, with six PCs
accounting for over 80% of the variation. The control soil and the
soil treated with 1,3-D were separated along PC1, and their
coefficients were positively correlated to the right of PC1. Analysis
of MeBr-treated communities did not show any pattern of
groupings except that communities from the first week of treatments
were positively correlated along PC2 and grouped with MITC after weeks
8 and 12. Pairwise comparison showed that the MeBr-treated communities
were significantly different (P < 0.05) from the
control and 1,3-D-treated communities.
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FIG. 1.
PCA performed on Biolog GN fingerprints of soil extracts
treated with MeBr, MITC, 1,3-D, and CP and of nonfumigated soil (C).
Numbers after the abbreviations indicate week 1, 8, or 12, and those
after the hyphen indicate concentrations of fumigants used in the
experiment as listed in Table 1. Since most of the 1,3-D samples
clustered together along PC1, they are not differentiated by time and
concentrations.
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The Biolog assay showed that functional characteristics of the control
and 1,3-D treatments were similar when compared to those for the other
three fumigants. The problem with the Biolog system is that color
development in the Biolog plates is due first to the respiratory
activities of fast-growing heterotrophic bacteria, resulting in the
stimulation or reduction of the catabolism of 95 carbon substrates
(6). The shifts in microbial communities observed in the
Biolog assays are due to organisms that grow rapidly because of their
high population in a sample. For example, Pseudomonas species, which are found in most of the samples in this study, respond
well in Biolog assays (9, 10, 14). Analysis of the
microbial communities of the Biolog GN microplates by DGGE has been
shown to confirm that carbon source utilization profiles obtained with
Biolog GN plates do not necessarily discriminate the numerically
dominant members of the microbial community used as the inoculum
(6, 32).
PLFA analysis.
Analysis of PLFA profiles for all four
treatments from weeks 1, 8, and 12 was carried out in triplicate. The
content of individual biomarker peaks ranged from a minimum of 1.3 nmol
per g (dry weight) for the four fumigants in week 1 to a maximum of 55 nmol per g (dry weight) for the 1,3-D- and CP-treated samples in week
12 (Fig. 2). Biomass
contents as indicated by total PLFA were significantly different at different time points for some treatments
(P < 0.05). At week 1, biomass contents in
MeBr-amended microcosms were significantly lower than those at weeks 8 and 12 (Fig. 2A) and were also lower than those for microcosms amended
with the three other fumigants (Fig. 2B, C, and D). There was
also a decrease in biomass of some gram-negative biomarkers (cy17:0,
15:0, and 18:1
7c) and fungal biomarkers (18:26c) with the
increase in MeBr concentration during the first week of the experiment
(Fig. 2A). However, there was a significant increase in biomass for
gram-positive bacteria (a17:0 and i17:0), fungi (18:26c), and
actinomycetes (10me16:0) in weeks 8 and 12. The effects of MITC
followed the same trend as those of MeBr, except that the recovery of
gram-negative bacterial biomass did not occur during week 8 (Fig. 2B).
1,3-D and CP had the strongest effects on actinomycetes, resulting in a
significant decrease in biomass for most treatments (Fig. 2C and D).
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FIG. 2.
Biomass contents (nanomoles of PLFA per gram [dry
weight] of soil) of samples collected after weeks 1, 8, and 12 from
MeBr (A)-, MITC (B)-, 1,3-D (C)-, and CP (D)-fumigated soils
(n = 3). Numbers after the abbreviations indicate
times and concentrations as described in the legend to Fig. 1. Error
bars represent standard deviations.
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Analysis of microbial community structure by PLFA.
The
community structures of fumigant-treated microcosms from weeks 8 and
12, measured by PLFA analysis, shifted away from those during the first
week of sampling. The shift was greatest with MeBr, which showed a
47.5% variation in component one and a 26.8% variation in component
two (Fig. 3A). The major difference in
the PLFA profiles between the MeBr-treated and the control microcosms
was that the MeBr-treated microbial communities contained significantly
more branched-chain PLFA (specifically, a17:0, i17:0, a15:0, and i15:0
[P < 0.05]), indicative of gram-positive bacteria (15, 34, 37). For all microcosms treated with MeBr, the relative proportion of PLFA indicative of fungal biomass
(13), specifically, 18:26c and 18: 36c, increased
over time. Correspondence analysis of the PLFA profiles showed similar
results, with all of the samples in the week 12 treatment forming a
distinct cluster (Fig. 3A). Analyses comparing PLFA profiles of MITC-,
1,3-D-, and CP-treated microcosms to those of the control samples
consistently revealed the same patterns. At week 1, the profiles were
furthest away from the control; at weeks 8 and 12, PLFA profiles of
samples treated with the three fumigants more closely resembled each
other and that of the control sample (Fig. 3B, C, and D).
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FIG. 3.
Scatter plots of the results from correspondence
analysis of the PLFA contents described in Fig. 2. Con, control.
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Cluster analysis of the PLFA profiles (complete squared Euclidean
distance with mean moles percent data) showed the major trends within
the four treatments during 12 weeks of the study (Fig.
4). MeBr-amended microcosm samples from
week 1 clustered according to time and concentrations. 1,3-D- and
CP-treated samples did not show any pattern of clustering throughout
the 12 weeks of the study. In fact, for all of the fumigants except
MeBr, bacteria recovered from the initial effects after 12 weeks
and clustered closer to the control.
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FIG. 4.
Cluster analysis of the PLFA contents (complete squared
Euclidean distance with moles percent data) described in Fig. 3.
Numbers after the abbreviations indicate times and concentrations as
described in the legend to Fig. 1. Cont, control.
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Advantages of PLFA analysis, in comparison to other techniques, are
that PLFA analysis has been regarded as an indicator of total microbial
biomass (37, 45) and that certain PLFA can be used as
biomarkers for specific groups of microorganisms (34, 39).
The presence of large proportions of branched-chain fatty acids (a15:0,
i15:0, a17:0, and i17:0), which are markers for gram-positive bacteria
(28), suggests that gram-positive bacteria were less
affected by the impact of these fumigants than gram-negative bacteria.
This is in agreement with the work of Zelles et al. (47),
who found that gram-positive bacteria were less injured by chloroform
fumigation; they attributed this to protection by the cell wall
structure of the bacteria, formation of spores, and ability to adapt to
fumigant vapor more quickly.
Analysis of soil microbial community structure by PCR-DGGE.
DGGE analysis of 16S rDNA fragments was used to examine the effects of
the four fumigants and the control on soil microbial communities.
Figure 5 shows cluster analysis
of DGGE patterns of the 16S rDNA fragments (primers
P338f and P518r) amplified from the four fumigated soils and control
soil at 1, 8, and 12 weeks after fumigation. The most drastic effect
occurred in the first week of the experiment. During this period, the
MeBr treatments clustered away (97% similarity index) from the
treatments with the other three fumigants and the control (Fig. 5A).
This observation was confirmed by the DGGE banding pattern in Fig. 5A,
which shows no dominant bands for MeBr-treated samples during the first
week of the experiment. At week 8 there was a significant shift in microbial community structure. The microcosm that was treated with the
highest concentration of MeBr clustered away from the other two
treatments, the three fumigants, and the control. As shown in Fig. 5B,
more bands, which did not occur during the first week after fumigation,
began to appear in the MeBr treatments. There was also a decrease in
the number of bands as the concentration of MeBr increased (MeBr8-1 to
-8-3). At 12 weeks, the microbial communities for all concentrations of
MITC, 1,3-D, and CP and the lowest concentrations of MeBr were similar
to those for the control (Fig. 5C).
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FIG. 5.
DGGE analysis of 16S rDNA fragments of pooled soil
samples, at different times after fumigation, collected from triple
microcosms treated with different fumigants. Amplified products were
separated on a gradient gel of 30 to 70% denaturant. All labeled bands
were excised from the gel, reamplified, and subjected to sequence
analysis. These reamplification products were cloned and screened as
described in the text. (A) Community structures at week 1, 7 days after
the initiation of the experiment. Significant differences between the
community structures of microcosms treated with MeBr, the highest
concentration of MITC, and control treatment had been induced at this
time, because all of the major bands in the MeBr samples were
undetected, while the samples treated with the other fumigants and the
control treatment continued to maintain complex banding patterns. (B)
Community structures after 8 weeks of treatment. Most of the samples
treated with the other fumigants continued to maintain complex banding
patterns, while new community structures emerged with the MeBr-treated
samples. (C) Community structures after 12 weeks of treatment. The
banding patterns in the MeBr-treated samples continued to emerge, and
the communities reverted almost completely to that seen with the
control or other fumigants. Numbers 1 through 13 in parentheses refer
to the lane numbers in the DGGE gels.
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To compare DGGE patterns for the three sampling points, Dice's indices
were determined for comparisons of all profiles, and UPGMA was used to
create a dendrogram describing pattern similarities (Fig.
6). This analysis clearly showed the
impact of the four fumigants during the first week of the experiment
and distinguished the effects from those at weeks 8 and 12. All of the
week 1 samples were grouped together as most similar, separating them
from week 8 and 12 samples.
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FIG. 6.
UPGMA tree with Dice's coefficient, representing the
genetic similarity of the microbial community profiles obtained by
PCR-DGGE. Numbers after the abbreviations indicate times and
concentrations as described in the legend to Fig. 1. Cont, control.
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To quantitatively examine the relative similarities of the communities,
the 16S rDNA band profiles were analyzed by peak fitting. Due to
artifacts associated with DGGE analysis of images of completely different gels, in which there are subtle differences in the gel gradients, running times, and DNA staining procedures, we decided first
to analyze our samples based on time from extraction of DNA from
pooled soil samples. This resulted in a direct comparison of the effect
of each compound at one time point in one gel. This was done after
running gels with triplicate microcosm soil samples that showed banding
patterns to be identical (data not shown). The ordination diagrams in
which the effects of the four fumigants were compared by correspondence
analysis after fumigation (Fig. 7A)
showed separation of the treatments from the control. The data
explained 42% variability on the first component and 24% on the
second component during the first week of the study (Fig. 7A).
Variability in the data was smaller at weeks 8 and 12 (Fig. 7B and C).
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FIG. 7.
Correspondence analysis of microbial communities
generated by the analysis of DGGE 16S rDNA PCR patterns at 1 week (A),
8 weeks (B), and 12 weeks (C) after MeBr, MITC, 1,3-D, and CP
treatment.
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The second method for determination of the structural diversity was the
calculation of the Shannon-Weaver index of diversity (H)
from the DGGE banding pattern of the samples. H was
calculated on the basis of the number and relative intensities of bands
on a gel strip. By avoiding the bias of cultivation and by direct extraction of DNA from fumigated soil samples, H was used as
a parameter that reflects the structural diversity of the dominant microbial community. The four fumigant-treated samples showed different
levels of diversity (Table 2), ranging
between 0.11 and 1.26 at different sampling times. Gel analysis of the
four treatments at the three sampling times revealed that MeBr exerted the most significant effects on the structural diversity of the soil.
One week after MeBr application, the DGGE banding patterns revealed
dramatic changes in the structure of the microbial community (Table 2
and Fig. 5). The number of bands decreased from 16 in the control to
almost undetectable numbers in MeBr-treated soil, which established a
new cluster far away from the control (Fig. 7A). This indicated the
collapse of the microbial community due to the acute toxicity of MeBr.
The parameter H decreased from 1.26 in the control to 0.11 in the treatment with the highest concentration of MeBr. The
H value increased slightly during week 8 and subsequently,
in week 12, to about 0.75 but remained clearly below the average
control value (H = 1.26). Communities from samples treated with MITC, 1,3-D, and CP were not as severely affected, although their H values were still below that of the control
(Table 2).
In this study we applied the Shannon-Weaver index of diversity to 16S
rDNA DGGE from total community DNA separated according to sequence
heterogeneity. This approach had been successfully used by Eichner et
al. (5) to describe the community structure in activated
sludge. Those authors noted that the number and intensities of bands do
not equal the number and abundance of species within the microbial
community due to features of 16S rDNA-based phylogeny and bias inherent
to PCR amplification from complex template mixtures. The reasons for
most of the limitations are that DGGE banding patterns are subjected to
PCR bias due to DNA extraction methods, potential preferential
amplification, and the formation of chimeras (40). Other
problems may result from one organism producing more than one DGGE band
because of multiple, heterogeneous rRNA operons (4, 22,
26). Also, for some phylogenetic groups of bacteria, 16S rDNA
sequences do not allow discrimination between species, so one DGGE band
may represent several species with identical rDNA sequences
(36).
In a community DNA mixture such as soil, the maximum number of
different rDNA fragments separated by DGGE may be vastly
underestimated. Torsvik et al. (33) found that there might
be as many as 104 different genomes present in
soil samples. This shows that DGGE cannot separate all of the 16S rDNA
fragments obtained from soil microorganisms, but only the dominant
species (20, 21). Therefore, the banding patterns obtained
in this study reflect the most abundant rDNA types in the community. In
this study the Shannon-Weaver index of diversity was used in
combination with the correspondence analysis of the DGGE banding
patterns based on the similarity coefficient to monitor a range of
community responses after the application of fumigants. First, the
changes in community structure within the laboratory microcosms during
the first week of the experiment were documented, and the major bands
that emerged were identified. Second, the recovery in community
structure in the MeBr treatments to a community dominated by
gram-positive bacteria may be an indication of how bacteria respond to
chemical treatments.
Analysis of predominant bacterial species by PCR-DGGE.
The
analysis of predominant bacterial species was carried out with soil
samples pooled from triplicate microcosms at weeks 1, 8, and 12. Bands
selected for analysis are shown in Fig. 5. Table
3 shows the prominent bands recovered
from the DGGE gel. At week 1 (Fig. 5A) all samples generated very
complex banding patterns except samples treated with MeBr and the
highest concentration of MITC. In addition to the two prominent bands
(F1 and F2) observed during the first-week analysis, more than seven
visible bands were also present in most of the samples analyzed. The
derived sequences from these bands confirmed that F1 was 100% similar to Pseudomonas reactans and that F2 had 99% similarity to
Pseudomonas putida. At week 8 new bands appeared in the MeBr
treatments. Four dominant bands (F3 to F7) and three other bands (F16,
F17, and F31) were also present. Most of the prominent bands visible at week 8 in the other treatments remained strong within each treatment but were not present in the MeBr treatments. The appearance of novel
bands (F3 to F7 and F16, F17, and F31) in the MeBr treatment was an
indication of the dominance of new microbial species. Bands F3 and F6
showed relationships to the gram-positive species Heliothrix oregonensis and Bacillus subtilis, respectively. The
fragment designated F4 was one of the most dominant species in the MeBr treatment community that evolved 8 weeks after fumigation. Its sequence
closely matched (97%) that of an unclassified organism (Acidobacterium capsulatum) 16S rRNA gene. Bands F5 and F7
also appeared in the MeBr treatments and matched 100% with uncultured soil bacterium C0111. Three other novel bands (F16, F17, and F31) loosely related to the genus Sphingomonas in the alpha
subclass of the Proteobacteria were detected uniquely
in the MeBr-treated samples. There were no significant new bands at
week 12; therefore, the dominant bands that appeared in all of the
samples were analyzed for general information on the dominant species,
as shown in Table 3.
View this table:
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TABLE 3.
Sequence analysis of bands excised from DGGE gels derived
from bacterial 16S rDNAs extracted from fumigated and nonfumigated
soils
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|
In conclusion, our data have shown that the effects of MeBr and its
alternatives on soil microbial composition and function could be
evaluated by using a functional assay and biochemical and genetic
fingerprints. This study showed that the PLFA technique was the most
effective method in evaluating community structure after fumigant
treatment, followed by the DGGE approach and the Biolog assay. We have
also shown that gram-positive bacteria survived better after fumigant
treatment, with 1,3-D exerting the least effect on microbial community
structure. The behavior of microbes in the soil after fumigation could
be evaluated more accurately by using field samples. However, due to
many problems associated with sample collection immediately after
fumigation, a laboratory microcosm experiment can be used to give basic
results if fumigants are applied at concentrations that reflect field conditions.
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Abstract
Introduction
