applied soil ecology 40 (2008) 518–528

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Microbial community response to transition from

conventional to conservation tillage in cotton fields

Breana L. Simmons a,*, David C. Coleman b

a
Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80521-1499, USA
b
Odum School of Ecology, University of Georgia, Athens, GA 30602-2360, USA

article info abstract

Article history: In the southeastern United States, conservation tillage techniques are used to conserve soil
Received 20 July 2007 nutrients and structure, providing habitat and substrate for biota, which are largely
Received in revised form responsible for the mineralization of nutrients in the soil. A deterrent for growers con-
25 June 2008 sidering the transition to conservation tillage is the delay in soil response (e.g. increased soil
Accepted 7 August 2008 carbon, efficient nutrient cycling, impacts on yield) associated with the equilibration of the
soil food web. The objective of this study was to determine if the microbial community
composition and biomass changed with transition to conservation tillage. Soils sampled
Keywords: from five sites, representing a chronosequence of conservation tillage (from conventionally
Microbial community structure tilled to 30 year no-till), were collected for fatty acid analysis. Microbial communities were
Microbial biomass significantly different among sites. Fungi, characterized by 18:2v6, 18:1v9, and 18:3v6c fatty
EL-FAME acids, were typically lowest in the conventionally tilled soil, probably due to repeated
Conservation tillage disruption of the fungal hyphae associated with tillage. In all soils, soil nutrient concentra-
tions, moisture and microarthropod abundance were correlated with microbial structure.
Plots in conservation tillage were significantly different from the conventionally tilled plots,
but did not exhibit a clear linear pattern across the chronosequence. This evidence that
belowground food webs can respond quickly to a cessation in tillage suggests that the delay
in soil response may be due more to the time required to build organic matter than to a slow
response by the biota.
# 2008 Elsevier B.V. All rights reserved.

1. Introduction increases decomposition of crop residues and changes the

Conventional tillage practices, where crop residues are incor-
porated into the soil by plowing or disking, are used to aerate
soils, reduce compaction, and control herbaceous pests, thereby
increasing seedling germination and yield (Dickey et al., 1983;
Kettler et al., 2000; Raper et al., 2000). However in many cases
tillage results in soil erosion, loss of organic matter, decreased
water infiltration, loss of soil structure, decreased soil fertility
and a reduction in overall soil quality due to the destruction of
soil aggregates and structure (Parr et al., 1992; Paustian et al.,
1997; Allmaras et al., 2000; Nyakatawa et al., 2000). Tillage
structure of the soil food web by relocating food resources and
exposing protected carbon (Hendrix et al., 1986; Moore and
deRuiter, 1991; Beare et al., 1992; Wardle, 1995; Six et al., 2002).
Conservation tillage is used to conserve soil nutrients and
structure, increase sequestration of soil carbon, and to provide
habitat and substrate for biota (Hendrix et al., 1998; Lal et al.,
1998; Paustian et al., 2000; Holland, 2004). However, a deterrent
for growers considering the transition to conservation tillage is
the delay in soil response (e.g. increased soil carbon, efficient
nutrient cycling, impacts on yield) associated with the equili-
bration of the soil food web (Phatak et al., 1999).

* Corresponding author. Tel.: +1 970 491 3552; fax: +1 970 491 1965.
E-mail address: breana@nrel.colostate.edu (B.L. Simmons).
0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2008.08.003
 applied soil ecology 40 (2008) 518–528
Previous studies have shown that microbial community
composition can be altered by a change in management
practices (Visser and Parkinson, 1992; Schutter and Dick, 2002;
Liebig et al., 2006; Yao et al., 2006; Elfstrand et al., 2007),
substrate availability and composition (Wardle et al., 1993;
Grigera et al., 2006; Ha et al., 2008), soil type (Schutter et al.,
2001), soil moisture (Chen et al., 2007; Stromberger et al., 2007),
soil chemistry (Pankhurst et al., 2001), and seasonal variation
in soil nutrient status (Bardgett et al., 1999; Schutter et al.,
2001). Plant characteristics can also affect soil microbial
communities by changing the soil environment (Carney and
Matson, 2006; Chen et al., 2007), which may be especially
important for growers utilizing transgenic crops (Griffiths
et al., 2007; Widmer, 2007).
The activity of soil biota, responsible for the mineralization
of nutrients from the soil, is an important component of soil
function (Ingham et al., 1985; Hunt et al., 1987; Moore, 1988;
Beare et al., 1992). This is especially important in low input
sustainable agriculture, where increased microbial diversity is
expected to increase soil quality (Parr et al., 1992; Visser and
Parkinson, 1992). In systems where crop residue is buried or
where labile substrate is abundant, bacteria dominate, due to
their ability to break down labile carbon sources more
efficiently than saprophytic fungi (Coleman et al., 1983; Curl
and Truelove, 1986; Moore et al., 2003). In these systems, the
rates of decomposition and nitrogen mineralization are
accelerated (Moore and deRuiter, 1991; Doles et al., 2001).
Microbial diversity is expected to increase with a reduction in
tillage, as fungal species begin to dominate the system (Beare
et al., 1992; Frey et al., 2003). In systems where crop residue is
left on the surface, saprophytic fungi dominate, slowly
breaking down more resistant substrates (Hendrix et al.,
1986; Moore et al., 2003).
The ability of an ecosystem to withstand disturbance may
lie in the energy pathway, where bacterial-dominated systems
are more resilient than fungal dominated systems (Allen-
Morley and Coleman, 1989; Moore and deRuiter, 1991; Bardgett
and Cook, 1998). Moore et al. (2003) postulate that recovery
times of each energy channel to disturbance may be different,
and result in an alteration of the food web. Fungi, especially
arbuscular mycorrhizal (AM) fungi, may be particularly
sensitive to tillage (Drijber et al., 2000). Acosta-Martinez
et al. (2007) report an increase in fungi with a combination
of no-till and increased cropping intensity, potentially due to
greater residue quantity. In a study involving the transition
from conventional to alternative agriculture, Doran (1987)
found that microbial populations and activities were regulated
more by crop type and rotation than by soil physical proper-
ties. Gonzalez et al. (2003) observed an increase in humifica-
tion in soils in no-till as compared to those in reduced tillage,
indicating that microbial populations were probably influ-
enced by increases in organic matter. In contrast, Spedding
et al. (2004) showed no effect of tillage on the microbial
community but did find a significant effect of seasonality on
fungi.
The objective of this study was to determine if and when
the microbial community composition and biomass changed
with the transition to conservation tillage. Extraction of fatty
acid methyl esters (EL-FAME) from whole soil samples is
simpler and less time consuming than analysis of phospho-
519
lipid fatty acid (PLFA) profiles (Schutter and Dick, 2000; Grigera
et al., 2007). This method has been useful for identifying
microbial community composition in a number of studies
(Cavigelli et al., 1995; Pankhurst, 1997; Ibekwe and Kennedy,
1999; Schutter and Dick, 2000; Sullivan et al., 2006; Mechri
et al., 2007; Stromberger et al., 2007; Bünemann et al., 2008). We
hypothesized that plots in conventional tillage would be
dominated by bacteria, while plots in conservational tillage
would experience an increase in the abundance of fungal
groups that correlated with time in no-till.
2. Methods
2.1. Collection sites
Soil was collected from five cotton fields near Douglas in
Coffee County, Georgia (USA) at map coordinates 318 210 N, 828
520 W. These sites were selected based on previous positive
collaboration between Coffee County growers and University
of Georgia researchers (Adl et al., 2006). Monthly precipitation
in the area is 107 mm with a mean annual rainfall of nearly
1300 mm. Mean annual temperature is 18.7 8C. One field was in
conventional tillage, where crop residue was incorporated by
moldboard plow after both summer and winter crops. Another
grower reserved a portion of a conventionally tilled field for
transition to conservation tillage, where no incorporation of
residues took place and plantings were done using a no-till
seed drill. Three other fields had been in conservation tillage
for several years and had not been tilled in several years. All
fields are labeled by the number of years spent in conservation
tillage at the beginning of this study: 0,1,5,10, and 30.
The soils are heavily weathered Ultisols: loamy to fine-
loamy, kaolinitic, thermic Kanhapludults, Kandiudults and
Paleaquults, characterized as fine-loamy to loamy, with low
organic matter (SOM), high pH, and low fertility, with
moderate to slow permeability (Soil Survey Staff, NRCS-
USDA). All fields had been continuously cropped for at least
30 years, and were planted in cotton for the duration of this
study, except Site 30, which was cropped in no-till peanut
during Spring 2003. This crop requires plants to be pulled from
the soil and set to dry, a practice that disturbs the topsoil.
However, a one-time rotation of peanut has been shown to not
significantly affect microbial biomass or soil organic carbon
(SOC) in sandy soils (Acosta-Martinez et al., 2004), Fields in
conservation tillage maintained a cover crop of rye in the
winter to add organic matter, which was rolled in the spring.
Herbicides and fertilizers were used at the beginning of each
crop rotation. Lime was added approximately every other year
or as needed in most fields. Two sites (5, 10) were managed in
conjunction with broiler hen operations, and had previously
received applications of broiler litter after the cotton was
mowed.
This sampling design has received criticism for replicating
at the plot scale but not at the field scale. However, replication
at that scale in this study would have required the cooperation
of several more growers, and introduced several more sources
of potential error. Differences in equipment, land use history,
landscape characteristics, crop variety, fertilizer and/or
pesticide use, and general management techniques between
520 applied soil ecology 40 (2008) 518–528
fields of the same ‘‘age’’ create a suite of new variables that
have previously been shown to influence soil biological
properties (Visser and Parkinson, 1992; Schutter and Dick,
2002; Liebig et al., 2006). Therefore, we replicated within fields,
rather than replicating between fields and bulking the
samples. This design was successfully implemented in these
same fields in a previous study (Adl et al., 2006). Furthermore,
in many systems it’s not uncommon to sample across
gradients using replicate plots where it’s not possible to find
similar, representative landscape sequences, and several
recent studies have used this approach (DeLuca et al., 2008;
Doblas-Miranda et al., 2008; Liao and Boutton, 2008).
2.2. Sampling
Twelve replicate plots (2 m  3 m) were set up at each site, for
a total of 60 experimental plots across the five sites. Each plot
was at least 5 m from its nearest neighbor plot. Plots were set
up randomly but oriented perpendicularly to row direction so
that each pass of the equipment impacted the same number
and type of plots. Plots were located using a handheld GPS unit
because flags were inconvenient for growers. Sampling for site
characteristics and microbial biomass occurred four times
over 2 years, at the end of each growing season but before the
crop was mowed (sites 1, 5, 10, 30) or incorporated (site 0). In
Spring 2004, only 4 plots were sampled at site 5, due to an error
with the GPS unit.
Samples were taken for soil C/N from the upper 5 cm of soil
using a soil probe (dia = 2 cm). Nematodes were sampled using
a soil probe (dia = 2 cm) from the upper 5 cm of soil.
Microarthropods were sampled from the upper 5 cm of soil
using steel rings (dia 5 cm) in a specialized beveled metal
corer, and were wrapped in aluminum foil for transport. Soil
for FAME extractions were taken from each plot with a soil
corer (dia = 2 cm) to a depth of 5 cm. All samples were sealed in
plastic bags and transported back to the lab in a cooler.
2.3. Laboratory procedures
Total soil carbon and nitrogen were determined on a Carlo
Erba analyzer in the UGA Institute of Ecology Analytical
Laboratory on soil that was air-dried, ground, and weighed
into tin capsules. Soil moisture values were obtained
gravimetrically, by drying soil subsamples in an oven at
105 8C for 48 h. An estimate of soil organic matter (SOM) was
obtained by ashing soil subsamples at 450 8C for 4 h in a
muffle furnace. Microbial biomass estimates were obtained
from soils stored at 4 8C for no more than 48 h by chloroform
fumigation extraction using a constant value of 0.33 (Vance
et al., 1987).
Nematodes were extracted using the Baermann funnel
technique (Coleman et al., 2006). Nematodes were counted
under an inverted microscope and categorized into feeding
groups by morphological characters (Yeates et al., 1993).
Microarthropods were heat extracted over 5 days using
modified Tullgren type funnels (Crossley and Blair, 1991).
Animals were collected in 70% ethanol and identified to order,
suborder, or family under a dissecting microscope. Samples
were prepared for permanent storage by transferring animals
to 95% ethanol after identification.
2.4. FAME procedures
FAME profiles were compiled using a modification of the ester-
linked (EL) FAME method described by Schutter and Dick (2000).
For each sample 3.0 g of air-dried soil was placed in a 30-ml glass
centrifuge tube, 15 ml of methanol–KOH (0.2 M) was added to
each tube, and the tubes were capped using Teflon-lined screw
caps. The centrifuge tubes were place in a test tube rack in a
37 8C water bath for 1 h. Every 10 min the tubes were mixed for
20 s. After the water bath, 0.5 ml of 1N acetic acid was repeatedly
added to each tube until the pH of the solution was neutral.
10 ml of hexane was added to the soil suspension, and the
mixture was vortexed for 30 s. The tubes were centrifuged at
480  g for 20 min. Approximately 7 ml of the hexane layer was
transferred to a disposable test tube. The transferred hexane
was evaporated to complete dryness under a gentle stream of
nitrogen. The dried extract was re-suspended in 1.0 ml of
hexane and dried down again for shipment to an analytical lab
in Delaware. In the analytical lab, the samples were re-
suspended in a 1:1 mixture of hexane and tertiary-butyl methyl
ether. The solution was transferred to a 2.0-ml gas chromato-
graphy vial and capped. The extract was analyzed with a HP
5890 gas–liquid chromatograph equipped with a HP Ultra 2
capillary column (5%-diphenyl-95%-dimethylpolysiloxane,
25 m by 0.2 mm) and a flame ionization detector.
Fatty acid methyl esters described here use the standard
nomenclature for lipid markers, A:BvC. A is the number of
carbon atoms, B is the number of double bonds, and vC indicates
the number of carbon atoms from the aliphatic end of the
molecule and the first unsaturated bond. Isomers are denoted
with the suffixes c (cis) or t (trans). Methyl branching is described
by the prefixes i (iso) and a (anteiso), while methyl and cyclopropyl
groups receive the notations Me and cy, respectively.
Peaks were identified by the analytical lab using MIDI peak
identification software (Microbial ID, Newark, DE). Major
microbial groups were assigned as follows: bacteria (i13:0
OH, i15:0, a15:0, 15:0, 16:0 2OH, i16:0,16:1v7c, 16:1v9c i17:0,
a17:0, cy17:0, cy19:0,18:0, cy19:0) and fungi (18:1v9, 18:2v6,
18:3v6c) (Frostegård et al., 1993; Bossio et al., 1998; Zelles, 1999;
Schutter and Dick, 2000). Bacterial/fungal ratios based on fatty
acid biomarkers have been used in other studies to char-
acterize functional diversity in microbial communities (Sped-
ding et al., 2004; Carney and Matson, 2006; Mechri et al., 2007).
While multiple fatty acid markers may indicate single species,
plant material, or other organic matter (Zelles, 1999), differ-
ences among these markers indicate a difference in the
belowground community, and diversity analyses calculated
from fatty acid markers were used for comparative purposes
only (McCulley and Burke, 2004; Liebig et al., 2006; Sullivan
et al., 2006; Minoshima et al., 2007).
2.5. Analysis
Analysis of variance (ANOVA) was performed to determine if the
total quantity and composition of FAMEs differed among sites
(0,1,5,10, and 30 years in no-till). Data were log transformed to fit
assumptions of normality and analyzed for significant differ-
ences in percentages of mole fractions among sites using least
significant difference means separation test (P = 0.05). These
data were also used to determine if there were seasonal effects
 applied soil ecology 40 (2008) 518–528 521

on microbial abundance using a repeated measures ANOVA. All
ANOVAs were performed in SAS (SAS Institute, 1989). Percent
mole fractions were also summed for each microbial group
(known bacterial and fungal markers) and subjected to ANOVA
using the pdiff option in the MIXED procedure to test for
significant differences in functional groups among sampling
dates and field sites. These were also used to determine
bacterial: fungal ratios (Spedding et al., 2004; Carney and
Matson, 2006; Stromberger et al., 2007). The ratio of cy17:0 to
16:1v7c was used as an indication of microbial stress (Bossio
et al., 1998; Mechri et al., 2007; Stromberger et al., 2007). For
comparative analysis of the fields, 10 FAMES common to all
samples and representing a range of microbial groups were
chosen: gram-positive bacteria (i16:0, i17:0, a17:0), gram-
negative bacteria (16:1v7c, cy17:0, cy19:0), non-specific bacteria
(18:0) and fungi (18:1v9, 18:2v6, 18:3v6c). Non-metric multi-
dimensional scaling (NMS) was performed on the mole fractions
of these markers. Site characteristics (percent nitrogen, percent
carbon, C/N ratio, percent soil organic matter, soil moisture) and
soil faunal abundances (microarthropods and nematodes) were
log transformed and used to create a secondary matrix. This
secondary matrix was correlated with the main matrix and used
to evaluate the effects of environmental variables on the distri-
bution of microbial functional groups. All multivariate analyses
were calculated using PC-ORD (McCune and Mefford, 1999).
3. Results
3.1. Soil characteristics and climate conditions
Soil nutrient status varied seasonally and among sites, with
spring samples having greater C/N ratios compared to the fall
samples at all sites (Table 1). Percent SOM was always highest
at site 30, and typically lowest at site 5. Soil moisture values
varied among sites and were not consistently higher at any
one site (Table 1). Mean annual temperatures in Coffee
County, GA, for 2003 and 2004 were 19.3 8C and 19.6 8C,
respectively, which is slightly higher than normal (18.7 8C).
During sampling, mean monthly temperatures were 19.2 8C
(April) and 20.3 8C (October) in 2003, and 19.0 8C (April) and
22.1 8C (October) in 2004. Total annual precipitation in 2003
measured 1649 mm, and dropped to 1395 mm in 2004. Monthly
precipitation levels during sampling were 117 mm (April) and
149 mm (October) in 2003, compared to 39 mm (April) and
54 mm (October) in 2004.
3.2. Microbial community composition
Over 2 years, a total of 51 different FAMEs were extracted; 48 in
spring 2003, 45 in fall 2003, 33 in spring 2004 and 38 in Fall 2004.
Total number of fatty acids at each site varied seasonally and
among sites (Table 2). During the spring sampling periods,
there were no significant differences in the number of fatty
acids extracted among sites (Table 2). In fall 2003, site 10 had
significantly greater number of fatty acids compared to other
fields; sites 5 and 30 had the lowest numbers (Table 2). In Fall
2004, this trend was repeated, with site 30 experiencing an
increase in total fatty acids extracted (Table 2). Diversity of
fatty acids, as measured by Simpson’s diversity index, was
similar among the sites, and did not consistently increase with
time in no-till.
The NMS identified two axes that explained 90% of the
variance in the FAME data (stress = 16.15) (Fig. 1). Axis 1
explained 46% of the data variability and was most positively
correlated with nutrient content and soil organic matter

Table 1 – Soil characteristics (0–5 cm) from samples collected from five sites in 2003 and 2004 across a chronosequence of
conservation tillage (0, 1, 5, 10, and 30 years) in southern Georgia, USA
Date Site N C/N ratio %SOM Water (%)

Spring 2003 30 12 16.48  0.59bc 1.02  0.09a 11.75  0.62b

10 12 14.65  0.26c 0.86  0.08ab 14.73  0.59a
5 12 17.11  0.82b 0.64  0.07b 9.39  0.78bc
1 12 16.07  0.42bc 0.60  0.04b 7.68  0.35c
0 12 18.99  0.40a 0.68  0.04b 10.49  0.54bc

Fall 2003 30 12 14.39  0.10b 0.83  0.04a 7.57  0.73b

10 12 13.02  0.14b 0.66  0.05bc 15.18  1.30a
5 12 12.60  0.43b 0.49  0.06d 5.86  0.83b
1 12 13.86  1.32b 0.53  0.01cd 17.17  0.89a
0 12 17.42  0.32a 0.74  0.02ab 4.85  0.25c

Spring 2004 30 12 16.69  0.26ab 0.75  0.03a 5.87  0.81b

10 12 16.52  0.66ab 0.66  0.02b 5.49  0.47b
5 4 15.30  063b 0.43  0.19d 3.79  1.23b
1 12 18.07  0.72a 0.49  0.02cd 14.61  0.63a
0 12 18.43  0.17a 0.55  0.05c 6.22  0.17b

Fall 2004 30 12 13.25  0.11bc 0.82  0.06a 13.56  0.98b

10 12 12.65  0.14c 0.69  0.03ab 24.20  0.62a
5 12 13.43  0.35bc 0.49  0.01c 14.36  0.72b
1 12 13.78  0.23b 0.61  0.03bc 22.96  0.60a
0 12 16.41  0.36a 0.75  0.03a 12.90  1.35b

Values are means  standard error. Letters within columns denote significant differences between treatments for one sampling date (Tukey’s).
522 applied soil ecology 40 (2008) 518–528

Table 2 – Diversity (D0 ) and total number (total) of fatty Table 3 – Results from NMS analysis (stress = 16.15) of
acids extracted at five sites across a chronosequence of percent mol fractions of FAMEs extracted from five sites
conservation tillage (0–30 years) in southern Georgia, across a chronosequence of conservation tillage in 2003
USA and 2004
Date Site N D0 Total (A) Environmental variables Axis 1 Axis 2
(mean  S.E.) (mean  S.E.)
% Nitrogen 0.115 0.077
Spring 2003 30 12 0.87  0.02a 15.75  1.89a % Carbon 0.101 0.006
10 12 0.86  0.03a 14.80  1.38a C:N ratio 0.074 0.207
5 10 0.80  0.09a 13.60  2.08a Soil organic matter 0.120 0.036
1 12 0.90  0.01a 18.40  1.29a Soil moisture 0.049 0.199
0 12 0.90  0.01a 18.50  1.42a Microarthropod abundance 0.027 0.106
Fall 2003 30 12 0.90  0.01bc 20.16  1.29c Nematode abundance 0.041 0.069
10 12 0.91  0.01a 27.58  1.40a
5 12 0.89  0.01c 18.08  0.64c (B) Fatty acids Axis 1 Axis 2 Microbial marker
1 11 0.91  0.01ab 21.72  0.77bc
i16:0 0.236 0.428 Gram-positive bacteria
0 12 0.91  0.01ab 24.17  0.94bc
i17:0 0.440 0.201 Gram-positive bacteria
Spring 2004 30 12 0.90  0.01a 25.83  0.79a a17:0 0.070 0.557 Gram-positive bacteria
10 12 0.91  0.01a 27.33  0.42a 16:1v7c 0.596 0.095 Gram-negative bacteria
5 4 0.88  0.01a 22.33  2.69a cy17:0 0.513 0.225 Gram-negative bacteria
1 12 0.91  0.01a 23.66  1.40a cy19:0 0.648 0.039 Gram-negative bacteria
0 12 0.90  0.01a 28.50  0.42a 18:0 0.419 0.091 Non-specific bacteria
18:2v6c 0.722 0.129 Fungi
Fall 2004 30 12 0.91  0.01b 27.83  1.40b
18:3v6c 0.011 0.058 Fungi
10 12 0.92  0.01a 32.66  0.88a
18:1v9c 0.584 0.732 Fungi
5 12 0.90  0.01b 23.17  1.51c
1 12 0.91  0.01a 27.83  1.13b Values are Pearson correlations from first and second ordination
0 12 0.91  0.01ab 29.33  0.21ab axes for (A) environmental variables and (B) microbial fatty acid
markers and corresponding microbial groups.
Statistic D0 = Simpsons diversity index. Letters denote differences
at P < 0.05 within sampling dates only.

(Table 3A). Axis 2 explained 44% of the data and was negatively
correlated with CN ratio and positively correlated with soil
moisture and microarthropod abundance (Table 3A). Pearson
correlations for FAME groups were inconsistent across groups.
Axis 1 was most positively correlated with 16:1v7c and cy17:0,
gram-negative bacterial marker and 18:1v9, a fungal marker,
and was most negatively correlated with cy19:0, a gram-
negative bacterial marker, and 18:2v6, a fungal marker
(Table 3B). Axis 2 was most positively correlated with a17:0,
Fig. 1 – Non-metric multidimensional scaling (NMS)
analysis of FAMEs extracted at five sites across a
chronosequence of conservation tillage (0, 1, 5, 10, and 30
years in no till). Symbols represent mean
values W standard error for a 2-axis solution
(stress = 16.15) across all sampling dates. The variance
explained by each axis is shown in parentheses. N = 48
(sites 0, 10, 30); 47 (site 1); 38 (site 5).
a gram-positive bacterial marker and most negatively corre-
lated with 18:1v9, a fungal marker (Table 3B). Post hoc tests
revealed that Site 5 was significantly different from the other
sites (F = 9.97, P < 0.0001). Axis 1 was significantly affected by
years in no till (F = 6.49, P < 0.0001) while Axis 2 was affected by
years in no till (F = 9.97, P < 0.0001) and season (F = 12.95,
P < 0.0004).
Overall, relative abundances of fungi, but not bacteria, were
higher at sites in conservation tillage compared to site 0
(Table 4). Relative abundances of gram-positive and gram-
negative bacteria were highest in fall 2003 and lowest in spring
2004 (Table 4). Relative abundances of eubacterial anaerobes
was also lowest in spring 2004 but highest in spring 2003. In
contrast, the relative abundance of fungi was highest in spring
2004. The sites in conservation tillage had significantly higher
relative abundances of fungi than the conventionally tilled site
in 2003, but this trend did not continue into 2004 (Table 4). Site
5 had the highest relative abundance of gram-positive bacteria
and fungi, while site 30 had the highest relative abundance of
gram-negative bacteria (Table 4). There was no significant
difference in the relative abundance of fungi in Fall 2004, and
relative abundances of other taxonomic groups were variable
across sites (Table 4).
The ratio of bacterial to fungal FAMEs differed significantly
between sites and was highest in the conventionally tilled soil
(site 0), and lowest in sites 1 and 5 (Fig. 2A). This ratio also
differed significantly between seasons, and was lowest in April
2004 (data not shown). The relative % mole fraction of 16:1v5c
was significantly lower in the conventionally tilled soil (site 0)
compared to the other sites (Fig. 2B). This variable was also
significantly affected by season, and was highest in October
 applied soil ecology 40 (2008) 518–528 523

Table 4 – Differences in FAME % mole fraction of functional groups between five sites across a chronosequence of
conservation tillage (0–30 years) in 2003 and 2004
Date Site N Gram-positive Gram-negative Eubacterial anaerobes Fungi

Spring 2003 30 9 8.81  0.41a 8.92  0.24ab 6.87  0.29ab 17.74  1.67a
10 12 8.19  0.37ab 8.51  0.90ab 7.01  0.66ab 16.01  0.64a
5 9 6.61  0.63bc 9.38  0.46a 8.10  0.59a 19.67  1.08a
1 12 5.76  0.36c 8.66  0.36ab 5.61  0.32b 19.38  0.99a
0 11 7.64  0.20bc 6.52  0.73b 6.15  0.68ab 9.08  0.48b

Fall 2003 30 12 8.84  1.01a 10.33  0.33a 7.50  0.45a 18.64  0.84a
10 12 8.41  0.25a 8.48  0.44bc 5.23  0.39bc 18.08  0.84a
5 12 9.41  0.29a 9.41  0.25ab 5.77  0.25bc 21.36  1.06a
1 11 6.98  0.21b 8.86  0.35bc 4.79  0.18c 18.75  0.67a
0 12 7.52  0.13a 7.85  0.35c 6.31  0.27b 11.90  0.36b

Spring 2004 30 12 7.46  0.13a 7.72  0.16a 4.44  0.19a 27.41  22.00ab
10 12 6.82  0.18b 6.96  0.12b 4.09  0.08a 28.02  1.59ab
5 4 7.25  00.28a 7.20  0.17b 3.34  0.38b 30.64  1.46a
1 12 4.72  0.21c 6.50  0.33c 3.08  0.17b 24.01  1.52b
0 11 6.60  0.18b 6.19  0.07c 4.49  0.10a 23.80  1.53b

Fall 2004 30 12 7.36  0.12b 6.56  0.26c 3.88  0.20a 15.51  0.35a
10 12 7.81  0.08a 7.49  0.11b 4.36  0.08b 17.13  0.31a
5 12 7.76  0.24a 8.09  0.18a 3.92  0.10a 21.81  0.51a
1 12 6.26  0.13d 7.67  0.27b 4.02  0.05a 16.01  0.91a
0 12 6.66  0.18c 6.63  0.13c 4.41  0.05b 15.87  0.88a

Values are means  standard error. Different letters denote significant differences between sites within date at P < 0.05. Differences in N
values reflect replicates that were lost due to either sampling or equipment error.

2003 (data not shown). The ratio of cy17:0 to 16:1v7C was
significantly higher in the conventionally tilled soil (site 0)
compared to the other sites (Fig. 2C), and was also significantly
affected by season, being lowest in April 2003 (data not
shown).
4. Discussion
Previous research in these fields indicated that microbial
biomass would be greatest in the spring, due to increased soil
carbon and residue from the winter cover crop exploited by
microbes (Adl et al., 2006). Current studies showed a decrease
in C/N ratio and an increase in total number of fatty acids in
the fall. However, microbial response to seasonality is
inconsistent (Paul and Clark, 1996; Bardgett et al., 1999;
Spedding et al., 2004; Hamel et al., 2006), and may be
influenced by a complex set of variables associated with
seasonality (e.g. timing of residue incorporation, daily tem-
perature and precipitation levels, continuous cropping or
periods of fallow) that interact with the microbial biomass to
produce a response. Because our samples were collected
before the crops were mowed or tilled, it is unlikely that a
standing cover crop would provide microbes with enough
residues to increase abundance and diversity. In the fall,
microbial biomass may benefit from increased soil nitrogen
from increased fertilization of the summer cash crop or slow
decomposition of surface residue from the winter cover.
However, those plots that did not have a cover crop for one or
more years (site 0) also experienced a seasonal increase in
total fatty acids, indicating that it may be an effect of soil
nutrient status rather than residue quantity or quality. The
low number of fatty acids extracted from site 5 during the
spring may be explained by consistently low amounts of SOM
throughout the study, which negatively affects the soil food
web (Linn and Doran, 1984). Environmental variables asso-
ciated with seasonality may have affected the ratio of
bacterial: fungal FAMEs, significantly lower in April 2004,
which was much drier than the previous year, resulting in low
soil moisture levels at all but one site.
Crecchio et al. (2007) argue that microbial response to
tillage is minimal and that incorporation of residues is
sustainable practice. However, our data show effects of tillage,
particularly on fungi. The relative abundances of fungi were
also consistently higher in sites in conservation tillage, which
has been shown in other studies (Drijber et al., 2000; Acosta-
Martinez et al., 2007; Minoshima et al., 2007; Roldan et al.,
2007). Similarly, while bacterial: fungal FAMEs did not
decrease linearly with age in conservation tillage, there was
still a significant difference between soils from the conven-
tionally tilled site compared to most other sites. This supports
research involving ‘‘fast’’ and ‘‘slow’’ energy channels in
agricultural systems, where soils in no-till evolve fungal
dominated, ‘‘slow’’ energy channels, while soils in conven-
tional tillage break down substrate via a bacterial dominated,
or ‘‘fast’’ energy channel (Coleman et al., 1983; Curl and
Truelove, 1986; Hendrix et al., 1986; Allen-Morley and Cole-
man, 1989; de Ruiter et al., 1998). These energy channels can
have an impact on the stability of soil food webs (Moore, 1994;
Rooney et al., 2006). However, because not all fatty acids were
used in the taxonomic analysis, and other FAMEs have not yet
been identified as specific microbial markers, it may be too
early to say whether ‘‘fast’’ or ‘‘slow’’ energy channels are
dominant in these five soils.
Fungi appear to be more sensitive than bacteria, and have
been shown to respond negatively to fire (Hamman et al.,
524 applied soil ecology 40 (2008) 518–528
Fig. 2 – The effect of age in conservation tillage on (A)
bacterial to fungal ratios as measured by FAME
biomarkers, (B) arbuscular mycorrhizal fungi (AMF)
expressed as the relative % mole fraction of 16:1v5c, and
(C) an indicator of microbial stress, cy17:0: 16:1v7c, from
soils (0–5 cm depth) at five sites across a chronosequence
of conservation tillage. Data shown includes standard
error; different letters denote significant differences at
P < 0.05 (Tukey’s).
2007), application of biosolids (Sullivan et al., 2006), soil
salinity (Pankhurst et al., 2001), tillage (Drijber et al., 2000;
Acosta-Martinez et al., 2007; Minoshima et al., 2007) and
fertilizer application (Bradley et al., 2006), while responding
positively to the use of green manure (Elfstrand et al., 2007)
and olive waste water (Mechri et al., 2007). Studies have also
shown that levels of fungi react to a cessation in disturbance
(Hedlund, 2002), increasing in number before declining again
during natural succession (Allison et al., 2005). Reduction in
AM fungi due to tillage (Drijber et al., 2000) may be particularly
important, due to the role of these fungi on stable aggregate
formation (Roldan et al., 2007). In our experiment, levels of
16:1v5c, a fatty acid used as an indicator of AM fungi (Drijber
et al., 2000; Bradley et al., 2006; Hamel et al., 2006; Sullivan
et al., 2006; Hamman et al., 2007; Stromberger et al., 2007) were
low, but still show a significant reduction in the convention-
ally tilled soil. Concentrations of this marker were highest in
soils from site 5, which was typically the driest site, with the
lowest levels of SOM. Studies have shown AMF to be
stimulated by applications of green manure (Elfstrand et al.,
2007), and soils with a high proportion of 16:1v5c may store
condensed forms of soil P, although this is still unclear and
requires further research (Bünemann et al., 2008). Frostegård
et al. (1993) correlated a three-fold increase in 16:1v5c with
increased pH, which in these soils is naturally high. Site 5 was
farmed in conjunction with a broiler hen operation, and
potential applications of broiler litter to the soils may have
bolstered levels of AM fungi.
It is possible that the fungal communities in the four
conservation tillage sites are generally higher due to lessened
disturbance rather than to the inherent benefits of reduced
tillage, such as increased SOM, water holding capacity, and
nutrient status. Greater relative abundance of fungi during
spring 2004 was likely due to low soil moisture and high C/N
ratios. These conditions may have allowed the fungi to out
compete the bacteria. Particulate organic matter (POM) from
residues may also play a role in the abundance and diversity of
microbes. Ha et al. (2008) report that different residues result
in different levels of POM, which cultivate distinct microbial
communities. Furthermore, bacteria have been positively
correlated with levels of fine POM, while fungi and mycor-
rhizae correlate positively with coarse POM (Grigera et al.,
2006). In this study we assume sites in conservation tillage to
have a higher amount of coarse POM, due to rolling of winter
cereals and mowing of summer cotton, compared to the
conventional site, where residues were incorporated or
removed from the field. In fact Adl et al. (2006) showed higher
amounts of debris, organic matter and aggregated organic
matter in these same soils, while soil from the site in
conventional tillage consisted only of loose mineral soil. If
conservation tillage did result in an increase in coarse POM,
then this could explain greater relative abundances of fungal
fatty acids.
In our study, the ratio of cy17:0 to the precursor 16:1v7c,
an indicator of bacterial stress (Bossio et al., 1998; Mechri
et al., 2007; Stromberger et al., 2007), was highest in the
conventionally tilled soil. This ratio has been linked to a
variety of stress factors, summarized in Stromberger et al.
(2007), including extreme environmental conditions. Bacter-
ial stress as measured in this experiment was significantly
 applied soil ecology 40 (2008) 518–528
higher in conventionally tilled soil in October 2003, and
continued to be the highest over the next two sampling
dates, although not significantly so. Environmental condi-
tions in 2004, higher air temperatures and lower precipita-
tion, appeared to favor fungi, as the ratio of bacterial to
fungal FAMEs decreased over the course of the experiment,
and was lowest in April 2004, the driest sampling date.
Using these two ratios, we found that fungi increased
concurrently with an increase in bacterial stress in all soils.
Harsher ambient conditions resulted in lower soil moist-
ures, supporting the idea that hotter, drier environmental
conditions may select for fungi over bacteria, despite
management regimes.
Surprisingly, nematodes did not appear to influence soil
microbial populations. Nematodes are important microbi-
vores and can stimulate microbial populations by grazing
(Ingham et al., 1985; Moore and deRuiter, 1991). This may be
the result of their relatively low abundance at these sites (Adl
et al., 2006). Axis 2 was influenced by microarthropods,
indicating that microarthropod abundance may have a
substantial influence on microbial communities. This is
further supported by the negative weights of fungal FAMEs
found on Axis 2. It is possible that microarthropods, such as
oribatids and collembola, affected the fungi by both predation
and competition. However, it is uncertain whether the
microarthropods and fungi are directly or indirectly affecting
one another or simply responding similarly to environmental
variables, such as soil moisture and %SOM. Our sampling
scheme provided a ‘‘snapshot’’ of the soil community at the
end of each growing season. Frequent sampling throughout
each crop cycle would yield more vigorous data for correlating
changes in the soil microbial community with a range of
biotic and abiotic soil characteristics, but would be cost-
prohibitive.
Overall, using FAMEs to compare fields across a chron-
osequence of tillage showed little difference in community
composition with age. Except for Site 5, which had the
lowest overall number of fatty acids and was significantly
different from the other sites, all the other sites, including
the site in conventional tillage, had comparable levels of
fatty acids and microbial functional groups throughout the
course of the study. If this is accurate, then growers wishing
to reduce tillage may not have to wait as long as anticipated
for nutrient cycling to equilibrate if proper soil conditions
are met (Phatak et al., 1999). However, despite the ability of
microbial communities to respond rapidly to changing
resources, it may be unrealistic to expect soil microbial
communities to recover to pre-agricultural levels, even with
a cessation in tillage (Buckley and Schmidt, 2001). Further-
more, microbes are not the entire story – other critical
members of the food web, such as protozoa and micro-
arthropods, have been shown to recover slowly from
disturbance in these same soils (Adl et al., 2006). More
research is needed to decouple the effects of land manage-
ment and the environment on the entire soil community,
especially in heavily managed systems where the effects of
crop rotation, cropping intensity, tillage, and fertilizer use
may select for certain microbial groups but are confounded
by environmental variables such as soil type, moisture, and
fertility.
525
5. Conclusions
In this study, the abundance and diversity of microbial fatty
acids were most heavily influenced by soil nutrient concen-
trations, moisture and microarthropods, although environ-
mental conditions such as air temperature and precipitation
may play a large role in community structure. Most sites in
conservation tillage were not significantly different from one
another in terms of microbial abundance and diversity. This
is encouraging, because it implies that the microbial com-
munity in the transitional field does not lag behind the other
no-till sites, and this will benefit soil nutrient cycling.
However, both the NMS and the ANOVA show consistent
differences among the four sites in conservation tillage and
the site in conventional tillage. Season, crop type, and
management regime all play a significant role in soil nutrient
status, %SOM, and soil microclimate, which in turn have a
significant effect on the stability and resilience of the
soil food web. More studies involving complete food
webs and more complete habitat data should be done to
separate the effects of land management from the effects of
environment.
Acknowledgements
We thank Max Carter, Mike Nugent, Mark Vickers, Ricky
Smith, and Adam Lott for the use of their fields, and to Rick
Reed and Ed McGriff of the Coffee County Extension Office for
their assistance. We also thank Mary Gresham, Sunny Shanks,
and Amy Whitehead for field and laboratory assistance, Dr.
Peter Hartel for use of lab space and equipment, and Dr. Jeff
Furhmann for analysis of our FAME samples. We are grateful
for the comments of five anonymous reviewers on an earlier
version of this manuscript.
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