Effects of tillage on microbial populations associated to soil aggregation in dryland spring wheat system

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Abstract
a b s t r a c t Tillage may influence the microbial populations involved in soil aggregation. We evaluated the effects of no till (NT) and conventional tillage (CT, tillage depth about 7 cm) continuous spring wheat system on cul-turable heterotrophic bacterial communities predominant in microaggregates (0.25e0.05 mm) and on soil-aggregating basidiomycete fungi in aggregate-size classes (4.75e2.00, 2.00e0.25, and 0.25e0.05 mm) at 0e20 cm depth of a Williams loam (fine-loamy, mixed, Typic Argiustolls) in dryland Montana, USA. Enzyme-linked immunosorbent assay used to quantify antigenic response to basidiomycete cell walls, was higher in NT than in CT in 4.75e2.00 mm size class in 2007 and higher in all classes and years at 0e5 cm depth, but was not different between tillage, years, and classes at 5e20 cm. The culturable bacteria from microaggregates were subjected to a soil sedimentation assay to determine their soil binding capability. The proportion of isolates which can function as soil aggregators was higher in NT than in CT at 0e5 cm but was not different at 5e20 cm. Our results provide a first insight into the beneficial effects of dryland NT compared to CT in reducing soil disturbance and residue incorporation and enriching the proportion of microorganisms responsible for aggregation, especially at the soil surface. Published by Elsevier Masson SAS.
Original article
Effects of tillage on microbial populations associated to soil aggregation
in dryland spring wheat system
TheCan Caesar-TonThat
*
, Andy W. Lenssen, Anthony J. Caesar, Upendra M. Sainju, John F. Gaskin
United States Department of Agriculture-Agriculture Research Service, 1500 North Central Avenue, Sidney, MT 59270, USA
article info
Article history:
Received 23 June 2009
Received in revised form
2 December 2009
Accepted 7 December 2009
Available online 18 December 2009
Handling editor: Christoph Tebbe
Keywords:
Soil aggregation
Predominant culturable bacteria
Basidiomycetes
Dryland
Spring wheat
Tillage
abstract
Tillage may inuence the microbial populations involved in soil aggregation. We evaluated the effects of no
till (NT) and conventional tillage (CT, tillage depth about 7 cm) continuous spring wheat system on cul-
turable heterotrophic bacterial communities predominant in microaggregates (0.25e0.05 mm) and on
soil-aggregating basidiomycete fungi in aggregate-size classes (4.75e2.00, 2.00e0.25, and 0.25e0.0 5 mm)
at 0e20 cm depth of a Williams loam (ne-loamy, mixed, Typic Argiustolls) in dryland Montana, USA.
Enzyme-linked immunosorbent assay used to quantify antigenic response to basid iomycete cell walls, was
higher in NT than in CT in 4.75e2.00 mm size class in 2007 and higher in all classes and years at 0e5cm
depth, but was not different between tillage, years, and classes at 5e20 cm. The culturable bacteria from
microaggregates were subjected to a soil sedimentation assay to determine their soil binding capability.
The proportion of isolates which can function as soil aggregators was higher in NT than in CT at 0e5 cm but
was not different at 5e20 cm. Our results provide a rst insight into the benecial effects of dryland NT
compared to CT in reducing soil disturbance and residue incorporation and enriching the proportion of
microorganisms responsible for aggregation, especially at the soil surface.
Published by Elsevier Masson SAS.
1. Introduction
Planting in the no till (NT) system is characterized by sowing of
crops directly in the soil without tillage as opposed to conventional
tillage (CT) where planting is done in tilled soil with most of the
plant residues incorporated into the soil. Interaction between soil
aggregation and carbon dynamics in different tillage managements
has been intensively investigated; however, little is known about
the composition and proportion of microorganisms in aggregates.
Soil disturbance from tillage causes rapid disruption of macroag-
gregates (>0.25 mm size class) resulting in less accumulation of
crop-derived C in free microaggregates (0.25e0.02 mm size class)
that are formed within macroaggregates [51]. In NT, reduced soil
disturbance and accumulation of crop residue at the soil surface
result in improved soil structure and aggregation compared with
CT. The slower macroaggregate turnover leads to more C seques-
tration in NT because of higher C concentration in macroaggregates
than in microaggregates since macroaggregates are composed of
microaggregates and labile organic matter, such as roots, fungal
hyphae, and young plant residues [53]. The encapsulation of ne
particulate organic matter by mineral particles and microbial
products confers stability to the microaggregates under NT which
can be bound together by transient, labile organic matter to form
new macroaggregates [24].
Bacteria can survive in aqueous solutions within soil aggregate
pores where they get energy and nutrients from encapsulated plant
residues [60]. It has been reported that bacteria can bind soil
particles and maintain soil aggregate stability by producing in situ
extracellular polymeric substances (EPS) which are produced as
capsular material and peripheral slime with adhesive properties
[2,17]. The microbial basis of soil aggregation is likely similar to the
process described by Mueller [39]: (1) microbiological degradation
or alteration of organic materials, (2) attachment of cells to soil
particles, (3) production of EPS, and (4) concerted construction of
biolms by microbial communities, resulting in aggregation of soil
particles. The greater structural stability of the microaggregates
than macroaggregates has been partly attributed to the presence of
bacterial cells embedded in capsules of EPS often surrounded by
a layer of clay particles [20]. Numerous studies have been focused on
the effects of tillage on microbial biomass [22], microbial enzymatic
activity [28], microbial diversity and community structure [34,42],
but little information exists on culturable bacterial species that are
predominant in the microaggregates or the proportion of isolates
that can act as soil aggregators in relation to tillage system.
Several studies have reported the effects of tillage on arbuscular
mycorrhizal fungi [35] or non-mycorrhyzal fungi [37] but little is
*Corresponding author. Tel.: þ1 406 433 9415; fax: þ1 406 433 5038.
E-mail address: thecan.caesar@ars.usda.gov (TheCan Caesar-TonThat).
Contents lists available at ScienceDirect
European Journal of Soil Biology
journal homepage: http://www.elsevier.com/locate/ejsobi
1164-5563/$ esee front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejsobi.2009.12.004
European Journal of Soil Biology 46 (2010) 119e127
known about tillage effects on saprotrophic basidiomycete fungi
responsible for soil aggregation and nutrient cycling. Many sapro-
trophic basidiomycete fungi inhabiting fragmentary soil residues
are important in decomposition and nutrient-release processes
[6,43] because they secrete extracellular ligninolytic enzymes
(laccases, manganese peroxidases, and cellulases) that attack the
principal plant polymers lignin, cellulose or hemicellulose [59].
Electron micrographs show fungal hyphae can form extensive
networks in soil and are often covered with extracellular poly-
saccharides which hold microaggregates together [21]. Besides
their function in nutrient cycling, saprotrophic basidiomycetes can
bind soil particles into aggregates through physical enmeshment
and production of EPS [10] with adhesiveproperties [13]. Basid-
iomycete fungi have been demonstrated to play a role in aggrega-
tion of clay particles [19] and stabilization of soils [2,8]. Polyclonal
antibodies raised against cell walls of a russuloid basidiomycete
fungus isolated in agricultural cropland in eastern Montana, USA,
were demonstrated to cross react signicantly with many other
russuloid species that were efcient soil stabilizers [9]. They were
used to develop an enzyme-linked immunosorbent assay (ELISA)
which can sensitively detect minute amount of antigens derived
from the cell walls of these specic soil aggregating basidiomycete
fungi in cropland soil, as low as 200 ng g
1
soil [9]. In this study, we
report information on the survival and growth of saprotrophic
basidiomycete fungi in soil aggregates under dryland spring wheat
as inuenced by tillage in the northern Great Plains.
We hypothesized that at the beginning of the growing season,
the abundance of microbial populations functioning as soil aggre-
gators would be greater in dryland continuous spring wheat after
3 years under NT compared to CT. Our objectives were to (1) eval-
uate the effects of tillage at 0e5 and 5e20 cm soil depths on soil
aggregation and stability, (2) quantify the amount of soil aggre-
gating basidiomycete fungi from different aggregate-size classes
(4.75e2.00, 2.00e0.25, and 0.25e0.05 mm) using enzyme-linked
immunosorbent assay (ELISA); (3) isolate the predominant
heterotrophic bacteria in microaggregates (0.25e0.05 mm) by
cultivation and use fatty acid methyl ester (FAME) proling for their
taxonomic identication, (4) procure adequate biomass of these
isolates by cultivation and use soil sedimentation and photometry
to determine their ability to aggregate soil, and (5) use DNA
sequencing to conrm the identication of the soil aggregating
culturable bacterial species.
2. Materials and methods
2.1. Site description, treatments, and soil sampling
The experimental site was located about 8 km northwest
of Sidney, Montana, USA. Soil at the location was mapped as
a Williams loam (ne-loamy, mixed, superactive, frigid Typic
Argiustolls) [18]. Mean annual precipitation at the site is 320 mm,
with about 80% occurring from April through September. Prior to
initiation of this experiment, the site had been in a cereal grain-
summer fallow rotation under fall and spring tillage for several
decades.
This report provides results from a tillage treatment from
a larger study investigating the interactions of tillage and cultural
management systems on four crop rotations. The experiment was
initiated in 2004 in a randomized complete block design with three
replications. The whole plot treatment was tillage system,
conventional tillage and no tillage. Split-plot treatments were four
crop rotations with two cultural management systems. For this
study, only the effect of tillage on microorganism communities and
population under spring wheat with conventional cultural
management was considered. Individual split-plot size was 12 m by
12 m. Nitrogen fertilizer was broadcast to spring wheat at
78 kg N ha
1
as urea in mid-April each year, prior to tillage. Preplant
conventional tillage was conducted in tilled plots with a eld
cultivator equipped with C-shanks hooked to a 45 cm wide sweeps
and coil-tooth spring harrows with 60 cm bars. Conventional tillage
depth of about 7 cm was controlled by stabilizer wheels on the eld
cultivator frame. Spring wheat Reederwas planted at 78 kg seeds
ha
1
in mid-late April with a 3.1 m wide drill in a row spacing of
20.3 cm. The drill was equipped with double-shoot Barton (http://
www.exicoil.com/barton.asp) disk openers for low soil distur-
bance, single-pass seeding and fertilization. Phosphorus fertilizer
as monoammonium phosphate (11% N and 52% P) was applied at
56 kg P ha
1
and K fertilizer as muriate of potash (60% K) was
applied at 48 kg K ha
1
to spring wheat at planting in a band about
5 cm below and to the side of the seed row. Spring wheat residue
was evenly spread with a combine equipped with a chopper-
spreader at harvest each year.
Soil samples were collected with a hand probe (5 cm inside
diameter) from 5 places to a depth of 20 cm in the central rows of
each plot after removing surface residues in early spring of 2005,
2006 and 2007, separated into 0e5 cm and 5e20 cm depths, and
composited within a depth. Samples were processed within
24e48 h for determinations of soil aggregation and microbial
communities. Aggregates were separated by dry sieving of moist
soil after drying at 4
C[36] and the aggregate proportion
(g aggregate kg
1
soil) was measured in 4.75e2.00, 2.00e0.25, and
0.25e0.05 mm aggregate-size classes. Mean weight diameter
(MWD), used as an index of aggregate stability, was calculated
according to the procedure described by Kemper and Rosenau [29].
2.2. Isolation of bacteria
Culturable bacteria occurring at the highest population levels in
the microaggregates were isolated using spiral plating technique
according to [11] with slight modications. Briey, microaggregates
(1 g) were agitated in MgSO
4
buffer (0.1 M, pH 7.3) with glass beads
(0.5 mm) for 6 h at 4
C on a shaker at 200 rpm to release the
bacteria. Soil suspensions were diluted by a factor of 100 with
buffer and spiral plated on low nutrient R2A medium (Difco
Laboratories, Sparks, MD, USA) [44]. R2A medium was previously
used to grow a wider spectrum of bacteria to grow without sup-
pressing the slow-growing species, thereby achieving a higher
recovery of bacteria [34], and was also used to isolate surfactant
producing bacteria [40]. The basic concept of spiral plating was to
continuously deposit a known volume of sample on a rotary agar
plate in the form of an Archimedes spiral [23,26]. The amount of
sample decreased evenly while the dispensing stylus was moved
from the center to the edge of the rotating agar plate. From the
beginning to the end of the spiral at the periphery of the plate
represented a 3 logarithmic unit dilution [26] when 150
m
l aliquots
of soil suspensions were plated, thus 10
5
cells g
1
soil represented
a typical population level at which the colonies which were
considered the predominant isolates were obtained. For each year
and each soil depth (0e5cmor5e20 cm), a total of 66 colony-
forming units growing at the end of the spiral at the periphery of
the rotary agar plates were collected for each treatment (NT and CT)
after 48e72 h of incubation at 28
C. A total of 792 isolates were
cultured and puried for the two tillage treatments at the two soil
depths (0e5 and 5e20 cm) in 2005, 2006 and 2007. Among the 792
isolates, only 747 were identied by (FAME) proling (see 2.4) and
were assayed in soil sedimentation to determine their potential to
aggregate soil. Only the proportion of isolates which showed soil
aggregating ability was compared between the two tillage treat-
ments at the two different soil depths. All isolates were stored at e
80
C in LuriaeBertani medium amended with 15% glycerol.
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127120
2.3. Sedimentation assay
A sedimentation assay was used under controlled laboratory
conditions to screen the culturable bacterial isolates that showed
the ability to aggregate soil. Pure cultures of isolates (100 mg)
grown for 24e48 h on 1% TSBA (trypsin soy broth agar, Difco
Laboratories, Sparks, MD, USA) were washed once with deionized
water [32] in order to eliminate residual nutrients from the culture
medium. Cells were counted using a hemacytometer before adding
to glass tubes (20 150 mm, Corning, NY) containing 10 ml of
deionized water and 1.25 g of sieved soil (<0.05 mm size-class) to
obtain nal cell concentrations of 10
6
cells ml
1
. The cell concen-
tration of 10
6
cells ml
1
was used to amend soil for assay the
minimum cell concentration at which sedimentation of soil parti-
cles could occur, as established in preliminary studies with a range
of bacteria species. Soil collected along a stream bank at Sidney,
Montana (14% clay, 14% silt, and 72% sand) was used for the
aggregation assay with the bacteria because of its low shrink/swell
capacity [3]. Triplicate samples were prepared for each individual
identied isolate. A control without bacteria was prepared in
a similar manner for each individual isolate.
Tubes were vortexed for 10 s at 2250 rpm (Vortex Genie 2,
Scientic Industries, USA) and the mixture was allowed to settle for
5 min at room temperature. Images of the reected light (Universal/
Hi-vision uorescent light F32T8/TL735, Philips, NY, USA, light
intensity 7.435 0.064 Rad [watt/m
2
]) for multiple samples
were captured using a digital camera (Nikon, model D-80, Japan)
with night vision settings (near infrared, 800e1000 nm) at
24.78
C0.746. The captured images were calibrated by referring
white (255 in gray-scale value) and black (0 in gray-scale value)
image spots. Adobe Photoshop (version 7.0) was used for the
conversion of the images in a gray-scale value of each target solu-
tion into the reectance (expressed in %) that directly correlates to
relative differences of the solution density; 100% reectance for
maximum density and 0% for minimum density. A reectance ratio
(% reectance of the solution with bacterial cells added divided by
the reectance of control solution without cells added) was
calculated for each isolate and values were averaged for all the
isolates for each species. The solution with reectance ratio >1.5
was established as a threshold which represents species that
aggregate soil. Ratio below 1.5 represented the activities of species
that were inconsistent or marginal in aggregating ability. To assure
that identication made by FAME proling corroborates with
molecular-based methods of identication, the most efcient soil
aggregating species identied by FAME [species with a reectance
ratio >1.5 and highest similarity index (SIM)] were further pro-
cessed for DNA sequencing to conrm their identity and position in
a phylogenetic tree having strain types from the Ribosomal Data-
base Project [16].
2.4. Identication by FAME and DNA analyses
All predominant culturable bacterial isolates from micro-
aggregates were identied using fatty acid methyl ester (FAME)
proling [7]. FAMEs were obtained by saponication, methylation
and extraction following the MIDI system (Microbial Identication
System, Inc. Newark, NJ, USA). MIDI Microbial Identication Soft-
ware (Sherlock Aerobic Bacterial TSBA50 Library; Microbial ID Inc.)
was used for the identication of the isolates. Bacillus maroccanus
(ATCC # 25099) and Stenotrophomonas maltophilia (ATCC # 13637)
were used as references. Only scores of SIM 0.500 were consid-
ered a good match [41]. Isolates having SIM <0.500 or that were
unidentiable by FAME due to lack of information in MIDI Aerobic
Bacterial Library TSBA50 were not investigated in this study.
To corroborate identications made by MIDI, molecular-based
methods of identication were additionally used. DNA from the
isolates was extracted using a Qiagen (Valencia, CA, USA) DNeasy
Tissue kit. Polymerase chain reaction (PCR) amplication of the 16S
rRNA gene region used primers16S-27f (5
0
-GAGTTTGATCCTGGCT-
CAG-3
0
)[31] and 16S-960r (5
0
-GCTTGTGCGGGYCCCCG-3
0
)[45] with
the following cycling conditions: 95
C (10 min); 25 cycles of 94
C
(30 s), 56
C (30 s), 72
C (2 min); and then 72
C (2 min). A 50
m
L
reaction was performed for each isolate, and PCR products were
puried using a QIAquick PCR Purication kit (Qiagen, Valencia, CA,
USA). Puried templates were sequenced in two directions with an
ABI 3130 automated sequencer (Applied Biosystems, Foster City,
CA, USA), using the same primers listed above. Isolate DNA
sequences generated in this study are available from the corre-
sponding author, and were aligned using CLUSTALW [58].
Maximum Parsimony (MP) analysis of the data set was performed
using PAUP* v. 4.0b8 [57]. The heuristic MP search employed 500
random taxon addition sequences and the tree-bisection-recon-
nection (TBR) branch-swapping algorithm. All characters were
weighted equally and insertion/deletion events, regardless of their
length, were treated as one mutational event [50]. A 10,000 repli-
cate ‘‘fast’’ stepwise-addition bootstrap analysis was conducted to
assess clade support.
2.5. ELISA
The ELISA used for the detection and quantication of the
amount of specic soil aggregating basidiomycete fungi in soil has
been described previously in details according to Caesar-TonThat
et al. [9]. Soil samples (12.5 mg) were prepared in 1 mL of carbonate
buffer (20 mM NaHCO
3,
28 mM Na
2
CO
3
, pH 9.6). Absorbance was
read at dual wavelengths of 450/655 nm with a microplate reader
(Bio-Rad, Hercules, CA, USA). The amount of fungi was expressed in
m
gg
1
soil as determined from a standard curve generated from
known amounts of antigens from basidiomycete cell walls. The
aggregate samples were analyzed at least 3 times.
2.6. Statistical analysis
The Honestly Signicant Difference test of TukeyeKramer in
ANOVA procedure was used to analyze data on soil aggregate
distribution, soil sedimentation assay, and fungal quantication at
0e5 and 5e20 cm depths, with signicance level evaluated at
P0.05 using the JMP statistical software package (version 6.0,
2005, SAS Institute Inc., Cary, NC, USA). Tillage was considered as
the main plot, aggregate-size class as the split-plot, and year as the
repeated measure treatment for the analysis. The PROC GLIMMIX
(SAS Institute Inc., Cary, NC, USA) statistical procedure that utilizes
a logit model [33] was used to t the proportion of the predominant
aggregating soil bacterial isolates under the 2 tillage managements
(CT and NT) over 3 years (2005e2007) and 2 soil depths (0e5 and
5e20 cm). Tillage management and cropping year wereanalyzed as
factors affecting the proportion of culturable bacterial isolates that
can function as soil aggregators using a generalized linear model. To
calculate the species diversity identied by FAME proles, the
Shannon and Weaver [48] index H ¼P(ni/N) (log ni/N) was
used, in which ni is the number of individuals observed for each
species and N is the total number of individuals observed in each
treatment. Rarefaction curves were constructed to compare the
richness of the soil aggregating culturable species of the 2 tillage
managements (CT and NT) over 3 years (2005e2007) and 2 soil
depths (0e5 and 5e20 cm). Rarefaction calculations were done
using the software Species Diversity and Richness III, version 3.03
[55]. The program uses the rarefaction equations described by Heck
et al. [25].
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127 121
3. Results
The distribution of soil among the different size classes of
aggregates showed that at 0e5 cm soil depth, aggregate proportion
in the 4.75e2.00 mm size class was in general greater in NT than in
CT in all 3 years but was only signicantly greater in the third
year of the study (2007), with a subsequent decrease in the
2.00e0.25 mm size class (Table 1). The mean-weight diameter
(MWD) was in general greater in NT than in CT in all 3 years, but
was only signicantly greater in 2007. Overall, aggregate propor-
tion was 1.6 times greater in NT (333.74 g kg
1
) than CT
(209.00 g kg
1
) and greater in 2005 and 2007 than in 2006 in the
4.75e2.00 mm size class. Similarly, MWD was overall greater in NT
(1.86 mm) than in CT (1.57 mm) and greater in 2005 and 2007 than
in 2006. At 5e20 cm depth, aggregate proportion and MWD were
not inuenced by tillage (Table 2). Both aggregate proportion in the
4.75e2.00 mm size class and MWD were greater in 2007 than 20 05
and 2006.
Table 3 indicates the amount of soil aggregating basidiomycete
fungi detected by ELISA in aggregate-size classes of 4.75e2.00,
2.00e0.25, and 0.25e0.05 mm from soil collected under CT and NT
in spring 2005, 2006, and 2007 at 0e5 and 5e20 cm depth. At
0e5 cm soil depth, the amount of fungi was greater in NT than in CT
in 2006 and 2007 in the 4.75e2.00 mm class; for example, there
was 1.3 times increase in 2006 (254.65
m
gg
1
under NT versus
192.60
m
gg
1
under CT) and a1.6 times increase in 2007
(290.22
m
gg
1
under NT versus 186.73
m
gg
1
under CT). In 2007,
the amount of fungi was greater under NT than CT in the
2.00e0.25 mm class (331.30
m
gg
1
under NT versus 201.76
m
gg
1
under CT) and in the 0.25e0.05 mm class (236.32
m
gg
1
under NT
versus 190.16
m
gg
1
under CT). Overall, fungi amount was greater
in NT than in CT and greater in 2006 than in 2005 in 2.00e0.25 and
0.25e0.05 mm size classes. At 5e20 cm depth, basidiomycete
amount was not different between tillage, aggregate-size classes,
and years.
The in vitro soil sedimentation assay has allowed the culturable
bacterial isolates (747) to be characterized for their capability to
aggregate soil. Results show evidence that tillage and year signi-
cantly inuenced the proportion of the predominant culturable soil
aggregating bacteria in microaggregates and that the interactions
tillage soil depth and year soil depth were signicant (Table 4).
Tests for the interaction of tillage management and soil depth
indicate a tillage effect at 0e5 cm soil depth but not at 5e20 cm
(Fig. 1); at 0e5 cm soil depth, the estimated proportions of the
culturable soil aggregating bacteria were 0.09 under CT and 0.29
under NT. Tests for the interaction of year and soil depth indicate
a year effect at 5e20 cm and not at 0e5 cm depth (Fig. 2); at
5e20 cm, the proportions of the culturable soil aggregating bacteria
were 0.07 in 2005 and 0.31 and 0.27 in 2006 and 2007 respectively.
Among the 747 predominant culturable bacterial isolates from
the two tillage treatments (CT and NT) at two soil depths (0e5 and
5e20 cm) in 2005, 2006 and 2007 which were identied to species
by FAME proling, only 160 isolates were selected because they
demonstrated efciency to aggregate soil particles by the soil
sedimentation assay. Table 5 indicates the distribution of these
culturable soil aggregating isolates (species abundance) into 12
different gram-negative species (
a
,
b
, and
g
Proteobacteria, and
Flavobacteria) and 14 gram-positive species (Bacilli including
family Bacillaceae, Brevibacillaceae, Planococcaceae, Paenibacilla-
ceae, and family Microbacteriaceae of Actinobacteria). At 0e5cm
depth, the number of soil aggregating culturable species found in
microaggregates under NT was 2.2 times higher than in CT and at
5e20 cm depth,1.3 times more species were found in CT than in NT.
At 0e5 cm soil depth, species diversity indices of NT 0e5cmwas
greater than CT 0e5atP¼0.089, but at 5e20 cm depth, there was
no difference between the two tillage treatments. In the compar-
ison between soil depths, diversity index of NT 0e5 cm was greater
than NT 5e20 cm at P0.05 and there was no signicant difference
between CT 0e5 cm and CT 5e20 cm. Fig. 4 indicates the rarefac-
tion curves created for the treatments NT 0e5 cm, CT 0e5 cm, NT
5e20 cm, and CT 5e20 cm. Rarefaction curves indicate that for all
treatments, a plateau was approached and that species accumula-
tions are nearly an asymptote (zero or low slope). Comparison of
the treatments shows that the species number was the highest in
NT 0e5 cm and the lowest in CT 0e5 cm.
To corroborate the identication by FAME of the soil aggregating
species (Table 5), the 16S rRNA amplication region of all the
species were DNA sequenced and analyzed. Fig. 3 indicates the
position of these isolates in the maximum parsimony tree showing
the relationship to reference gram-negative and gram-positive
bacteria. In general, DNA analysis of the isolate sequences matched
with the FAME analysis. Among the discrepancies found between
the two approaches of identication, DNA analysis placed the
members of families Paenibacillaceae (isolate 06ZCL1B7 and
07TCL1B3) and Brevibacillaceae (06ZCL2C12 and 06ZCL1B6) in the
Bacillaceae family cluster.
Table 1
Effects of tillage on soil aggregate proportion and mean-weight diameter of aggre-
gates at the 0e5 cm depth.
Year Tillage
a
aggregate proportion in size class (g Kg
1
soil) Mean-weight
diameter (mm)
4.75e2.00 mm 2.00e0.25 mm 0.25e0.05 mm
2005 CT 319.43ab
b
658.27cd 20.83ab 1.82bc
NT 411.77a 569.77d 16.17ab 2.04ab
2006 CT 68.73d 881.10a 48.67a 1.23e
NT 159.13cd 821.63ab 17.83ab 1.46de
2007 CT 238.83bc 738.50bc 20.83ab 1.64cd
NT 430.33a 552.83d 14.50b 2.08a
Means
2005 365.60a 614.02b 17.77a 1.93a
2006 113.93b 851.37a 33.25a 1.35b
2007 334.58a 645.67b 17.67a 1.93a
CT 209.00b 759.09a 29.62a 1.57b
NT 333.74a 648.08a 16.17a 1.86a
Signicant Difference procedure of Tukey and Kramer at P0.05.
a
Tillage: CT, conventional till; NT, no tillage.
b
Within a set in a columm, numbers followed by different letters are signicantly
different by the Honesty.
Table 2
Effects of tillage on soil aggregate proportion and mean-weight diameter of aggre-
gates at the 5e20 cm depth.
Year Tillage
a
Aggregate proportion in size class (g Kg
1
soil) Mean-weight
diameter (mm)
4.75e2.00 mm 2.00e0.25 mm 0.25e0.05 mm
2005 CT 335.33ab
b
631.67ab 20.33a 1.85ab
NT 317.67ab 661.67ab 19.00a 1.80ab
2006 CT 182.57b 805.33a 8.70b 1.60b
NT 211.13b 782.03a 6.80b 1.53b
2007 CT 442.47a 554.87b 0.80b 2.12a
NT 440.43a 557.03b 0.80b 2.11a
Means
2005 326.50b 646.67b 19.67a 1.83b
2006 196.85c 793.68a 7.75b 1.56c
2007 441.45a 555.95b 0.80b 2.12a
CT 320.12a 663.96a 9.94a 1.83a
NT 323.08a 666.91a 8.87a 1.84a
Signicant Difference procedure of Tukey and Kramer at P0.05.
a
Tillage: CT, conventional tillage; NT, no tillage.
b
Within a set in a columm, numbers followed by different letters are signicantly
different by the Honesty.
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127122
4. Discussion
Our data indicate that large macroaggregate (4.75e2.00 mm
class) formation and mean-weight diameter (MWD) (Table 1) were
in general higher in NT than in CT in all three years and there was an
increasing trend for the amount of basidiomycete fungi (Table 3)
and proportion of aggregating predominant culturable bacteria
(Fig. 2) in aggregates under NT than CT at 0e5 cm soil depth. This
suggests that NT spring wheat system in dryland eastern Montana,
USA, improves soil aggregation and aggregate stability and
increases the amount/proportion of soil aggregating microorgan-
isms in aggregates at the soil surface. In similar dryland conditions
in the Northern Great Plains, Sainju et al. [47] investigated tillage
effects on dryland spring wheat residue and soil carbon fractions
and proposed that lower precipitation, shorter growing season and
slower decomposition of residue in the soil could cause minimum
effect of tillage on crop residue production and soil organic C (SOC)
content in aggregate-size fractions, at 0e5 cm. However, they
demonstrated that macroaggregate formation (4.75e2.00 mm
class) and soil particulate organic carbon (POC) concentrations in
macroaggregates are higher in NT than in CT at the 0e5 cm.
Although SOC and POC concentrations were not measured in our
study, we also expect that POC is higher in macroaggregates under
NT than CT. At the subsurface layer (5e20 cm), there was no
inuence of tillage in soil aggregation and aggregate stability
between NT and CT in all three years (Table 2) and tillage had no
effect on the amount/proportion of the soil aggregating basidio-
mycete fungi and culturable bacteria, probably because of the
limited tillage depth (about 7 cm) in CT that reduced fresh residue
incorporation into the soil and deep soil disturbance.
The greater proportion of saprotrophic basidiomycete fungi in
soil aggregates in NT than in CT at 0e5 cm depth suggests that NT
cultivation induced a means of survival for soil basidiomycete fungi
better than CT systems. An NT system with inputs of high lignin
content wheat residues (225 g kg
1
)[30] would provide a good
source of nutrients for these fungi that are known to produce lig-
ninolytic degrading enzymes to break down lignied plant mate-
rials for their utilization. They are sensitive to soil disturbance due
to tillage [9], as tillage decreases basidiomycete populations in
response to increase in mitosporic species, such as Penicillium spp.
and Fusarium spp., which can withstand soil disturbance by
increasing the production of survival spores [56] and which also
have much smaller mycelial individuals often occupying single
organic particles or seeds in soil. They can also probably survive and
grow better in NT than in CT in the semi-arid regions dryland
cropping system in eastern Montana, since NT generally provides
a more favorable environment for fungi to grow by increasing water
Table 3
Effect of tillage on the amount of basidiomycete fungi in aggregate-size classes at 0e5 and 5e20 cm soil depth.
Year Tillage
a
Amount of basidiomycetes in aggregate size class (
m
gg
1
aggregates)
0e5cm 5e20 cm 0e5cm 5e20 cm 0e5cm 5e20 cm
4.75e2.00 mm 2.00e0.25 mm 0.25e0.05 mm
2005 CT 197.47c
b
169.26a 174.49c 151.80a 186.60b 149.23a
NT 228.60bc 187.72a 211.56bc 172.41a 223.23ab 174.42a
2006 CT 192.60c 175.20a 222.15bc 181.65a 209.27ab 171.41a
NT 254.65ab 200.40a 298.88ab 200.47a 247.06a 182.15a
2007 CT 186.73c 163.11a 201.76c 183.44a 190.16b 153.16a
NT 290.22a 201.90a 331.30a 194.39a 236.32a 173.56a
Means
2005 213.04a 178.50a 193.03b 162.11a 205.01b 161.82a
2006 223.62a 187.80a 260.52a 191.06a 228.17a 176.78a
2007 238.47a 182.50a 266.53a 188.91a 213.24ab 163.36a
CT 192.27b 169.19a 199.47b 172.69a 195.41b 157.93a
NT 257.82a 196.68a 280.58a 189.09a 235.54a 176.71a
Signicance
Year * * * * * *
Tillage *** * *** * *** *
year tillage * * ** * * *
*Signicance at P0.05; **signicant at P0.01 and ***signicant at P0.001.
a
Tillage: CT, conventional tillage; NT, no till.
b
Within a set in a column, numbers followed by different letters are signicantly different by the Tukey and Kramer Honestry Signicant Difference test.
Table 4
Analysis of variance for the proportion of the predominant soil aggregating bacteria
isolated from microaggregates.
Source F value Pr >F
Tillage
a
28.20 <0.0001
Year
b
5.07 0.02
Tillage year 0.76 0.48
Soil depth
c
1.03 0.32
Tillage soil depth 15.51 0
Year soil depth 5.83 0.01
Year Tillage soil depth 0.8 0.46
a
Tillage: no till and conventional tillage.
b
Cropping years: 2004e2007.
c
Soil depth: 0e5 cm and 5e20 cm.
Fig. 1. Proportion of culturable soil aggregating bacteria as affected by tillage (no till,
NT and conventional tillage, CT) and soil depth (0e5 and 5e20 cm) interaction.
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127 123
content compared with CT in the dryland cropping system [27,46].
It has been reported that some basidiomycetes are adapted to
desiccating environments by their ability to produce copious
amount of mucilage allowing them to grow at moisture potentials
as low as 4to8 MPa [61].
Saprotrophic basidiomycetes are well documented to control
the mineral and energy cycling of plant litter and to function as
regulators for the release of pulse nutrients to the soil, in both time
(immobilization of nutrients into fungal biomass [14]) and space
(translocation and redistribution of carbon and nutrients through
hyphal assemblages called rhizomorphs or strands [12]). Under NT
dryland spring wheat system where residue inputs are frequent (i.e.
yearly) and the soil at 0e5 cm depth is not disturbed, the increase of
basidiomycete populations in all aggregates size classes suggests
that, during the course of residue decomposition, the fungi forms
an extensive branching mycelial network [1] which can entangle
soil particles to form aggregates where they probably reallocate
and slowly redistribute nutrients immobilized within their tissues.
These activities could provide more easily available carbon and
minerals to bacteria inhabiting the microaggregates for their
growth and may favor the growth and survival of specic bacterial
populations involved in soil aggregation. Although no evidence was
provided in this study to support the hypothesis that more carbon
resources and minerals were transported to the microaggregates in
NT treatment than CT by basidiomycete fungi, our data are in
accordance with Six et al. [52] who demonstrated that there is
greater accumulation of crop-derived C in free microaggregates in
NT than in CT.
Under CT with crop residues incorporated every year into the
soil, the contact area between soil and organic matter increases and
wheat residues decompose at higher rates with a more rapid loss of
nutrients [15] and mineral leachate [54].Saprotrophicbasidiomycete
populations could suffer under CT because they rely on complex
plant debris for a major part of their diet, contrary to shorter-lived
and ephemeral molds that prefer simple carbohydrates from
readily decomposed materials for consumption which are more
Fig. 2. Proportion of culturable soil aggregating bacteria as affected by year
(2005e2007) and soil depth (0e5 and 5e20 cm) interaction.
Table 5
Distribution of the predominant culturable soil aggregating bacterial isolates from microaggregates of soil cropped to continuous spring wheat from 2004 to 2007 under
different tillage
a
management, at 0e5 and 5e20 cm soil depth.
Class Identication by FAME CT 0e5
a
NT 0e5CT5e20 NT 5e20 Reectance ratio
b
Identication by
DNA sequencing
c
Gram negative
a
-Proteobacteria Rhizobium radiobacter 0 1 0 0 2.065 5ZCL1a1-1 (0.766)
Brevundimonas vesicularis 0 0 3 0 1.543 6TCL2b23 (0.466)
Novosphingobium capsulatum 0 0 4 1 2.926 6ZCL2b20 (0.627)
Sphingomonas sanguinis 0 0 0 2 2.001 6ZCL2c8 (0.699)
b
-Proteobacteria Variovorax paradoxus 0 2 0 0 1.657 7ZCL1a14 (0.510)
Burkholderia cepecia 0 0 1 0 2.077 6TCL2b7 (0.671)
Vogesella indigofera 0 0 1 0 1.579 5TCL1a3-3 (0.502)
g
-Proteobacteria Pseudomonas uorescens biotype A 0 2 0 0 1.599 6ZCL1a15 (0.629)
Flavobacteria Zobellia uliginosa 0 1 0 0 1.548 7ZCL1c9 (0.404)
Chryseobacterium balustinum 0 3 0 1 1.966 7ZCL1c22 (0.693)
Chryseobacterium indoltheticum 2 7 1 2 1.991 7ZCL1c21 (0.682)
Flavobacterium hydratis 0 1 0 0 1.974 5ZCL1a3-5 (0.379)
Gram positive
Bacilli (Bacillaceae) Bacillus atrophaeus 2 4 1 1 4.310 5TCL1a10 (0.890)
Bacillus cereus 0 1 0 0 6.075 6ZCL1b10 (0.591)
Bacillus exus 0 2 3 6 1.799 6TCL2a3 (0.585)
Bacillus niacini 1 2 0 1 2.280 NA
Bacillus pumilus 2 7 4 6 1.943 7ZCL2b18 (0.710
Bacilli (Brevibacillaceae) Brevibacillus choshinensis 3 9 16 18 2.592 6ZCL2c12 (0.867)
Brevibacillus parabrevis 1 4 5 3 5.282 6ZCL1b6 (0.723)
Bacilli (Planococcaceae) Kurthia sibirica 0 1 0 0 2.856 6ZCL1b18 (0.481)
Bacilli (Paenibacillaceae) Paenibacillus azotoxans 0 0 1 0 1.909 7TCL2a15 (0.671)
Paenibacillus macerans 1 0 0 0 4.337 7TCL1b3 (0.809)
Paenibacillus pabuli 5 3 1 0 1.954 6TCL1b9 (0.833)
Paenibacillus validus 0 2 4 3 4.620 6ZCL1b7 (0.752)
Actinobacteria (Microbacteriaceae) Microbacterium hominis 0 1 0 0 1.953 5ZCL1a2-2 (0.723)
Microbacterium lacticum 0 0 1 0 1.671 6TCL2a6 (0.926)
Diversity index
d
1.92b* 2.60 a* 2.18 ab 1.90 b
*P¼0.089, otherwise P0.05.
a
Management: CT 0e5 and NT 0e5, conventional till and no till at 0e5 cm soil depth; CT 5e20 and NT 5e20, conventional till and no till at 5e20 cm soil depth.
b
Average of reectance ratio (reectance measurement of soil suspension containing 106 cells/mL after 5 min of sedimentation time divided by reectance measurement of
soil suspension without bacteria added) obtained from all isolates of each soil aggregating species.
c
Isolates from species that have the highest similarity index and the reectance ratio >1.5 were DNA sequenced and analyzed. Relationship of unknown isolates and type
specimens are shown in Fig. 1. The similarity index indicated between parentheses was based on MIDI Aerobic Bacterial Library TSBA50; NA, not available.
d
ShannoneWiener's diversity index.
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127124
available under CT. The rapid decomposition of residues in CT in the
surface soil layer could result in low storage of nutrients in
microaggregates, thus affecting the growth and survival of bacterial
communities that require sufcient energy resources to produce
extracellular components acting as soil binding agents. A possible
explanation for higher proportions of soil aggregating bacteria in CT
5e20 cm compared to CT 0e5 cm (0.17 versus 0.09) could be that
below tillage depth (approximately 7 cm), the negative impact of CT
treatment observed in the upper soil layer (0e5 cm soil depth) is
alleviated by slow decomposition and the rhizosphere effect (rhi-
zodeposits), which provide necessary nutrient resources for soil
aggregating bacteria to proliferate. In addition, the non signicant
difference in the amount of basidiomycetes in aggregates of NT
5e20 cm and CT 5e20 cm (Table 3) was probably because these
saprotrophic fungi are more frequently found in plant debris at or
near the soil surface than in greater depth. However, saprotrophic
fungi associated to water stable aggregates could be found colo-
nizing dead roots of Pinus radiata D. Don. planted on sandy soils to
a depth of several feet [5].
The relatively higher proportions of culturable soil aggregating
bacteria in NT compared with CT at 0e5 cm depth in micro-
aggregates (Fig. 1) suggests that NT increased the population of
these specic bacteria at the surface soil probably by increasing
particulate organic carbon (POC) content that constitutes the
source of energy and nutrients for microorganisms [4,52].As
a result, specic bacterial species probably increased mucilage
production responsible for increased aggregation under NT.
Mendes et al. [36] reported that the greatest rates of enzyme
activities tended to be found inside the microaggregates where the
microorganisms are biologically active and signicantly involved in
processing soil C [27].
Rarefaction analysis created for each of the tillage treatments
over 3 years at the 2 soil depths (Fig. 4) revealed that the culturable
soil aggregating bacterial species analyzed in this study were
sufcient to describe the bacterial diversity at the species level. The
rarefaction curves showed indication of reaching nearly an
asymptote, thus demonstrating that the number of culturable
bacteria was sufciently sampled and that no species can be
expected if additional isolates were to be analyzed. The slope of CT
0e5 cm curve was shallower than those of the curves obtained for
NT 0e5 cm, NT 5e20 cm and CT 5e20 cm, suggesting that a small
number of species were dominating in the treatment CT 0e5 cm.
Many culturable bacteria identied by FAME and demonstrated
to have high potential for soil aggregation from this study were also
found in other studies. Sessitsch et al. [49] demonstrated the
presence of Rhizobium and Shingomonas spp of the class of
a
Pro-
teobacteria in soil particle fractions of various size using cultiva-
tion-independent techniques. Miller et al. [38] found Pseudomonas
spp in soil aggregates from grassland and arable cropping systems.
Bacillus cereus,Bacillus pumilus,Brevibacillus choshinensis, and
species from genera Pseudomonas and Chryseobacterium were also
found previously in soil aggregates in a previous study [11].
Fig. 3. Single most parsimonious tree, 2878 steps in length, resulting from the analysis of 16S rRNA gene sequences (838 aligned bases; 364 of these parsimony informative) from 12
soil-aggregating culturable bacteria and 17 known type strains of related bacteria. Isolates from this study are shown in bold, while taxa used as phylogenetic placeholders (shown
in italics, with GenBank accession numbers following specic epithet) are from the Ribosomal Database Project II Hierarchy Browser collection of sequenced Type Strains [16].
Bootstrap values (>50%) are shown above branches. CFB, Cytophaga-Flavobacterium-Bacteroides group.
Fig. 4. Rarefaction curves constructed for the different tillages treatments (NT, no
tillage and CT, conventional tillage) at different soil depths (0e5 cm and 5e20 cm)
showing the expected number of species as a function of the number of soil aggre-
gating culturable bacteria sampled.
Caesar-TonThat et al. / European Journal of Soil Biology 46 (2010) 119e127 125
In recent years, culture-independent methods used to study
bacterial communities have received particular attention because
the majority of soil bacteria, i.e., more than 99%, are not accessible
by cultivation methods. However, molecular approaches are not yet
fully feasible to establish the potential to form macroaggregates of
a microbial community. Our approach using predominant cultur-
able isolates from microaggregates combined with measurements
of their soil aggregative ability appears to be a good starting point
to provide information on microbial populations that can function
as soil aggregators, since no molecular method is available to test
for soil aggregation as this metabolic potential can have different
genetic backgrounds. In addition, we acknowledge that the present
study is on a single dryland soil and that further investigations on
a variety of dryland soils are necessary. Also, more information on
the inuence of seasonal change on the abundance of specic
microorganisms associated with aggregation is necessary to
understand the variations in their communities under different
tillage, crop rotation, and systems in semi-arid environments.
Acknowledgments
The authors express their sincere appreciation to L. L. Solberg for
technical assistance and to Dr. Mark West for his statistical analysis
assistance. We also acknowledge Dr. J. Jabro, Dr. W. Shelver, and
Dr. Tim Bourett for their constructivecomments on the manuscript.
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