microbial community diversity in agroforestry and grass vegetative filter strips
TRANSCRIPT
Microbial community diversity in agroforestry and grassvegetative filter strips
Irene M. Unger • Keith W. Goyne •
Robert J. Kremer • Ann C. Kennedy
Received: 12 April 2012 / Accepted: 9 August 2012 / Published online: 18 August 2012
� Springer Science+Business Media B.V. 2012
Abstract Vegetative filter strips (VFS) have long
been promoted as a soil conservation practice that yields
many additional environmental benefits. Most previous
studies have focused primarily on the role of vegetation
and/or soil physical properties in these ecosystem
services. Few studies have investigated the soil micro-
bial community of VFS. Therefore, we examined
potential differences in soil microbial community
characteristics of claypan soil planted to VFS with
differing vegetation and a traditional row-crop system in
a maize–soybean rotation. Samples were tested for soil
microbial function and community structure using
dehydrogenase and fluorescein diacetate (FDA)
hydrolysis enzyme assays and phospholipid fatty acid
(PLFA) analysis, respectively. The grass VFS soil
exhibited the greatest dehydrogenase activity levels and
FDA activity was greater in the grass and agroforestry
(i.e., tree–grass) VFS soils relative to the cropland soil.
The PLFA analysis revealed community structural
differences underlying these functional differences.
The agroforestry VFS soil was characterized by a
greater proportion of total bacteria, gram-negative
bacteria, anaerobic bacteria and mycorrhizal fungi than
the cropland soil. The grass VFS soil shared some
characteristics with the cropland soils; but the grass VFS
supported greater mycorrhizal fungi and protozoa
populations. This work highlights differences in soil
microbial function and community structure in VFS
relative to cropland soil 12 years post VFS establish-
ment. It also enhances our fundamental knowledge
regarding soil microorganisms in VFS, which may aid in
explaining some ecosystem services provided by VFS
(e.g., decomposition of organic agrichemicals).
Keywords Dehydrogenase enzyme activity �Fluorescein diacetate hydrolysis enzyme activity
(FDA) � Phospholipid fatty acid analysis (PLFA) �Soil microbial community �Vegetative filter strips (VFS)
Introduction
Vegetative filter strips (VFS) have long been promoted
as a soil conservation practice that yields many
I. M. Unger (&)
Department of Biology and Environmental Science,
Westminster College, 501 Westminster Avenue, Fulton,
MO 65251, USA
e-mail: [email protected]
K. W. Goyne
Department of Soil, Environmental and Atmospheric
Sciences, University of Missouri, 302 ABNR Bldg.,
Columbia, MO 65211, USA
R. J. Kremer
USDA-ARS, Cropping Systems and Water Quality Unit,
University of Missouri, 302 ABNR Bldg., Columbia,
MO 65211, USA
A. C. Kennedy
USDA-ARS, Land Management and Water Conservation
Unit, Washington State University, 231 Johnson Hall,
Pullman, WA 99164, USA
123
Agroforest Syst (2013) 87:395–402
DOI 10.1007/s10457-012-9559-8
additional environmental benefits. Key ecosystem
services of VFS may be attributed to the soil microbial
community; however, little is actually known of how
these communities are structured or of how their
community structure relates to the functions they
provide. Likewise, the soil microbial community of
claypan soils has not been studied extensively. Clay-
pan soils are characterized by an abrupt argillic subsoil
horizon, dominated by smectite clay, which restricts
and alters water flow patterns; these soil properties
provide a unique and potentially stressful environment
for soil microorganisms. Installation of upland VFS
within claypan agroecosystems may create a more
favorable microbial environment and thus enhance
nutrient cycling in these systems. Previous soil
microbial community studies of our system (i.e.,
claypan planted to VFS), have focused primarily on
community function as identified by soil enzyme
assays (Udawatta et al. 2008; 2009). These studies
have found differences in enzyme activities between
claypan soils under no-till crop rotation and claypan
soils with VFS and suggest the potential of enhanced
nutrient cycling in the claypan soils planted to VFS
(Udawatta et al. 2008; 2009).
Despite the importance of the soil microbial
community and its role in nutrient cycling, many
studies of VFS systems focus instead on the influence
of vegetation and associated soil physical properties
on other ecosystem services provided by these
systems. For example, perennial vegetation and its
enhanced root systems are implicated in reduced
erosion, increased assimilation of fertilizers and
pollutants (Borin et al. 2010; Lovall and Sullivan
2006; Schultz et al. 2000), degradation of herbicides
(Lin et al. 2011), and riparian and stream habitat
quality (Osborne and Kovacic 1993). Vegetative filter
strips have also been shown to increase biodiversity,
provide wildlife habitat and corridors, and have
positive effects on stream habitat (Lovall and Sullivan
2006) and water quality (Lerch et al. 2005; Udawatta
et al. 2002).
Vegetative filter strips are an important conserva-
tion measure whether planted along riparian corridors
or integrated into upland agroecosystems. They indeed
may be quite valuable for agricultural practices in the
claypan soil region. The objective of this research is to
study soil microbial community characteristics of
agroforestry (i.e., tree–grass) VFS, grass VFS, and
row-crop (i.e., corn–soybean rotation under no-till
management) systems in claypan soils. We broaden
the focus of previous research on this system to
determine if soil microbial community structure, like
soil microbial community function, differs among
these systems. We hypothesize that both VFS systems
will support more robust microbial communities than
the row-crop system. In addition, we anticipate that the
microbial community in the agroforestry VFS will be
structured differently than that of the grass VFS and of
the row-crop system.
Materials and methods
Study site
Soils for this study were collected from the paired
watershed study site at University of Missouri’s
Greenley Memorial Research Center, in north-central
MO, USA (40�010N, 92�110W). This site consists of a
1.65 ha control watershed that lacks VFS and two
watersheds where VFS have been implemented: (1) a
4.4 ha watershed with agroforestry VFS and (2) a
3.16 ha watershed with grass VFS (Fig. 1). The three
watersheds are north facing with a 0–3 % slope; they
have been planted in a corn–soybean rotation with no-
till management since 1991. Previous research at the
site indicates no effect of landscape position on soil
organic C, total N (Veum et al. 2011) or water-stable
aggregates (Veum et al. 2012). Upland filter strips
(4.5 m width) were established along the contour in
1997 and no-till cropping continues to occur in
23–36 m strips between the VFS. Vegetative filter
strips were planted with a grass-legume mixture
consisting of redtop (Agrostis gigantea Roth), brome
grass (Bromus spp.) and birdsfoot trefoil (Lotus
corniculatus L.). The agroforestry VFS watershed
also features a mixture of oak species (i.e., pin oak
(Quercus palustris Muenchh), swamp white oak (Q.
bicolor Willd.) and bur oak (Q. macrocarpa Michx.))
planted in alternating fashion 3 m apart in the center of
the filter strips. Soils at the site have formed from loess
overlying weathered glacial till (Watson 1979). Put-
nam silt loam (fine, smectitic, mesic Vertic Alba-
qualfs) and Kilwinning silt loam (fine, smectitic,
mesic Vertic Epiaqualfs) have been mapped by the
USDA in the summit/shoulder and backslope/foots-
lope positions, respectively (Watson 1979). A distinc-
tive feature in soils at this site is presence of an abrupt,
396 Agroforest Syst (2013) 87:395–402
123
well-developed argillic horizon (i.e., claypan) occur-
ring at a depth 20–62 cm below the soil surface
(Udawatta et al. 2006). For further study site infor-
mation see Veum et al. (2009) and references therein.
Soil collection and microbial analysis
Soil samples (three per watershed at 0–10 cm depth)
were collected in October 2009 from all watersheds at the
shoulder landscape position (Fig. 1). In the VFS water-
sheds, soils were collected from the second filter strip
from the top of the watershed and soils were sampled at an
equivalent landscape position in the control watershed.
Samples from the agroforestry VFS were collected
approximately 0.6 m from the base of the trees to avoid
root-wad soil from the nursery and weed matting that
extends up to 0.5 m from the base of each tree.
Each soil sample (n = 9; three per watershed) was
moist sieved (\2 mm) and then partitioned into seven
subsamples for soil microbial analysis. Dehydroge-
nase and fluorescein diacetate (FDA) hydrolysis
enzyme assays were used to quantify soil microbial
community function. Dehydrogenase was used to
approximate the respiratory activity for soil microor-
ganisms (Tabatabai 1994). Procedures for this assay
generally follow Kremer and Li (2003). Fluorescein
diacetate, which is a general substrate for several
hydrolytic enzymes, including esterases, lipases and
certain proteases (Dick 1997), was used to estimate
general hydrolytic activity necessary for decomposi-
tion (i.e., C mineralization). Procedures for this assay
follow Schnuurer and Rosswall (1982) as modified by
Kremer and Li (2003).
The soil microbial community structure was deter-
mined using PLFA analysis. Procedures generally
follow Bligh and Dyer (1959) as described by Petersen
and Klug (1994). Total lipid extracts were fractionated
and the polar-lipid fractions were transesterified with
mild alkali to recover PLFA as methyl esters (Ibekwe
et al. 2002). The PLFAs were separated, quantified and
identified on a gas chromatograph fitted with flame
ionization detection, and peak chromatographic
responses were translated into molar responses using
an internal standard. Each sample peak was compared
against a database of known microbial fingerprints.
Standard markers (Pritchett et al. 2011) were used to
determine responses attributed to total bacteria, gram-
positive bacteria, gram-negative bacteria, anaerobic
bacteria, total fungi, mycorrhizae, and protozoa
(Table 1) (Unger et al. 2009). Total biomass was
determined from mole response calculations; for these
the mole responses for each sample were summed and
then multiplied by an extraction efficiency factor
based on the internal standards added to each run
(Bailey et al. 2002). The ratio of saturated to
monounsaturated fatty acids (MFA) were calculated
as shifts in this ratio are known to occur in some
bacteria under physiologically stressful conditions
(i.e., low C, low O2, high acidity, and high temper-
ature) (Bossio and Scow 1998); these results were
grouped and reported as saturated:monounsaturated
fatty acid ratio (S:MFA) (Fierer et al. 2003; Bossio and
AgroforestryVFS
Grass VFS
ControlWatershed
Fig. 1 Paired watershed study site at University of Missouri’s
Greenley Memorial Research Center, Novelty, MO, USA and
watershed map (courtesy K. Veum). Grey bands indicate
location of grass or agroforestry vegetative filter strips (VFS);
stars indicate sampling locations
Agroforest Syst (2013) 87:395–402 397
123
Scow 1998; Kieft et al. 1997). In addition, the
bacteria:fungi ratio (B:F) was calculated as described
in Unger et al. (2009).
Data analysis
Enzyme assay data were analyzed using ANOVA
(SAS Proc Mixed) to test for differences in microbial
community function in the agroforestry VFS, grass
VFS and row-crop soils; enzymes were analyzed
separately. Mol response data from the PLFA analysis
were analyzed similarly. Each PLFA response vari-
able (i.e., total biomass, total bacteria, gram-positive
bacteria, gram-negative bacteria, anaerobic bacteria,
total fungi, mycorrhizae, protozoa, B:F ratio, and
S:MFA ratio) was examined separately to determine
differences in soil microbial community structure for
the three soil types. The linear statistical model
compares the three systems (i.e., agroforestry VFS
vs. grass VFS vs. row-crop soils); subsamples were
pooled within the site replication (n = 3 per site) and
rep within sample was used as the error term. For all
analyses, differences between means were determined
using t-tests. All analyses were conducted using SAS
software (SAS Institute 1999. SAS Institute Inc., Cary,
NC, USA).
Results
Variation in soil microbial community function and
structure were observed among the three treatments
(Table 2). Functional differences amongst the treat-
ments were most disparate for dehydrogenase activity.
Activity of this enzyme was significantly greater in the
grass VFS relative to the cropland soil (Fig. 2a),
corresponding to a 103 % increase, but dehydrogenase
activity was only nominally greater in the agroforestry
VFS compared to the cropland. The activity of FDA in
the agroforestry VFS and grass VFS soils was not
significantly different (Fig. 2b); however, soil microbial
communities in these buffer systems exhibited 24–25 %
greater FDA activity than the cropland soil (Fig. 2b).
Total microbial biomass was not significantly
different amongst the treatments; however, commu-
nity composition differed between the agroforestry
VFS, grass VFS and cropland soils. The agroforestry
VFS soil was characterized by 15 % more total
Table 1 Fatty acid markers and the associated categories of organism used in this study
Microbial group Markers
Bacteria 10:0B
12:0b
12c alcohol
14:0i
15:00
15:00 all
15:0a
15:0i
15:1cy
16:0
16:0Me 10
16:1x7
16:1x7t
16c alcohol
17:0a
17:0cyc
17:0i
17:1x7i
17:0me10
17:1x6
18:1x7
18:1x7c
19:0cyc19:0cyc
Gram-negative bacteria 10:0B
12:0b
12c alcohol
17:0cyc
18:1x7c 19:0cyc
Gram-positive bacteria 15:00
15:00 all
15:0a
15:0i
16:0Me 10
16c alcohol
17:0a
17:0i
Aerobic bacteria 16:1x7 16:1x7t 18:1x7
Anaerobic bacteria 15:1cy 17:0cyc 19:0cyc
Actinomycetes 16:0Me 10 17:0me10
Mycorrhizae 16:1x5 18:2x6,9
Fungi 16:1x5
18:1x9
18:1x9c
18:2x6,9
18:3x3
18:3x6
18:3x6c
Protozoa 20:3x6 20:4x6
Sulfate 17:1x6 17:1x7i
Monounsaturated fatty acids 14:0
15:0
16:00
17:0
18:0
19:0
20:0
398 Agroforest Syst (2013) 87:395–402
123
bacteria (Fig. 3), 21 % more gram-negative bacteria,
23 % more anaerobic bacteria, and 35 % more
mycorrhizal fungi than the cropland soil (Fig. 4).
The grass VFS supported larger communities of
mycorrhizal fungi and protozoa than the cropland
(44 and 43 % greater, respectively) (Fig. 4). However,
grass VFS supported the same amount of total bacteria
(Fig. 3), gram-negative bacteria and anaerobic bacte-
ria as the cropland (Fig. 4). The agroforestry VFS and
the grass VFS supported the same amounts of total
bacteria (Fig. 3), mycorrhizal fungi (Fig. 4), gram-
positive bacteria and total fungi (Table 2). All soils
were similar in the stress response PLFA indicators
(i.e., S:MFA ratio and MFA) (Table 2); however, the
B:F ratio was significantly greater in the cropland soils
than either of the VFS soils (B:F ratios are as follows:
cropland = 3.26, agroforestry VFS = 2.8, and grass
VFS = 2.45; p = 0.006).
Discussion
As hypothesized, both VFS systems supported more
robust microbial communities than the row-crop
system.
The agroforestry VFS had greater FDA activity,
while the grass VFS measured higher activity for both
enzymes over the cropland soils. Enhanced enzyme
activity in the VFS systems may be related to absence
of tillage disturbance (Bandick and Dick 1999); greater
Table 2 Results from ANOVA analyses to test for differences
in microbial community characteristics in agroforestry VFS,
grass VFS and row-crop soils
Community variable F-value Pr [ F
Enzyme activity
Dehydrogenase 9.80 0.01
FDA 5.87 0.04
Microbial group
Total biomass 0.77 0.50
Bacteria:Fungi ratio 13.92 0.006
Total bacteria 5.52 0.04
Gram-Negative bacteria 5.61 0.04
Gram-Positive bacteria 2.10 0.20
Anaerobic bacteria 5.63 0.04
Fungi 8.83 0.17
Mycorrhizae 8.05 0.02
Protozoa 10.62 0.01
Stress indicators
Saturated:Monounsaturated fatty acids 3.80 0.09
Monounsaturated fatty acids 1.53 0.29
Signifant p values are in bold
0
10
20
30
40
50
60
70
80
Agroforestry VFS Grass VFS Cropland
Treatment
0
10
20
30
40
50
60
70
80
Agroforestry VFS Grass VFS Cropland
(µg
g-1
so
il)
Treatment
a
b
a
a a
bD
ehyd
rog
enas
e A
ctiv
ity
FD
A A
ctiv
ity
(µg
g-1
so
il)
A
B
Fig. 2 Dehydrogenase (a) and FDA (b) activity of the soil
microbial community in the vegetative filter strips (VFS) and
cropland. Bars with same letter are not significantly different
(a\ 0.05)
0.17
0.175
0.18
0.185
0.19
0.195
0.2
0.205
0.21
0.215
0.22
Agroforestry VFS Grass VFS Cropland
To
tal B
acte
rial
Treatment
a
b
ab
Res
po
nse
(m
ol %
)
Fig. 3 Total bacteria response of the soil microbial community
in the vegetative filter strips (VFS) and cropland. Bars with same
letter are not significantly different (a\ 0.05)
Agroforest Syst (2013) 87:395–402 399
123
aggregate stability (Helgason et al. 2010; Udawatta
et al. 2009); or more complex and varied organic matter
inputs (Dornbush, 2007; Macdonald et al. 2009).
Bandick and Dick (1999) found greater enzyme
activity in systems that had continuous cover (grass,
pasture or cover crops) than those that were cultivated.
They attributed these differences to the absence of
tillage and to the rhizosphere effect. Systems with
continuous cover maintain an extensive root system;
these root systems contribute carbon resources to the
soil microbial community and thus support greater
microbial biomass and diversity. Tilling affects aggre-
gation which is an important process for the develop-
ment of microsites for microbial activity. Helgason
et al. (2010) observed more macroaggregates in no-till
systems as opposed to conventionally tilled systems.
Aggregates from no-till sites had higher total carbon
and total nitrogen than aggregates from conventionally
tilled sites; correspondingly, no-till aggregates had
higher total, bacterial and fungal biomass than con-
ventionally tilled aggregates.
Despite the fact that the row-crop system in our
study is under no-till management, cultivation related
disturbances may be contributing to the differences we
observed in soil microbial function between the two
VFS systems and the cropland soils. Notably, soils
from the agroforestry VFS and grass VFS at our study
site were previously found to have significantly more
water-stable aggregates than cropland soil and enzyme
activities were positively correlated with the number
of pores, porosity and macroporosity (Udawatta et al.
2009). In a more recent study of our system, Veum
et al. (2012) substantiated the differences in water-
stable aggregates observed by Udawatta et al. (2009);
in addition, they observed greater water extractable
organic C and greater C:N ratios under the agrofor-
estry VFS and grass VFS systems than in the cropland
systems. Thus, macroaggregates in the VFS systems,
with their associated pore space and available nutri-
ents, are providing a more suitable habitat for micro-
organisms than the cropland soils; this effect is
enhanced by litter inputs and root exudates (i.e.,
carbon resources) from the grass and tree components
of the filter strips.
Differences in soil microbial community function
are expected to be related to differences in soil
microbial community structure. Interestingly, PLFA
analysis indicated that the vegetative treatments did
not differ in total microbial biomass but they did differ
in microbial community composition. Specifically we
observed greater total bacteria, anaerobic bacteria,
gram-negative bacteria, and mycorrhizal fungi in the
agroforestry VFS soil than the cropland soil and
greater mycorrhizal fungi and protozoa in grass VFS
soil than the cropland soil. We also observed differ-
ences between the agroforestry VFS and grass VFS
systems with the agroforestry VFS supporting greater
populations of gram-negative bacteria and anaerobic
bacteria but less protozoa than the grass VFS systems.
As stated above lack of disturbance, greater aggregate
stability and organic matter inputs may explain
differences between soil microbial community struc-
ture of the two VFS systems and the cropland soils.
Differences in organic matter inputs are the most
likely explanation for the variances between the
agroforestry VFS and grass VFS systems. While these
systems are planted in the same grass species, the
addition of trees to the agroforestry VFS creates a more
diverse litter input. Tree and grass litter vary in
chemical composition as well as C:N ratio and tree and
grass roots are expected to produce different exudates.
Diverse organic matter inputs are expected to support a
more diverse microbial community. Other studies lend
support to this explanation. In a study to determine the
effects of plants, plant litter and their interaction on
microbial biomass and soil enzyme activity, Dornbush
(2007) found litter addition, as opposed to root
exudates, to have the greatest effect on enzyme activity
and microbial biomass. However, other studies have
demonstrated the influence of rhizosphere exudates
and root turn-over on soil microbial communities with
bacteria populations decreasing with increasing
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
Gram-NegativeBacteria
Anaerobic Mycorrhizae Protozoa
Mic
rob
ial R
esp
on
se (
mo
l%)
Microbial Group
Agroforestry Buffer
Grass Buffer
Control
a a
bb abb
b
b
a
ba
Bacteria
Fig. 4 Response of the soil microbial community in the
vegetative filter strips (VFS) and cropland. Bars with same
letter are not significantly different (a\ 0.05)
400 Agroforest Syst (2013) 87:395–402
123
distance from roots (Buyer et al. 2002; Kennedy 1999).
Mungai et al. (2005) and Macdonald et al. (2009)
further demonstrate the effect that tree litter inputs and
root exudates have on soil microbial community
characteristics. In their study of temperate alley
cropping systems, Mungai et al. (2005) found higher
enzyme activities in the tree row versus in the middle of
the alley. The authors attribute these differences to the
availability and quality of organic substances (i.e.,
litter quality reflected by C:N values), soil temperature,
and soil water content (Mungai et al. 2005). Mean-
while, Macdonald et al. (2009) demonstrated land use
changes, specifically afforestration, can affect soil
microbial community composition. In this case, con-
version from pasture to pine (Pinus radiate D. Don)
resulted in higher fungi:bacteria ratios (i.e., lower B:F
ratios); the authors attribute this shift to higher C:N
ratios under pines rather than pasture (Macdonald et al.
2009). Thus the addition of trees and the subsequent
enhanced organic inputs may explain the differences
between the agroforestry VFS and grass VFS systems.
Further differences are expected to arise in the future as
the trees in the agroforestry VFS mature, since stand
age is known to affect soil microbial community
structure (Macdonald et al. 2009).
It is interesting to note differences in observed
enzyme activities between this and previous studies at
our site. Similar trends were observed for dehydroge-
nase activity; both our study and Udawatta et al.
(2008) found greatest dehydrogenase activity in the
grass VFS system, with no differences for this enzyme
in the agroforestry VFS and cropland soils. However,
we observed much lower dehydrogenase activity than
Udawatta et al. (2008). Even greater differences were
observed for FDA. In this case we found much higher
activity levels than Udawatta et al. (2008). In addition,
slightly different trends were observed. Udawatta et al.
(2008) found the lowest FDA activity under row-crop
conditions and greatest FDA activity under the grass
VFS treatment. In contrast, our results show no
significant difference in FDA activity between the
two VFS treatments; however, both of these systems
had greater FDA activity than the cropland soils.
These differences may be due to season and year of
sample collection: Udawatta et al. (2008) collected
soils in spring 2006 whereas we collected in autumn
2009. Soil microbial communities are known to
fluctuate with seasonal conditions (Bandick and Dick
1999). In addition the three years between sampling
dates have allowed the trees in the agroforestry VFS to
increase in size and thus influence over the soil
microbial community via increased litter inputs and
better developed root structures.
Conclusions
As expected, differences in microbial community
characteristics were observed in soil collected from
agroforestry VFS, grass VFS and no-till cropland. The
agroforestry VFS had greater FDA activity, while the
grass VFS measured higher activity for both enzymes
over the row-crop soils. Likewise, the agroforestry
VFS and grass VFS (albeit to a lesser extent)
supported a different assemblage of microorganisms
in the soil community than the cropped system.
Greater bacterial biomass, and a greater proportion
of gram-negative bacteria, anaerobic bacteria and
mycorrhizal fungi, in the agroforestry VFS, may be
related to the ability of such conservation measures to
retain nutrients or mitigate the potentially harmful
effects of organic agrochemicals (e.g., pesticides).
Further analysis of the soil microbial community
structure within VFS and its relatedness to ecosystem
services is warranted.
Acknowledgments This work was funded through the Center
for Agroforestry at the University of Missouri under Cooper-
ative Agreements 58-6227-9-059 with the USDA-ARS. Any
opinions, findings, conclusions or recommendations expressed
in this publication are those of the author(s) and do not
necessarily reflect the view of the USDA. The authors wish to
thank Jeremy Hansen (USDA-ARS) for assistance with PLFA
analyses, and Laura Gosen (University of Missouri) for
assistance with enzyme assays.
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