microbial community diversity in agroforestry and grass vegetative filter strips

8
Microbial community diversity in agroforestry and grass vegetative 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 Ó 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

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Page 1: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 2: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 3: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 4: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 5: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 6: Microbial community diversity in agroforestry and grass vegetative filter strips

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

Page 7: Microbial community diversity in agroforestry and grass vegetative filter strips

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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