applied soil ecology 41 (2009) 239–248

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/apsoil

Impact of biotic and abiotic interaction on soil microbial

communities and functions: A field study

Brajesh K. Singh *, Lorna A. Dawson, Catriona A. Macdonald 1, Sarah M. Buckland

Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, United Kingdom

article info abstract

Article history: Interactions between plants, soils and microbes regulate terrestrial ecosystem functioning.
Received 18 April 2008 Biotic and abiotic interactions can strongly affect the community structure which in turn
Received in revised form will impact on ecological processes. Plant species with different ecophysiological traits can
7 October 2008 exert strong effects on soil biological properties. Our objective was to investigate and
Accepted 9 October 2008 identify the effects of different biotic and abiotic variables on soil microbial community
structure and functions and to examine if plant species with different physiological traits
support different microbial communities in soils. Here, we show that 3 years of the presence
Keywords: of plants had direct impacts on soil function in terms of total heterotrophic respiration and
Biotic and abiotic factors on microbial biomass and microbial community structure. However, the plant species-
Ecological interactions specific impact on bacterial community structure was weak, and differences were mainly
Plant physiological traits driven by sample field location. The fungal community analysis gave similar results, with
Fungal community soil location being the most important factor driving fungal community structure. The effect
Bacterial community of plant species on fungal community structure was weak but statistically significant. There
Plant species was a strong concordance between bacterial and fungal communities (P < 0.001) which
suggested that the bacteria and fungi have an influence on shaping the structure of each
other’s community. Among the abiotic factors, moisture had a comparatively higher impact
on bacterial communities compared to soil N and C. However, the fungal community was
not affected by the soil moisture but soil N and C had a stronger impact than on the bacterial
community. These results indicate that the microbial community structure in the natural
environment is influenced by interactions between both biotic and abiotic factors.
# 2008 Elsevier B.V. All rights reserved.

1. Introduction position, which may represent a positive association where

Microbial community structure and ecological functions are
influenced by interactions between above and belowground
biota (Wardle et al., 2002). These interactions can have
positive, negative or neutral impact on diversity and commu-
nity structure of plants and soil microbes (Singh et al., 2004). It
has been observed that many plants select beneficial groups of
microorganisms via rhizodeposition (loss of carbon due to root
exudation, leakage or root decomposition), and litter decom-
plants supply carbon for microbial growth and microorgan-
isms in return provide major elements such as N and P, as well
as protection against pathogen and parasite attacks (Singh
et al., 2004). It is believed that the carbon source made
available by plants drives many complex chemical and
biological interactions in the soil (e.g. Jones et al., 2004). These
include sustaining a complex food web of prokaryotes and
eukaryotes, the composition of which is thought to be
regulated by complex signalling (Phillips et al., 2003). However,

* Corresponding author. Tel.: +44 1224 498200; fax: +44 1224 498207.
E-mail address: b.singh@macaulay.ac.uk (B.K. Singh).
1
Present address: Rothamsted Research, Harpenden AL5 2JQ, UK.
0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2008.10.003
240 applied soil ecology 41 (2009) 239–248
it is not known if plants actively select for beneficial soil
microbial communities. Significant change in soil microbial
community structures has been reported with change in
vegetative cover. These vegetation-induced changes have
been observed both at the universal bacterial group level
(Grayston et al., 1998, 2004; Nusslein and Tiedje, 1999) as well
as at the functional group level (Singh et al., 2007a). However,
another study reported similar microbial community struc-
ture in soils which shared similar agricultural management
practices despite differences in above-ground community
composition (Buckley and Schmidt, 2001). Similarly, Girvan
et al. (2003) found that soil type is the primary determinant of
the composition of the total bacterial community in arable
soils. Most of the previous studies focused only on the
bacterial community (Buckley and Schmidt, 2001; Girvan
et al., 2003; Nunan et al., 2005), while a few exclusively studied
universal fungal communities (Smit et al., 1999; Marcial-
Gomes et al., 2003). There are only few reports of investiga-
tions on the impact of both plant species and soil type on both
fungal and bacterial communities (Mougel et al., 2006; Costa
et al., 2006; Singh et al., 2007b). To our knowledge none has
investigated the concordance between bacterial and free-
living fungal communities. With the growing evidence for the
linked ecological functions of bacterial and fungal commu-
nities in processes such as decomposition and nutrient
cycling, it is important to understand whether both commu-
nities are influenced by the same environmental factors (Costa
et al., 2006).
The influence of the plant community on ecosystem
functions is well known (Horner-Devine et al., 2003; Tilman
et al., 2006). Furthermore, it is well established that plant
species which dominate different stages of succession have
different sets of ecophysiological traits (Aber et al., 1990; Van
Vuuren et al., 1992; Grime et al., 2007) and that these sets of
traits can exert strong effects on soil biological properties
(Wardle et al., 1998; Bardgett et al., 1999; Hobbie, 2002; Garnier
et al., 2004) such as selecting for decomposer food webs with
certain basic attributes (Wardle et al., 2002). There are
evidences that plant species differ considerably in the
composition of soil biota that they support (Grayston et al.,
1998; Wardle et al., 1998; Marilley et al., 1998; Bardgett et al.,
1999; Johnson et al., 2003; Vandenkoornhuyse et al., 2003).
Other studies found mainly mycorrhizal fungi (Van der
Heijden et al., 1998a,b) or bacteria (Van der Heijden et al.,
2006) to be key determinants of ecosystem diversity and
functions. Although soil ecologists have long recognised the
need to understand the mechanisms by which community
structure is shaped and ecosystem functions are maintained,
study on multi-community and multi-trophic interactions
between above and belowground communities and their
impact on ecosystem function is lacking. In the present study,
we studied the impact of grass species with differential
physiological traits (in monoculture and as a natural mixed
community) and soil abiotic factors (soil C, N and moisture) on
soil bacterial and fungal communities under field conditions.
We also investigated the interactions between bacterial and
fungal communities and the impacts on shaping microbial
community structure. Soil functional capabilities under
different grass species were also measured in terms of
respiration rate and total microbial biomass.
2. Materials and methods
2.1. Site, soil properties and plant species
This study was carried out at an experimental site at
Sourhope, Scotland (558280 3200 N 28140 4300 W), a semi-natural
Festuca ovina–Agrostis capillaries–Galium saxatile grassland,
determined as a Luzula multiflora–Rhytidiadelphus loreus sub-
community (National Vegetation Classification (NVC) U4d),
also the location of the recent NERC Soil Biodiversity
Programme (Usher et al., 2006). The site is a long-term
(>200 year old) grassland, and in 1998 a fence was erected
to exclude grazing animals (e.g. livestock, deer, rabbits and
hares). It is situated at 309 m above sea level and varies in slope
from 88 at the top to 48 at the foot and has a northerly aspect,
mean annual rainfall is 954 mm. The soils are developed on a
drift locally derived from andesitic lavas of old red sandstone
and are characterised as acid brown forest soil belonging to
the Sourhope series (SH 74711) (pH 4.5–5.0) (Grayston et al.,
2004).
In 1999, the site was fenced to prevent sheep grazing and
set out in five replicate blocks, each block being placed along
the contour of the slope. Experimental plots (1 m2) were
arranged in a randomised block design with nine treatments
in each of the five blocks. Nine treatments involved creating
monocultures, eight of which were common herbaceous
species at the Sourhope site: Agrostis capillaris (Ac), Anthox-
anthum odoratum (Ao), Festuca ovina (Fo), Festuca rubra (Fr),
Nardus stricta (Ns), Luzula multiflora (Lm), Trifolium repens (Tr),
Rumex acetosa (Ra); and a ninth species which was present in
the improved areas of pasture, Lolium perenne (Lp). These
species also represent a broad range of physiological and
functional types, illustrated by the contrasted values in key
plant traits (Table 1). The monoculture plots were created by
removing the surface 2–3 cm of turf and inserting vegetative
specimens collected from the surrounding natural vegetation
contained in the Sourhope enclosure, washing and brushing
off as much soil as possible, before transplanting into their
respective plots. Monocultures of L. perenne were established
by sowing in seed (60 g per plot). A 10th treatment included
plots of undisturbed, natural (N) turf, to provide data on the
natural soil community in the absence of turf removal. A 11th
treatment comprised bare (B) plots, to provide data on the
effect of turf-stripping without reintroduction of transplants.
The plots were maintained by regular hand-weeding to
promote ongoing establishment of the monocultures and to
prevent vegetation colonization on the bare plots. Plots were
mown monthly between May and September, following the
management design implemented on the NERC soil biodiver-
sity experiment.
2.2. Measurement of soil respiration, microbial biomass,
soil N, soil C and moisture
Three years after the treatments were established, soil
samples were collected from each field-block by a (3.5 cm
diameter  8 cm depth) corer. Roots were removed by hand
picking. From each plot, four cores were taken and stored in
plastic bags at 4 8C until analyses were completed. For
molecular work, sub-samples were frozen at 20 8C until
 applied soil ecology 41 (2009) 239–248 241

Table 1 – Plant species data for traits considered to have a significant influence on the rate of nutrient cycling.
Plant spp RGRa SLAb DMCb Leaf thicknessc Leaf toughnessc Leaf Nd Leaf Pd
(mm) (N mm1) (% d. wt) (% d. wt)

Agrostis capillaris 1.36 30.8 25.1 0.012 1.16 2.33 0.22

Anthoxanthum odoratum 0.94 27.3 22.0 0.098 5.86 2.04 0.15
Festuca ovina 1.00 10.9 41.0 0.300 7.79 1.46 0.09
Festuca rubra 1.18 17.7 26.7 0.179 6.32 1.69 0.22
Lolium perenne 1.30 26.4 21.2 0.134 1.15 3.27 0.44
Luzula multiflorae 1.01 21.6 21.4 0.222 0.97 1.95 0.20
Nardus stricta 0.71 10.2 38.8 0.256 11.72 1.59 0.17
Rumex acetosa 1.71 28.1 10.3 0.312 0.31 3.37 0.32
Trifolium repens 1.26 38.8 17.9 0.131 0.10 NA NA

Abbreviations: RGR, relative growth rate (unit: week1); SLA, specific leaf area (unit: mm2 mg1); DMC, dry matter contents (%); (N mm1),
Newton per millimeter.
a
Data source: Grime and Hunt (1975).
b
Data source: Grime et al. (2007).
c
Data source: Hodgson, personal communication.
d
Data source: Thompson et al. (1997).
e
Data have been taken from L. campestris, a closely related spp.

DNA extraction. Gravimetric soil moisture content was
determined on field fresh soil (ISO, 1997). The total soil C
and N contents of the each sample was determined by
automated Duma Combustion using a Carlo-Erba Elemental
Analyser (Carbo-Erba Instruments, Milan, Italy; Grayston
et al., 2004).
Soil respiration was determined on 20 g soil samples (after
sieving though a 4 mm mesh) following a conditioning period
of 7 days in the dark at 25 8C and constant humidity and CO2
concentration. The head space of the soil jars was sampled
(1 ml) at 0, 2, 4 and 6 h. The CO2 was determined by GC analysis
and respiration rates calculated (Sparling, 1981). Soil microbial
biomass was measured by chloroform-fumigation extraction
(Brookes et al., 1985).
2.3. DNA extraction and PCR
DNA was extracted from 0.5 g of each soil sample by using soil
DNA clean kit (Mo Bio, Carlsbad, California) according to the
manufacturers’ instructions. Extracted DNA was PCR amplified
using universal bacterial 50 FAM (6-carboxyfluorescein) labelled
forward primer 63F (50 -AGG CCT AAC ACA TGC AAG TC-30 ) and
reverse primer 1494R (50 -TACGG YTACC TTGTT ACGAC-30 )
(Operon, Germany). Each 100 ml reaction mixture contained
10 ml 10 PCR buffer (Bioline, London, UK), 2 ml dNTP (0.2 mM),
8 ml MgCl2 (25 mM), 2 ml BSA (Roche Diagnostic, Lewes, UK), 2 ml
of forward and reverse primers (20 mM), 1 ml of Taq polymerase
(Bioline) and 1 ml of soil DNA template. PCR conditions consisted
of an initial denaturation at 95 8C for 5 min followed by 30 cycles
of 95 8C for 30 s, 55 8C for 30 s, 72 8C for 60 s, and a final extension
at 72 8C for 10 min. Fungal DNA was PCR amplified using
primers specific to fungal ITS. A forward ITS1F-FAM labelled (50 -
CTT GGT CAT TTA GAG GAA GTA A-30 ) (White et al., 1990) and a
reverse ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 ) (Gardes and
Bruns, 1993) were used. The PCR mixture and thermocycling
conditions were the same as described above. To check purity
and size of PCR amplicons, 5 ml of each reaction mix was run on
1% agarose gel (w/v). The PCR products were purified using
GeneEluteTM PCR clean-up kit (Sigma–Aldrich, Dorset, UK)
following the protocol provided by the manufacturer.
2.4. TRFLP—analysis for bacterial and fungal communities
Microbial community fingerprint patterns (based on the 16S
rRNA gene for bacterial communities and the ITS gene for fungal
communities) were generated by TRFLP (Terminal Restriction
Fragment Length Polymorphism) analysis. Initially, a range of
restriction enzymes (RsaI, HaeIII, HhaI, MspI and TaqI; Promega,
Southampton, UK) were used on randomly selected samples to
screen for the best enzyme for TRFLP analysis. HhaI and TaqI
produced best results for bacterial and fungal communities,
respectively, on the basis of number, consistency and even
distribution of terminal fragments and, therefore, all further
analyses were carried out with these two restriction enzymes.
Approximately 1000 ng of purified DNA was digested either with
HhaI (for bacterial) or TaqI (for fungal). Each 20 ml reaction mix
consisted of the appropriate volume of DNA (for 1000 ng) mixed
with 2 ml of appropriate enzyme, 2 ml of 10 buffer and 0.2 ml of
BSA and the volume was made up by addition of sterile water.
Samples were then incubated for 3 h at optimal temperature
(37 8C for bacterial DNA and 65 8C for fungal DNA) as per enzyme
supplier’s instructions on a PCR machine followed by a 15 min
deactivation period at 95 8C. Aliquots (1 ml) of digested PCR
products were mixed with 12 ml of formamide loading dyes (ABI,
UK) and 1 ml of internal size standard (2500 TAMARA, ABI) before
denaturing for 5 min at 95 8C. TRFLP analysis was carried out on
an automated sequencer, ABI PRISM1 3130xl Genetic Analyzer
(Applied Biosystems, Warrington, UK). Terminal restriction
fragments (TRFs) generated were analysed using GeneMapper
3.7 (ABI, UK). Only TRFs at position between 25 and 500 bp were
considered for further analysis in order to avoid fragments
formed by the primer dimmers. The overall structure of
bacterial and fungal communities among the different habitats
(soils) and treatments (the presence of plants) were compared
by analysing binary (presence/absence) data using a multi-
variate approach (principal coordinate analysis).
2.5. Cloning and sequencing
Bacterial 16S rDNA gene amplicons from samples (pooled
DNA from selected treatments) were obtained using the
242 applied soil ecology 41 (2009) 239–248

same set of primers (unlabelled) described in Section 2.3 and
were cloned in E. coli using TOPO cloning kit (Invitrogen,
Paisley, UK). About 25 clones were selected for each sample
for sequencing. The correct insert size in each clone was
checked by vector targeted PCR (primer M13 F and M13 R) and
gel electrophoresis. PCR amplicons were purified by ethanol
precipitation. Sequencing was performed with Big Dye
Terminator cycle sequencing reaction kit (ABI, UK) using
vector specific T3 or T7 primers on an automated DNA
sequencer (Prism 3130xl Genetic Analyser, ABI, UK). The
quality of sequence was checked by the Sequence analysis
software (ABI) and approximately 500 bp of good quality
sequence was obtained for each clone. Analysed sequences
were compared with 16S rDNA using FASTA-3 search of the
EMBL database (http://www.ebi.ac.uk/fasta33). Fasta3 along
with ribosomal database of the Michigan State University
(http://rdp8.cme.msu.edu/html/) and NCBI (http://
www.ncbi.nlm.nih.gov) were used to classify individual
sequences into different groups. Sequences were then
grouped into different phyla and their distribution was
presented in a histogram.
Soils from the Fo treatment had the highest amount of
microbial biomass. A similar observation was made for soil
respiration values, where the presence of plants had a highly
significant effect on the respiration rate (P < 0.01). Again, the
rate of respiration was lowest in the bare soil and highest in
soil from the Fo treatment, which was significantly different
from all the other treatments (P < 0.05; Fig. 1b). When plant
was used as a treatment (considering all plant treatments
together), there was no apparent effect of the presence of
plants on soil moisture. However, the moisture in the bare soil
treatment was comparatively lower than in the soil under Fo,
Ns and Tr plants (P < 0.05; Fig. 1c). Biological and physico-
chemical properties of the field-blocks are listed in Table 2.
There was certain variation between different field-blocks for
all measured parameters but they were not statistically
significant.

2.6. Statistical analysis

Treatment effects (plant or field-block) on soil N, C and

moisture and on biological (respiration and microbial bio-
mass) were analysed using analysis of variance (ANOVA) for
randomised blocks. Significant differences between treat-
ments were established using least significant difference (LSD)
values. The overall structure of the bacterial and fungal
communities in the different locations (field-blocks) and
treatments (plant species) was compared by analysing binary
data (presence/absence) using principal coordinates (PCO)
analysis and a Euclidian/Jaccard similarity matrix. Principal
coordinates analysis uses a matrix of similarities or dissim-
ilarities between the samples to produce a low-dimensional
representation of the data in such a way that the distances
between points are as close as possible to the original
dissimilarities. For each data set we carried out an ANOVA
on PCO scores for the first five dimensions as a randomised
complete block design. To identify overall pattern of commu-
nity distribution, PCO scores from the first 10 PCO dimensions
were used for canonical variate analysis (CVA). An ordination
diagram was obtained from the scores from CV axis 1 and CV
axis 2 to elucidate the effect of plant species or field-block on
soil microbial communities. To examine the correlation
between different abiotic and biotic factors and microbial
community pattern, regression analysis was carried out. All
statistical analyses were carried out using GenStat version 8
(VSN International Ltd.).

3. Results
3.1. Effects of plant species on soil moisture, respiration,
microbial biomass, soil N and soil C
The effect of the presence of plants on microbial biomass was
highly significant (P < 0.001; Fig. 1a). Bare soil had the lowest
microbial biomass compared to all planted soils (P < 0.05).
Fig. 1 – Microbial biomass (a), respiration (b), moisture of
fresh soil (c) and C:N ratio (d) in soils with different plant
species. Error bars represent W standard errors. Treatment
acronyms are Agrostis capillaris (Ac), Anthoxanthum
odoratum (Ao), bare soil (B). Festuca ovina (Fo), Festuca rubra
(Fr), Nardus stricta (Ns), Lolium perenne (Lp), natural turf (N)
and Trifolium repens (Tr).
 applied soil ecology 41 (2009) 239–248
Table 2 – Biological and physico-chemical properties of the field-blocks. Values in brackets represents standard errors of
replicates.
Unit Block 2 Block 3
Soil N (%) 1.1 (0.09) 1.0
Soil C (%) 13.8 (1.1) 13.2
Moisture (%) 453.6 (2.6) 47.8
Microbial biomass mg/g of soil 1894.4 (315.9) 2093.8
Respiration mg/g of soil 2.3 (0.28) 2.2
Regression analysis on these characteristics revealed an
interactive response. Moisture showed a slight positive
correlation with respiration (P < 0.001, R2 = 0.27) and microbial
biomass (P < 0.05, R2 = 0.15). This relationship was strength-
ened when multiple regression was employed using plant type
as a grouping factor. The relationship between soil respiration
and microbial biomass was strong and statistically highly
significant (P < 0.001, R2 = 0.55). When both moisture and
biomass were used as explanatory variates to identify impact
on soil respiration, it gave a very high correlation value
(P < 0.001, R2 = 0.63). Similarly, moisture alone had compara-
tively smaller impacts on microbial biomass and the relation-
ship was weak (R2 = 0.15), however this relationship was
substantially increased when regression was carried out with
plants as a grouping factor (P < 0.001, R2 = 0.40). The C:N ratio
in soils planted with different species tended to be different,
although this effect was not statistically significant.
3.2. TRFLP analysis of the bacterial community
TRFLP profiles of the bacterial community were obtained for
all samples. On average between 20 (for the natural mixed
community) and 38 (for Ao) well-resolved TRFs were detected
(Fig. 2a). A one-way ANOVA on TRFs had suggested that there
was no effect of plant species on the number of TRFs (P > 0.05).
When the number of TRFs was analysed for presence in each
field-block, between 19 (for field-block 3) and 38 (for field-block
7) TRFs were detected. ANOVA suggested that field-block had a
significant effect on the number of TRFs (P < 0.01). Further
analysis of the data suggested that field-block 3 had
significantly lower numbers of TRFs than all the other field-
blocks while field-blocks 2 and 4 had significantly lower
numbers of TRFs than field-block 7 (Fig. 2b).
The TRFLP data from all samples were analysed by PCO
analysis. Scores of the first 10 dimensions were obtained.
ANOVA on the first five PCO scores suggested that none of the
PCO dimensions were significantly influenced by plant species
(Table 3). However, when the ANOVA was carried out
including the location as a block effect, the bacterial commu-
nity in the bare soil was significantly different from all other
soils in PCO dimension 4 (data not shown). However there was
no difference in the bacterial communities between soils with
different plant species. PCO scores from the first 10 dimen-
sions were further analysed by CVA, which confirmed that the
soil bacterial community in the bare soil treatment was
different from all other soil treatments (Fig. 2c). However, CVA
analysis also suggested that there was a weak effect of plant
species on soil bacterial communities. Here some soils, with
particular plant species treatments were separated from all
other soils on CV 2, although the data were dispersed and
243
Block 4 Block 6 Block 7
(0.06) 1.1 (0.03) 1.3 (0.08) 1.1 (0.08)
(0.64) 14.4 (0.4) 17.6 (1.2) 13.9 (0.93)
(0.99) 44.4 (3.2) 50.5 (1.9) 47.4 (3.93)
(256.8) 2064.4 (152.3) 1475.4 (97.4) 1947.2 (434.7)
(0.16) 2.1 (0.16) 2.3 (0.23) 2.4 (0.35)
variation within replicates was large. For example, Fr was
separated from all other soils except Ac. Similarly, Tr was
different from all except N (Fig. 2c).
However, when the TRFLP data were analysed for the
effects of field-blocks on community structure, all first three
PCO dimensions were significantly affected by the block
location (Table 3). Further analysis of the data by CVA
suggested that the bacterial community of field-block 1 was
different from all other field-blocks. Similarly field-blocks 3
and 4 were different from field-blocks 6 and 7 (Fig. 2d).
Regression analysis on the first five PCO dimensions, sug-
gested that bacterial community structure was not correlated
with respiration, but the third dimension was significantly
influenced by the moisture content (Table 3). There was a
strong correlation between microbial biomass and bacterial
communities in PCO dimension 2 (P < 0.001). PCO dimension 4
of the bacterial community was significantly influenced by soil
N (P < 0.05) and soil C (P < 0.01) (Table 3).
3.3. Cloning and sequencing of the bacterial community
Clone libraries for 16S rRNA genes were constructed for the
bacterial community from soil treatments B, Ac, Ao, N, Tr and
Lp. For each library, 25 clones were selected randomly for
sequencing of approximately 500 bases. In all, 107 good
quality partial sequences were obtained and analysed. Based
on their FASTA 3, RDP II and NCBI database matches, all
sequences were grouped under 11 phyla; alpha-, beta-,
gamma- and delta-Proteobacteria, Firmicutes, Actinobac-
teria, Acidobacteria, Gemmatimonadetes, Bacteroidetes,
Chloroflexi, and unclassified domains. Gemmatimonadetes
and Bacteroidetes were present in only one library (N and Ao,
respectively). The distribution of other phyla of bacteria in
each sample is presented in Fig. 3. All libraries contained
representative from Acidobacteria which varied from 14% (B
soil) to 53% (Tr soil) of the total clones. Similarly alpha-
Proteobacteria were present in all samples and their relative
proportions varied from 29% (in Ao soil) to 56% (in Lp soil).
gamma-Proteobacteria were present in all libraries except Tr.
The Firmicutes were present only in the B soil but they
constituted a substantial proportion of clones (14%) in this
library. Actinobacteria were another major group which were
present in all samples except B and Lp libraries. A substantial
proportion of the unclassified bacteria was also detected in B
and Lp soils. Overall, the soil without any plants (B) was
dominated by Bacilli, Clostridia (Firmicutes), alpha-Proteo-
bacteria and an unclassified group. It has the lowest number
of Acidobacteria in comparison with other libraries and was
devoid of beta-Proteobacteria, Actinobacteria and Chlorflexi
groups. Soil samples with growing plants were dominated by
244 applied soil ecology 41 (2009) 239–248
Fig. 2 – Number of bacterial terminal restriction fragments
(TRFs) obtained from soils (a) under different vegetation
(plant species) and (b) from different field-blocks.
Ordination plots of the first and second dimensions of
canonical variate analysis (CVA) scores for the soil
bacterial community obtained for different plant species
(c) and field-blocks (d). Error bars are Wstandard errors.
Numbers in parentheses are percentage variation
explained by the CV. Treatment acronyms are Agrostis
capillaris (Ac), Anthoxanthum odoratum (Ao), Festuca ovina
(Fo), Festuca rubra (Fr), Nardus stricta (Ns), Luzula multiflora
(Lm), Trifolium repens (Tr), Rumex acetosa (Ra), Lolium
perenne (Lp), natural turf (N) and bare soil (B).
Proteobacteria and Acidobacteria. However the difference in
clone distribution between plant species was smaller (Fig. 3).
3.4. TRFLP analysis of fungal communities
Like the bacterial community, TRFLP profiles for fungal
communities were obtained successfully from all soil sam-
ples. ANOVA suggested that there was no effect of plant
species on the number of TRFs (P > 0.05), which varied from 46
(for Fr) to 79 (for Lp) (Fig. 4a). However, the average TRFs for
different field-blocks varied from 46 (for field-block 2) to 71 (for
field-block 7) (Fig. 4b) and the effect of field-blocks on the
number of TRFs was significant (P < 0.05).
PCO analysis of fungal binary data of TRFs suggested that
the first three dimensions of the PCO scores were not
affected by plant species. However, the fourth dimension
was significantly influenced by plant species (P < 0.05)
(Table 4). In this dimension, fungal communities in B soil
were different from all other samples except Lm, Ns, Ra and
Tr (P < 0.05). However, in this dimension, the fungal
communities from Ac and N samples were also different
from Ra and Lm (P < 0.05), suggesting an impact of plant
species on the soil fungal community. Data were further
analysed by CV analysis on the PCO scores from the first 10
dimensions and an ordination diagram was obtained (Fig. 4c
and d) which confirmed that the fungal community in B soil
was different from all other samples. B soils were separated
from other samples on CVA axis 1 which accounted for 42%
of variability in the dataset. Fungal communities from Fo, Fr,
Lm, Lp and Tr treatments were separated from other plant
species treatments on axis 2 suggesting some impact of
plant species on fungal community structure in soil (Fig. 4c).
When the data were analysed for the impact of field-block
on the fungal communities, the first two dimensions of the
PCO score showed a significant correlation (P < 0.01 and
<0.001, respectively). This observation was further sup-
ported by the CVA analysis of the first 10 PCO dimensions
where fungal community from field-blocks 2, 3 and 4 were
separated on CVA axis 1. Field-blocks 6 and 7 were
separated from Field-block 2 on CVA axis 2, while from 3
and 4 on both axes 1 and 2 (Fig. 4d).
Regression analysis on PCO scores from the first five
dimensions suggested a comparatively weak but significant
correlation between soil respiration and fungal communities
in PCO dimension 5 and a strong correlation between
microbial biomass and fungal communities in PCO dimension
2 (P < 0.001). Further analysis of data suggested a strong
influence of soil N (P < 0.05) and soil C (P < 0.01) on fungal
communities in PCO dimension 1 (Table 4).
3.5. Interaction between bacterial and fungal
communities
To investigate the interactions between soil bacterial and
fungal communities, regression analysis of the first five
dimensions of bacterial communities was carried out
against the first five dimensions of fungal communities.
With the exception of dimension five, all dimensions of
bacterial communities showed a significant correlation
between bacterial and fungal communities (P < 0.05). PCO
 applied soil ecology 41 (2009) 239–248
Table 3 – Significance (P-values) of the effects of location (field-blocks), plant species and selected environmental variates
on the soil bacterial community. The number in parentheses under the PC dimension column represents % variation
explained by individual dimensions.
PC dimensions Plant Field-block Respiration
1 (29.3%) 0.72 0.001 0.22
2 (8.13%) 0.71 0.04 0.09
3 (5.9%) 0.94 0.004 0.15
4 (4.9%) 0.12 0.059 0.199
5 (4.37%) 0.2 0.85 0.22
dimension one of the bacterial community was significantly
correlated with PCO dimensions 1, 2 and 5 of the fungal
communities. PCO dimensions 2, 3, and 4 of bacterial
communities were also correlated with PCO dimensions, 2, 4
and 1 of the fungal communities. When PCO scores of
dimension 2 of bacterial and fungal communities were
plotted against each other a significant correlation was
observed (P < 0.001) (Fig. 5). The degree of concordance
between the two microbial communities was further
examined by Procrustean analysis of the first five dimen-
sions of fungal and bacterial communities. Procrustean
analysis was chosen over the more commonly used
Mantel test because it is more powerful in detecting
matrix association (Peres-Neto and Jackson, 2001). A
permutation procedure for Procrustean rotation (PROTEST;
Jackson, 1995) was used to evaluate the significance of the
association between fungal and bacterial data matrices
obtained by T-RFLP. Here too, a significant correlation was
found (M1/2 = 0.39; P < 0.001) between bacterial and fungal
communities. When procrustean residuals were plotted
against soil moisture, N, and C no significant correlation
was found.
Fig. 3 – Frequency distribution of phylogenetic taxa at
class-level in clone libraries of amplified 16S rRNA genes
of bacteria recovered from bare soil and soils with
different plant species. Treatment acronyms are Agrostis
capillaris (Ac), Anthoxanthum odoratum (Ao), Festuca ovina
(Fo), Festuca rubra (Fr), Nardus stricta (Ns), Luzula multiflora
(Lm), Trifolium repens (Tr), Rumex acetosa (Ra), Lolium
perenne (Lp), natural turf (N) and bare soil (B).
245
Moisture Biomass Soil N Soil C
0.19 0.12 0.09 0.16
0.45 <0.001 0.75 0.93
0.023 0.41 0.3 0.1
0.084 0.33 0.03 0.005
0.78 0.84 0.38 0.5
4. Discussion
Plants create positive feedbacks to nutrient cycling because of
species differences in carbon deposition and competition with
microbes for nutrients. It has been argued that plant
physiological trait effects can be as or more important than
abiotic factors such as climate, in controlling ecosystem
functions (Verville et al., 1998). Physiological traits such as
relative growth rate (RGR) and specific leaf area (SLA) have
been linked to the success of plant growth (Zou et al., 2007),
evolutionary specialisation (Hunt and Cornelissen, 1997), and
to soil biota (Wardle et al., 1998). Data from this study revealed
that the presence of plants resulted in a significant increase in
soil moisture content, microbial biomass and respiration. This
observation suggests that plant presence not only supports
growth in soil microbial communities but also influences soil
abiotic properties. Although not presented in this manuscript,
we also found different plant species produced different
quality (C:N ratio, dry weight/wet weight ratio) and quantity of
plant biomass (shoot, root and litter). However, plant biomass
did not correlate with microbial biomass, respiration or soil
moisture content. Multivariate analysis of the microbial
community suggested that the bacterial communities are
highly influenced by sample location (field-block) and to a
lesser extent, by the presence of plants (Fig. 2). The plant
species-specific effect on the bacterial community was weak
(Fig. 2; Table 3). However, CVA analysis did suggest a weak
impact of a few plant species on soil microbial communities
after the 3 years of growth. Species-specific impacts of plants
on soil biota have been attributed to specific plant physiolo-
gical traits, independent of environmental factors. It has been
argued that plants with differential ecophysiological traits will
exert an impact on soil biota through differential rhizodeposi-
tion and quality of litters (Bardgett et al., 1999). However,
several other studies suggested that soil characteristics
(Girvan et al., 2003; Singh et al., 2006) and spatial distribution
(Nunan et al., 2005; Ritz et al., 2004) are more important factors
in shaping the microbial community structure. Our data
supports the later observation. Among the abiotic factors,
multivariate analysis of data suggested that the bacterial
community was influenced by soil moisture (dimension 3) and
the impact of soil C and N appeared only in dimension 4
(Table 3).
The objective of cloning and sequencing data was to
confirm our observation of TRFLP for the bacterial community
and that is why only limited clones were sequenced for this
study. These data support our TRFLP results in the sense that
the presence of plants causes change in the bacterial
community but the plant species-specific effect on the
246 applied soil ecology 41 (2009) 239–248
Fig. 4 – Number of fungal TRFs obtained from soils (a) under
different vegetation (plant species) and (b) from different
field-blocks. Ordination plots of the first and second
dimensions of CVA scores for the soil fungal community
obtained for different plant species (c) and field-blocks (d).
Error bars are Wstandard errors. Numbers in parentheses
are percentage variation explained by the CV. Treatment
acronyms are Agrostis capillaris (Ac), Anthoxanthum
odoratum (Ao), Trifolium repens (Tr), Lolium perenne (Lp),
natural turf (N) and bare soil (B).
bacterial community is not prominent. The results showed
that the bare soil was dominated by Firmicutes and Proteo-
bacteria, while soils with different plant species were mainly
dominated by the Acidobacteria and Proteobacteria. However,
this observation should be interpreted with caution as the
number of clones on which this finding was based was limited.
Nonetheless, agreeing with this, a few reports suggest that the
Proteobacteria and Acidobacteria dominate in rhizosphere
locations at the expense of other Gram-positive bacteria
(Singh et al., 2007b; Lu et al., 2006). In a study of the grassland
soil adjacent to the present site at Sourhope, Griffiths et al.
(2006) found the upper organic layer dominated by alpha-
Proteobacteria and the mineral layer by Acidobacteria. McCaig
et al. (1999) showed that clone libraries from the adjacent plot
of the same site comprised approximately 40% alpha-
Proteobacteria and 8% Acidobacteria. Our results are in
accordance with these findings.
As with the bacterial community, the fungal community
was strongly influenced by block position and only weakly
influenced by plant species. However, PCO dimension 4
showed a significant impact of plant species on fungal
community structure, suggesting a relatively stronger effect
in comparison to the bacterial community. Further analysis of
data by CVA suggested that soils with Fo, Tr, Lp and Ac
harboured distinctly different fungal communities than the
other soils. This may suggest that plant species with faster
growth rates have distinct effects on the fungal community in
comparison to slower growing plant species such as Ns. The
fungal community responded differently to abiotic factors as
compared to the bacterial community. Soil moisture did not
have any influence on the fungal community while soil N and
soil C had a comparatively stronger influence in shaping the
fungal community structure; PCO dimension 1 was signifi-
cantly co-related with these properties.
The most important finding of this work is the interaction
between the fungal and bacterial community structure.
Although fungi and bacteria have been studied extensively
in isolation in various habitats, few studies have examined
them simultaneously (Singh et al., 2007b; Costa et al., 2006),
and none of them attempted to investigate the interaction
between these two microbial communities. Bacteria and fungi
live in close association in the soil environment. Due to this
close proximity, fungal–bacterial interactions could be nega-
tive, positive or neutral (Boer et al., 2005). Our data suggest a
strong correlation between fungal and bacterial communities.
The degree of concordance between these two communities
was further investigated by the Procrustean analysis of PCO
scores. This was also done to factor out plant-specific
influences. Our results suggest a significant concordance
between these two microbial communities (P < 0.001). The
procrustean residuals were not correlated with any soil abiotic
properties, suggesting that the relationship between bacterial
and fungal communities was independent of soil moisture
content, pH, C and N. However, our results should be
interpreted with caution as the present observation was
made for just one sampling regime. Nonetheless, the relation-
ships between AM fungi and bacteria have been reported
previously. In a laboratory study, a single AM fungus, Glomus
mosseae, was reported to have a direct impact in shaping the
bacterial community found on plant roots (Artursson et al.,
 applied soil ecology 41 (2009) 239–248
Table 4 – Significance (P-values) of the effects of location (field-blocks), plant species and selected environmental variates
on the soil fungal community. The number in parentheses under the PC dimension column represents % variation
explained by individual dimensions.
PC dimensions Plant Field-block Respiration
1 (12.3%) 0.42 0.005
2 (6.9%) 0.17 <0.001
3 (4.9%) 0.85 0.35
4 (4.4%) 0.03 0.5
5 (3.7%) 0.76 0.07
Fig. 5 – Relationship between fungal and bacterial
communities based on principal coordinate analysis
dimension 2 (PCO-2) scores associated with all field plots.
2005). Later, Singh et al. (2008) reported that that AM fungal
community influenced the bacterial community structure on
grass rhizoplanes. However, this study is the first time a
concordance between fungi and bacteria at the community
level and field scale has been shown. This observation
suggests that a complex multi-trophic interaction occurs
between above and belowground communities and the
microbial community structures are shaped both by several
abiotic and biotic interacting factors.
Acknowledgements
We thank Dr Naoise Nunan (CNR, France) for help in statistical
analysis, Jasmine Ross, Eileen Reid, Ruth Primrose, Brian Ord,
Stacey Munro for various analyses and Dr Dave Johnson
(University of Aberdeen) for critical reviewing of the manu-
script. This project was funded by the Scottish Executive
Environment and Rural Affairs Department.
references
Aber, J.D., Mellilo, J.M., MacClaugherty, C.A., 1990. Predicting
long-term patterns of mass loss, N dynamics and soil
organic matter formation from initial fine litter chemistry in
temperate forest ecosystems. Can. J. Bot. 68, 2202–2208.
Artursson, V., Finlay, R.D., Jansson, J.K., 2005. Combined
bromodeoxyuridine immunocapture and terminal-
restriction fragment length polymorphism analysis
highlights differences in the active soil bacterial
247
Moisture Biomass Soil N Soil C
0.4 0.83 0.31 0.002 0.01
0.16 0.75 <0.001 0.67 0.34
0.28 0.34 0.62 0.92 0.64
0.31 0.16 0.7 0.83 0.89
0.04 0.11 0.09 0.4 0.31
metagenome due to Glomus mosseae inoculation or plant
species. Environ. Microbiol. 7, 1952–1966.
Bardgett, R.D., Mawdsley, J.L., Edwards, S., Hobbs, P.J., Rodwell,
J.S., Davies, W.J., 1999. Plant species and nitrogen effects on
soil biological properties of temperate upland grasslands.
Funct. Ecol. 13, 650–660.
Boer, E.D., Folman, L.B., Summerbell, R.C., Boddy, L., 2005. Living
in a fungal world: impact of fungi on soil bacterial niche
development. FEMS Microbiol. Rev. 29, 795–811.
Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985.
Chloroform fumigation and release of soil nitrogen—a rapid
direct extraction method to measure microbial biomass
nitrogen in soil. Soil Biol. Biochem. 17, 837–842.
Buckley, D.H., Schmidt, T.M., 2001. The structure of microbial
communities in soil and the lasting impact of cultivation.
Microbial Ecol. 42, 11–21.
Costa, R., Gotz, M., Mrotzek, N., Lottmann, J., Berg, G., Smalla, K.,
2006. Effects of site and plant species on rhizosphere
community structure as revealed by molecular analysis of
microbial guilds. FEMS Microbiol. Ecol. 56, 236–249.
Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced
specificity for basidiomycetes: application to the
identification of mycorrhiza and rusts. Mol. Ecol. 2, 118–133.
Garnier, E., Cortez, J., Billes, G., Navas, M., Roumet, C.,
Debussche, M., Laurent, G., Blanchard, A., Aubry, D.,
Bellman, A., Niell, C., Toussant, J., 2004. Plant functional
markers capture ecosystem properties during secondary
succession. Ecology 85, 2630–2637.
Girvan, M.S., Bullimore, J., Pretty, J.N., Osborn, A.M., Ball, A.S.,
2003. Soil type is the primary determinant of the
composition of the total and active bacterial communities
in arable soils. Appl. Environ. Microbiol. 69, 1800–1809.
Grayston, S.J., Wang, S.Q., Campbell, C.D., Edwards, A.C., 1998.
Selective influence of plant species on microbial diversity in
the rhizosphere. Soil Biol. Biochem. 30, 369–378.
Grayston, S.J., Campbell, C.D., Bardgett, R.D., Mawdsley, J.L.,
Clegg, C., Ritz, K., Griffiths, B.S., Rodwell, J.S., Edwards, S.J.,
Davies, W.J., Elston, D.J., Millard, P., 2004. Assessing shifts in
microbial community structure across a range of grasslands
of differing management intensity using CLPP PLFA and
community DNA techniques. Appl. Soil Ecol. 25, 2563–2584.
Griffiths, R.I., Bailey, M.J., McNamara, N.P., Whiteley, A.S., 2006.
The functions and components of the Sourhope soil
microbiota. Appl. Soil Ecol. 33, 114–126.
Grime, J.P., Hunt, R., 1975. Relative growth rate—its range and
adaptive significance in a local flora. J. Ecol. 63, 393–422.
Grime, J.P., Hodgson, J.G., Hunt, R., 2007. Comparative Plant
Ecology: A Functional Approach to Common British Species.
Castlepoint Press, UK.
Hobbie, S.E., 2002. Effects of plant species on nutrient cycling.
Trend Ecol. Evol. 7, 336–339.
Horner-Devine, M.C., Leibold, M.A., Smith, V.H., Bohannan,
B.J.M., 2003. Bacterial diversity patterns along a gradient of
primary productivity. Ecol. Lett. 6, 613–622.
Hunt, R., Cornelissen, J.H.C., 1997. Components of relative
growth rate and their interrelation in 59 temperate plant
species. New Phytol. 135, 395–417.
248 applied soil ecology 41 (2009) 239–248
International Standard Organisation, 1997. Soil Quality—
Determination of Water Content on a Volume Basis—
Gravimetric Method. ISO 11461. ISO, Geneva.
Jackson, D.A., 1995. PROTEST—a procrustean randomization
test of community environment concordance. Ecoscience 2,
297–303.
Johnson, D., Booth, R.E., Whiteley, A.S., Bailey, M.J., Read, D.J.,
Grime, J.P., Leak, J.R., 2003. Plant community composition
affected the biomass, activity and diversity of soil
microorganisms in reconstituted calcareous grassland. Eur.
J. Soil Sci. 54, 671–678.
Jones, D.L., Hodge, A., Kuzyakov, Y., 2004. Plant and mycorrhizal
regulation of hizodeposition. New Phytol. 163, 459–480.
Lu, Y., Rosencrantz, D., Liesack, W., Conrad, R., 2006. Structure
and activity of bacterial community inhabiting rice roots
and the rhizosphere. Environ. Microbiol. 8, 1351–1360.
Marcial-Gomes, N.C., Fagbola, O., Costa, R., Rumjanek, N.G.,
Buchner, A., Mendona-Hagler, L., Smalla, K., 2003.
Dynamics of fungal communities in bulk and maize
rhizosphere soil in the tropics. Appl. Environ. Microbiol. 69,
3758–3766.
Marilley, L., Vogt, G., Blanc, M., Aragno, M., 1998. Bacterial
diversity in the bulk soil and rhizosphere of Lolium perenne
and Trifolium repens as revealed by PCR restriction analysis
of 16S rDNA. Plant Soil 198, 219–224.
McCaig, A.E., Glover, L.A., Prosser, J.I., 1999. Molecular analysis
of bacterial community structure and diversity in
unimproved and improved upland grassland pastures.
Appl. Environ. Microbiol. 65, 1721–1730.
Mougel, C., Offre, P., Ranjard, L., Corbeand, T., Gamalero, E.,
Robin, C., Lemanceau, P., 2006. Dynamics of the genetic
structure of bacterial and fungal communities at different
developmental stages of Medicago truncatula
Gaertn.cv.jemalong line J5. New Phytol. 170, 165–175.
Nunan, N., Daniell, T.J., Singh, B.K., Papert, A., McNicol, J.W.,
Prosser, J.I., 2005. Links between plant and rhizoplane
bacterial communities in grassland soils characterized
using molecular techniques. Appl. Environ. Microbiol. 71,
6784–6792.
Nusslein, K., Tiedje, J.M., 1999. Soil bacterial community shift
correlated with change from forest to pasture vegetation in
a tropical soil. Appl. Environ. Microbiol. 65, 3622–3626.
Peres-Neto, P.R., Jackson, D.A., 2001. How well do multivariate
data sets match? The advantages of a Procrustean
superimposition approach over the Mantel test. Oecologia
129, 169–178.
Phillips, D.A., Ferris, H., Cook, D.R., Strong, D.R., 2003. Molecular
control points in rhizosphere food webs. Ecology 84,
816–826.
Ritz, K., McNicol, W., Nunan, N., Grayston, S., Millard, P.,
Atkinson, D., Gollotte, A., Habeshaw, D., Boag, B., Clegg,
C.D., Griffiths, B.S., Wheatley, R.E., Glover, L.A., McCaig, A.E.,
Prosser, J.I., 2004. Spatial structure in soil chemical and
microbiological properties in an upland grassland. FEMS
Microbiol. Ecol. 49, 191–205.
Singh, B.K., Millard, P., Whiteley, A.S., Murrell, J.C., 2004.
Unravelling rhizosphere–microbial interactions:
opportunities and limitations. Trend Microbiol. 12, 386–393.
Singh, B.K., Nazarie, L., Munro, S., Anderson, I., Campbell, C.D.,
2006. Use of multiplex terminal restriction fragment length
polymorphism for rapid and simultaneous analysis of
different components of the soil microbial community.
Appl. Environ. Microbiol. 72, 7278–7285.
Singh, B.K., Tate, K.R., Kapadia, G., Hedley, C.B., Macdonald,
C.A., Millard, P., Murrell, J.C., 2007a. Effect of afforestation
and reforestation of pastures on the activity and population
dynamics of methanotrophic bacteria. Appl. Environ.
Microbiol. 73, 5153–5161.
Singh, B.K., Munro, S., Potts, J., Millard, P., 2007b. Influence of
grass species and soil type on rhizosphere microbial
community structure in grassland soils. Appl. Soil Ecol. 36,
147–155.
Singh, B.K., Nunan, N., Ridgway, K.P., McNicol, J., Young, J.P.W.,
Daniell, T.J., Prosser, J.I., Millard, P., 2008. Relationship
between assemblages of mycorrhizal fungi and bacteria on
grass roots. Environ. Microbiol. 10, 534–541.
Smit, E., Leeflang, P., Glandorf, B., van Elsas, J.D.V., Wernars, K.,
1999. Analysis of fungal diversity in the wheat rhizosphere
by sequencing of cloned PCR-amplified gene encoding 18S
rRNA and temperature gradient gel electrophoresis. Appl.
Environ. Microbiol. 65, 2614–2621.
Sparling, G.P., 1981. Microcalorimetry and other methods to
assess biomass and activity in soils. Soil Biol. Biochem. 13,
93–98.
Thompson, K., Parkinson, J.A., Band, S.R., Spencer, R.E., 1997. A
comparative study of leaf nutrient concentration in a
regional herbaceous flora. New Phytol. 136, 679–689.
Tilman, D., Reich, P.B., Knops, J.M.H., 2006. Biodiversity and
ecosystem stability in a decade-long grassland experiment.
Nature 441, 629–632.
Usher, M.B., Sier, A.R.J., Hornung, M., Millard, P., 2006.
Understanding biological diversity in soil: The UK’s Soil
Biodiversity Research Programme. Appl. Soil Ecol. 33,
101–113.
Vandenkoornhuyse, P., Ridgway, K.P., Watson, I.J., Fitter, A.H.,
Young, J.P.W., 2003. Co-existing grass species have
distinctive arbuscular mycorrhizal communities. Mol. Ecol.
12, 3085–3095.
Van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis,
P., Streitwolf-Engel, R., Boller, T., Wiemken, A., Sanders, I.R.,
1998a. Mycorrhizal fungal diversity determines plant
biodiversity, ecosystem variability and productivity. Nature
396, 69–72.
Van der Heijden, M.G.A., Boller, T., Wiemken, A., Sanders, I.R.,
1998b. Different arbuscular mycorrhizal fungal species are
potential determinants of plant community structure.
Ecology 79, 2082–2091.
Van der Heijden, M.G.A., Bakker, R., Verwaal, J., Scheublin, T.R.,
Rutten, M., van Logtestijn, R., Staehelin, C., 2006. Symbiotic
bacteria as a determinant of plant community structure and
plant productivity in dune grassland. FEMS Microbiol. Ecol.
56, 178–187.
Van Vuuren, M.M.I., Aerts, R.F., Berendse, F., De Visser, W., 1992.
N mineralization in heathland ecosystems dominated by
different plant species. Biogeochemistry 16, 151–166.
Verville, J.H., Hobbie, S.E., Chapin, F.S., hooper, D.U., 1998.
Response of tundra Ch4 and Co2 flux to manipulation of
temperature and vegetation. Biogeochemistry 41, 215–235.
Wardle, D.A., Barker, G.M., Bonner, K.I., Nicholson, K.S., 1998.
Can comparative approaches based on plant
ecophysiological traits predict the nature of biotic
interactions and individual plant species effects in
ecosystems? J. Ecol. 86, 405–420.
Wardle, D.A., Bardgett, R.D., Klironomos, J.N., Setala, H., van der
Putten, W.H., Wall, D.H., 2002. Ecological linkages between
aboveground and belowground biota. Science 304, 1629–
1633.
White, T.J., Bruns, T.D., Lee, S., Taylor, J., 1990. Analysis of
phylogenetic relationship by amplification and direct
sequencing of ribosomal RNA genes. In: Innis, M.A., Gelfond,
D.H., Sainsky, J.J., White, T.J. (Eds.), PCR Protocol: A Guide to
Method and Applications. Academic Press, New York, NY.
Zou, J., Roger, W.E., Siemann, E., 2007. Differences in
morphological and physiological traits between native and
invasive populations of Sapium sebiferum. Funct. Ecol. 21,
721–730.