Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:
Tree Physiology 16, 1031--1038
© 1996 Heron Publishing----Victoria, Canada
Functional biodiversity of microbial communities in the rhizospheres
of hybrid larch (Larix eurolepis) and Sitka spruce (Picea sitchensis)
SUSAN J. GRAYSTON and COLIN D. CAMPBELL
Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen, AB15 8QH, U.K.
Received October 25, 1995
Summary The diversity of microorganisms associated with
trees and their different functional capabilities is thought to be
a consequence of variation in carbon compounds in the rhizosphere. We used the Biolog® system (Biolog Inc., Hayward,
CA), a redox-based test, to construct sole carbon source utilization profiles (metabolic fingerprints) of microbial communities from the rhizospheres and rhizoplanes of hybrid larch
(Larix eurolepis A. Henry) and Sitka spruce (Picea sitchensis
Bong. Carr.) taken from a farm woodland site and two secondrotation plantation forest sites. Canonical variate analysis
(CVA) of carbon utilization data differentiated among the
microbial communities from the three forest sites, with the
greatest discrimination between the farm woodland and the two
second-rotation forest sites. Carbohydrates and carboxylic acids were the substrates responsible for this discrimination.
Carbon profiles of the microbial communities from the rhizospheres of the two tree species also clustered when evaluated
by CVA, as a result of differences in utilization of carboxylic
acids and amino acids, suggesting that these tree species differ
in the exudates they produce. Isolation and enumeration of
organisms confirmed that there were qualitative and quantitative differences in the culturable populations of microorganisms at the different sites and between tree species. We
conclude that Biolog is a useful technique for evaluating the
functional diversity of microbial communities; however, to
interpret the results accurately, they must be assessed in conjunction with the actual carbon substrates available in the
particular ecosystem under study.
Keywords: Biolog, carbon utilization profiles, metabolic fingerprints, root exudates.
Introduction
Trees are grown for timber in a wide variety of soil types and
systems (e.g., coppice plantations, agroforestry and farm
woodland on former agricultural land). Because of increasing
emphasis on low-input sustainable production systems, nutrient availability is a key factor governing tree growth (Mahendrappa et al. 1986). Soil microorganisms can increase nutrient
availability through mineralization of soil organic matter and
the solubilization of soil minerals (Lee and Parkhurst 1992,
Sparling 1994). Although microbial growth in soil is carbon
limited (Wardle 1992), the presence of high concentrations of
carbon in the rhizosphere, as a result of rhizodeposition, makes
it an area of high microbial activity (Lynch and Whipps 1990).
It has been hypothesized that the diversity of microorganisms
present in different plant rhizospheres is due to variation in the
compounds exuded by plants (Curl and Truelove 1986, Bowen
and Rovira 1991, Bolton et al. 1992). Because soil microorganisms differ in their potential to perform nutrient transformations and other beneficial functions (e.g., antagonism of
pathogens), the composition of the microbial community in the
rhizosphere will have important consequences for tree growth
(Grayston et al. 1996). However, there has been little research
on the nature of tree root exudates (Leyval and Berthelin 1993)
or on the diversity of microorganisms in tree rhizospheres
(Rambelli 1973, Malacjzuk and McComb 1979, Linderman
1988).
The Biolog® system is a commercially available redoxbased sole carbon source utilization test (Biolog Inc., Hayward, CA) that is used to make metabolic fingerprints and
hence enable identification of pure cultures of microorganisms
(Biolog 1993). Recently, the Biolog system has been used to
assess differences in microbial communities from diverse
habitats (Garland and Mills 1991), soil types (Winding 1994)
and grasslands (Zak et al. 1994) and to quantify the impact of
heavy metals on microbial diversity (Campbell et al. 1995). In
all of these studies, multivariate analysis of the carbon substrate utilization profiles facilitated classification of microbial
communities based on their metabolic abilities. The technique
is, therefore, an ecologically relevant method to measure biodiversity, because it determines differences in community utilization of carbon, a key factor governing microbial growth in
soil. The community approach to measuring biodiversity may
provide more information than measuring the presence of
individuals in the population, which may not relate to the
function of the community as a whole.
The objective of this study was to determine the taxonomic
and metabolic diversity of microbial communities from the
rhizospheres and rhizoplanes of two tree species, hybrid larch
(Larix eurolepis L. Henry) and Sitka spruce (Picea sitchensis
Bong. Carr.) growing at three different forest sites, two secondrotation forest plantations and a farm woodland. We tested the
hypothesis that microbial communities from the rhizospheres
of different tree species and different sites vary in their rate of
1032
GRAYSTON AND CAMPBELL
utilization of C sources because of differences in community
composition.
Materials and methods
Sample collection
Rhizosphere soil samples were collected from three sites in the
Grampian Region in northeastern Scotland. Samples were
taken from monoculture stands of two tree species, hybrid
larch (Larix eurolepis) and Sitka spruce (Picea sitchensis) at
two second-rotation plantation forest sites, Durris (NO
772917, 2°22′32″ W 57°0′58″ N, Countesswells Series) and
Midmar (NJ 695005, 2°30′14″ W 57°8′22″ N, Countesswells
Series), and a farm woodland site at Lower Affleck (NJ
864239, 2°13′33″ W 57°18′20″ N, Thistlyhill Series). All tree
species were of the same age (7 years) and had been planted
four years previously. Trees at the forest site were planted at
2,500 stems ha −1, whereas trees at the farm forest at Lower
Affleck were planted at a density of 4,000 stems ha −1. The
ground vegetation consisted of grasses and herbs. At each site,
triplicate soil and root samples were collected to a depth of
15 cm with a soil corer (5 cm in diameter) and bulked for each
of three replicates for each tree species.
Carbon utilization profiles
Rhizosphere communities were extracted by shaking 10 g of
rhizosphere soil (soil adhering to roots) in 100 ml of 0.25strength Ringers solution (Oxoid, Unipath Ltd., Basingstoke,
U.K.) for 10 min. In a sequential process, the rhizoplane
communities were then extracted by shaking 5 g of the washed
roots in 50 ml of Ringers solution containing glass beads (20 g)
on a wrist action shaker for 10 min. Biolog GN microplates
(Biolog Inc., Hayward, CA) were used to determine the metabolic diversity of the microbial communities from these samples. The 96-well microtiter plate contained 95 different
carbon sources and a control well with no carbon source
(Biolog 1993). Each well also contained a redox indicator dye,
tetrazolium violet, and nutrients in dehydrated form, which
were rehydrated on addition of the sample (Bochner 1989). A
50-ml aliquot of a 10 −4 dilution of each rhizosphere and
rhizoplane sample was centrifuged at 750 g for 10 min to
remove any soil or root particles that might introduce extraneous carbon into the wells. The 10 −4 dilution was chosen to
minimize background color in the inoculum and was consistent for all samples to reduce confounding effects on rates of
color development caused by different inoculum densities
(Garland and Mills 1991). A 150-µl aliquot of the supernatant
from the centrifuged samples was added to each of the 96 wells
in the GN microplate. Plates were incubated at 25 °C and color
development was measured as absorbance at 590 nm every
24 h for 120 h with an optical density (OD) plate reader.
The data were evaluated by canonical variate analysis (CVA)
to differentiate samples based on the overall pattern of carbon
utilization and to identify which carbon sources influenced the
discrimination. Previous studies (Garland and Mills 1991,
Winding 1994) noted a correlation between inoculum cell
density and rate of color development that was corrected for,
when data were transformed, by dividing each well OD by the
average well color development (AWCD) for that sample (Garland and Mills 1991). The AWCD of different carbon substrate
groups (e.g., carbohydrates, carboxylic acids, amino acids,
amides, polymers and miscellaneous compounds) was calculated (as defined by Zak et al. 1994) and treatment effects
assessed by a three-way ANOVA. Canonical variate analysis
(CVA) and ANOVA were performed using Genstat 5.3 (Copyright 1992, Lawes Agricultural Trust, Rothamsted Experimental Station, U.K.).
Plate counts of microbial populations
The same rhizosphere and rhizoplane community samples
used in the carbon utilization profiles were serially diluted and
0.1 ml suspensions spread, in duplicate, on the following
selective media: Tryptone Soy agar (Oxoid, 0.1 strength)
plus cycloheximide (50 mg l −1) for enumeration of bacteria
and actinomycetes; Pseudomonas Isolation agar (Oxoid),
which is selective for populations of pseudomonads; and Rose
Bengal agar (Oxoid) for enumeration of yeasts and fungi. The
plates were incubated at 25 °C and colonies counted after
4 days for pseudomonads and fungi and after 14 days for
bacteria and actinomycetes. An ANOVA was used to determine
statistically significant treatment differences according to the
F-test, and the LSD for the 95% confidence interval (LSD0.05)
value was used in multiple comparisons. Isolates from the
Pseudomonas Isolation agar were identified using the Biolog®
system (Biolog 1993). Microlog software (Biolog Inc.) was
used to identify cultures based on their metabolic profiles.
Results
Carbon utilization profiles
Color development in the Biolog wells generally followed a
sigmoidal curve with incubation time but varied for different
groups of carbon sources (Figure 1). The order of highest
utilization after 120 h was carbohydrates > amino acids >
miscellaneous compounds > polymers > amides > carboxylic
acids. The AWCD for each group of carbon compounds
showed that there was significantly greater utilization of carbohydrates by microbial communities in rhizosphere soil compared with those on the rhizoplane (Table 1). Microbial
communities from the farm woodland site at Lower Affleck
tended to have greater rates of utilization of all six groups of C
sources than microbial communities taken from either of the
two second-rotation forest sites at Durris and Midmar (Table 1). The two tree species exhibited differences in carbon
utilization. At the Durris site, there was significantly higher
utilization of amino acids by the microbial communities from
the larch rhizosphere and rhizoplane than from the Sitka spruce
rhizosphere and rhizoplane (Table 1).
Multivariate analysis was performed on all data from each
incubation time to identify patterns of response among samples. The results presented are those after 24 h of incubation
because this incubation time resulted in the greatest discrimination among treatments. The different soils had distinct patterns of carbon utilization (Figure 2). The most distinctive
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1033
The discrimination between larch and Sitka spruce was mainly
a result of differences in utilization of some carboxylic acids
and amino acids (Table 2). Differences in utilization of carbon
sources between rhizosphere and rhizoplane samples from the
same tree species were only found at Midmar, where microbial
communities from the larch rhizoplane had significantly lower
utilization of all carbon compounds listed in Table 3 than
microbial communities from the larch rhizosphere.
Microbial populations
Figure 1. Average well color development profiles (OD 590 nm) for
rhizosphere and rhizoplane samples from Durris for each of the six
groups of carbon substrates in the Biolog GN plate ( m carbohydrates,
e carboxylic acids, h amino acids, r amides, ) polymers, n miscellaneous and j all substrates). Values are means of 12 replicate samples.
patterns were obtained with samples from the farm woodland
site at Lower Affleck, which had lower coordinate values on
canonical variate (CV)-1 (which explained 38.5% of the variance in the data) than the two second-rotation forest sites at
Midmar and Durris. The two forest sites showed some separation on the CV-2 axis (which explained 15% of the data
variance) with Midmar samples possessing a lower canonical
score than Durris samples (Figure 2). The samples from larch
at Durris were most distinct, being separated mainly on CV-1,
but with clear separation of the rhizosphere and rhizoplane on
CV-2 as well (Figure 2). The ANOVA of the AWCD of the
carbon compounds responsible for discrimination of CV indicated that microbial communities in the farm woodland had
significantly greater utilization of several carboxylic acids. In
addition, microbial communities from Durris showed significantly greater utilization of cellobiose than those from Midmar.
In addition to separation among the forest sites, there was
also clear clustering of samples between different tree species
and root sample zones (Figure 2). On CV-2, the separation of
microbial communities (e.g., Durris larch rhizosphere and
rhizoplane) was the result of differences in utilization of several carbohydrates (N-acetyl glucosamine, adonitol, cellobiose
and xylitol), carboxylic acids (D-galactonic acid lactone, itaconic acid and succinic acid) and the amide 2-amino-ethanol.
Rhizosphere and rhizoplane samples from the farm woodland
at Lower Affleck contained significantly higher populations of
all microorganisms than samples from the second-rotation
forest sites at Durris and Midmar (Figure 3). There were no
significant quantitative differences between populations of microorganisms associated with the different tree species, but
there were clear trends in associations (Figure 4). For example,
Sitka spruce had higher populations of fungi in its rhizosphere
and on its rhizoplane than larch, except for rhizoplane samples
from Midmar (Figure 4). There were more bacteria in the
rhizosphere and on the rhizoplane of larch than of Sitka spruce,
except for rhizosphere samples at Midmar. In addition to
quantitative differences in microorganisms between the two
tree species at the different sites, there were also qualitative
differences. At Lower Affleck, there were significantly greater
numbers and species of pseudomonads than at the two other
sites (Figure 5) and these pseudomonads constituted a high
proportion of the bacterial population. The Sitka spruce rhizosphere soils and rhizoplane contained significantly higher
populations of pseudomonads than those of larch at every site
except for the rhizosphere samples at Durris (Figure 5). Cultures of these pseudomonads included a mixture of fluorescent
(P. fluorescens Type G., P. corrugata, P. putida) and non-fluorescent species (P. cepacia) from the Sitka spruce rhizosphere
and rhizoplane, whereas non-fluorescent species dominated in
the larch rhizosphere.
Twenty fungal species were isolated from the three forest
sites. Nine species were present at Lower Affleck, one of which
was site specific. Sixteen species were found at the Durris site,
five of which were not isolated from the other sites. Twelve
species of fungi were present in the Midmar samples; three
were site specific. All sites appeared to have the same dominant fungal genera: Trichoderma sp., Penicillium sp., Fusarium sp., Mucor sp., Rhizopus sp. and Aspergillus sp. At all
sites, Trichoderma and Penicillium species dominated the rhizosphere of larch, and Aspergillus species dominated the rhizosphere of Sitka spruce.
Thirteen species of yeast were isolated from the three sites,
eleven from Lower Affleck, three of which were not found at
the other sites. Nine species were found at Durris, of which two
were site specific. Eight species were found at Midmar, all of
which were present at the other sites. In addition, two yeast
species were specific to larch and two were specific to Sitka
spruce.
1034
GRAYSTON AND CAMPBELL
Table 1. Average well color development in the six groups of carbon compounds in the Biolog GN plate by microbial communities from the
rhizosphere and rhizoplane of hybrid larch and Sitka spruce from three forest sites. 1
Carbon compound
Carbohydrates
Carboxylic acids
Amino acids
Amides
Polymers
Miscellaneous
1
Forest site
Hybrid larch
Sitka spruce
LSD (0.05)
Rhizoplane
Rhizosphere
Rhizoplane
Rhizosphere
Lower Affleck
Durris
Midmar
Mean (all sites)
0.715
0.654
0.244
0.537
0.739
0.857
0.567
0.721
0.789
0.485
0.326
0.533
1.012
0.548
0.583
0.715
Lower Affleck
Durris
Midmar
Mean (all sites)
0.535
0.378
0.239
0.384
0.497
0.469
0.405
0.457
0.584
0.325
0.310
0.407
0.591
0.304
0.372
0.422
Lower Affleck
Durris
Midmar
Mean (all sites)
0.797
0.464
0.232
0.498
0.666
0.675
0.414
0.585
0.769
0.370
0.343
0.494
0.701
0.378
0.426
0.502
Lower Affleck
Durris
Midmar
Mean (all sites)
0.427
0.238
0.082
0.249
0.332
0.256
0.210
0.266
0.444
0.131
0.169
0.248
0.329
0.156
0.203
0.229
Lower Affleck
Durris
Midmar
Mean (all sites)
0.546
0.416
0.209
0.390
0.523
0.364
0.349
0.412
0.497
0.325
0.260
0.361
0.592
0.309
0.293
0.398
Lower Affleck
Durris
Midmar
Mean (all sites)
1.101
0.815
0.347
0.754
1.051
1.067
0.546
0.888
1.050
0.680
0.304
0.678
1.253
0.743
0.706
0.901
0.385
0.160
0.182
0.091
0.261
0.131
0.197
0.093
0.186
0.091
0.439
0.209
Values are means of three replicate samples.
Discussion
We found quantitative and qualitative differences in the microbial populations of the rhizospheres of hybrid larch and Sitka
Figure 2. Canonical variate scores of soil samples based on sole carbon
source utilization profiles of microbial communities from either the
rhizosphere (filled symbols) or rhizoplane (open symbols) of hybrid
larch or Sitka spruce, respectively, from a farm woodland at Lower
Affleck (d, semi-circles) or second-rotation plantation forests at Durris(m, .) and Midmar (r, j). Symbols represent different samples.
spruce. These populations also varied depending on the soil
type where the trees were grown. We demonstrated that the
Biolog system can be used as a rapid screening technique to
discriminate among microbial communities from different tree
species and habitats based on metabolic profiles. The use of
differential utilization of a range of carbon compounds to
assess microbial diversity is an ecologically relevant method
for measuring diversity, because carbon is one of the main
influences on microbial growth in soil (Wardle 1992). However, the technique can only elucidate the causal relationship
in conjunction with information about what exudates are produced by trees. We found that the microbial communities from
the farm woodland system at Lower Affleck exhibited more
rapid utilization of all six groups of carbon compounds contained in the Biolog plate than the microbial communities from
the two second-rotation forest systems, indicating different
metabolic profiles between the farm woodland and second-rotation forest systems. The plate counts also confirmed that
there were qualitative and quantitative differences in the microbial communities between the forest sites.
The differences in metabolic and taxonomic diversity of the
microorganisms from Lower Affleck compared to the two
second-rotation forest sites probably reflect the previous land
use of this site that has affected the soil characteristics. Thus,
previous application of fertilizers at Lower Affleck, necessary
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1035
Table 2. Carbon compounds utilized differently by microbial communities in the rhizosphere of hybrid larch and Sitka spruce.
Carbon
compound
Community
with significantly
greater utilization
Carbohydrate
Cellobiose
Hybrid larch
Carboxylic acids
D-Galacturonic
acid
acid
D-Saccharic acid
Malonic acid
γ-Hydroxybutyric acid
Itaconic acid
Hybrid larch
Hybrid larch
Hybrid larch
Hybrid larch
Sitka spruce
Sitka spruce
Serine
γ-Aminobutyric acid
Hybrid larch
Sitka spruce
D-Glucoronic
Amino acids
in arable farming systems, likely removed any nutrient limitation to microbial growth in the soil. In addition, annual herbaceous plants exude more of their assimilated carbon into the
soil than trees (Leyval and Berthelin 1993, Grayston et al.
1996) and also exude a different spectrum of compounds
(Grayston et al. 1996), which would influence microbial community structure. The second rotation forest soils also had a
lower pH than the farm woodland system. An increase in pH
has been shown to increase root exudation in grasses (Meharg
and Killham 1990). In addition, bacteria prefer more neutral
soil conditions and therefore the low pH of the second-rotation
forest soils would select against these microorganisms.
The Biolog system assesses the metabolic diversity of the
culturable bacteria only, because fungal growth is too slow to
have an influence on the profile (Garland and Mills 1991).
Heuer et al. (1995) recently demonstrated that species of Enterobacter were mainly responsible for the color development
in Biolog wells by using 16S rRNA probes, after inoculation
with microbial communities from the phyllosphere and rhi-
Figure 3. Total populations of bacteria, actinomycetes, yeasts and
fungi in forest rhizosphere soils at Durris, Midmar and Lower Affleck.
Values are means of 12 replicate samples. Bars indicate LSD 0.05
values.
zosphere of potato plants. At Lower Affleck, a high proportion
of the bacterial population consisted of Pseudomonas species,
which likely accounts for the higher utilization of the carbon
sources in the Biolog plate by the microbial communities from
this site compared with those from the second-rotation forest
sites.
Another factor that could have influenced exudation patterns and hence microbial diversity is the presence of different
mycorrhizal symbionts (Molina et al. 1992). Native mycorrhi-
Table 3. Average well color development in the Biolog GN plates by microbial communities from a farm woodland site (Lower Affleck) and two
second-rotation forest sites (Durris and Midmar).1
Carbon compound
Forest site
Lower Affleck
Durris
Midmar
LSD (0.05)
Carbohydrates
N-Acetyl galactosamine
N-Acetyl glucosamine
Cellobiose
0.95
1.18
1.11
0.69
0.74
0.78
0.68
0.52
0.46
0.27
0.28
0.19
Carboxylic acids
D-Galactonic
acid lactone
acid
D-Galacturonic acid
γ-Hydroxybutyric acid
Malonic acid
Itaconic acid
Succinic acid
0.36
1.25
1.36
0.80
0.91
1.06
0.39
0.27
1.01
0.66
0.53
0.73
0.66
0.28
0.30
0.87
0.74
0.46
0.69
0.68
0.28
0.06
0.24
0.24
0.12
0.08
0.38
0.05
Serine
γ-Aminobutyric acid
1.14
0.65
0.80
0.38
0.68
0.37
0.20
0.10
D-Gluconic
Amino acids
1
Values are means of 12 samples.
1036
GRAYSTON AND CAMPBELL
Figure 4. Populations of (A) bacteria, (B) actinomycetes, (C)
fungi and (D) yeasts in the rhizosphere and on the rhizoplane of
Sitka spruce and hybrid larch at
Durris, Midmar and Lower Affleck. Values are means of three
replicate samples. Bars indicate
LSD0.05 values.
zal fungi present in the soil are thought to infect tree roots
(Bledsoe 1992). Therefore, the lack of previous tree growth at
Lower Affleck may have resulted in a poorer and less diverse
infection than at the second-rotation forest sites. Mycorrhizae
differ in their carbon storage patterns and their conversion of
plant sugars to metabolic intermediates (Finlay and Söderstrom 1992), and these differences could result in alterations in
the quality and quantity of carbon released from tree roots.
Leyval and Berthelin (1993) showed that beech (Fagus sylvatica L.) infected with Laccaria laccata exuded more sugars
and amino acids and a different range of organic acids than
non-mycorrhizal beech or mycorrhizal Scots pine (Pinus
sylvestris L.). Our results suggest that hybrid larch and Sitka
spruce may produce different exudates because the carbon
profiles of the microorganisms from the rhizosphere of these
two tree species vary, mainly as a result of differences in
carboxylic acid and amino acid usage. Previously, we found
that utilization of carboxylic acids was highly variable in
rhizosphere samples from different tree species growing in the
same soil type (Grayston et al. 1994). Although the carbon
compounds exuded by hybrid larch and Sitka spruce have not
been characterized, it is known that the quantity and character
of root exudates released by trees vary considerably among
species (see review by Grayston et al. 1996). A wide range of
organic acids, which are quantitatively the most important
organic component of tree root exudates (Smith 1976), are
produced by different tree species. For example, gluconic and
malonic acids, which showed differential utilization between
larch and Sitka spruce microbial communities (Table 2), are
produced by some Pinus species, but not by others (Grayston
et al. 1996). The types of amino acids exuded are also highly
variable; deciduous trees produce cystine and homoserine,
whereas these amino acids have not been isolated from
exudates of evergreens (Grayston et al. 1996).
Variation in metabolic diversity by microbial communities
from the rhizosphere of the two tree species was accompanied
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1037
zospheres of larch and Sitka spruce at different sites as a
consequence of variation in the species composition of the
different microbial populations. The identification of root
exudates from these tree species and inclusion of these carbon
sources in future utilization tests will improve the resolution of
the technique. Measurement of the production of different
types of exudates in the tree would allow us to link utilization
rates directly with exudation. The beneficial effects of rhizosphere microorganisms on tree growth highlight the need for
a greater understanding of microbial community structure and
function in the rhizosphere.
Acknowledgments
We thank D. Hirst for assistance with canonical variate analysis. C.M.
Cameron, M.S. Davidson, A. Norrie and E.J. Reid are thanked for
technical assistance. Forest Enterprises--Durris, in particular Sandy
Sandilands, are thanked for information and access to the second
rotation forest sites. This research was funded by the Scottish Office
Agriculture, Environment and Fisheries Department.
Figure 5. Populations of pseudomonads in the rhizosphere and on the
rhizoplane of Sitka spruce and hybrid larch at Durris, Midmar and
Lower Affleck. Values are means of three replicate samples. Bars
indicate LSD0.05 values.
by differences in the culturable populations present. There
appeared to be tree- and site-specific bacteria, actinomycetes
and yeasts. All the fungal species isolated were present at each
site and with both tree species, although the dominant fungal
types changed with tree species. However, all the fungal species isolated are prolific spore producers and therefore one
must be cautious when interpreting the results of these plate
count data. At Lower Affleck, a high proportion of the bacterial
population consisted of Pseudomonas species, whereas at the
second-rotation forest sites there was a greater diversity of
actinomycetes. At every site, the larch rhizosphere contained
fewer fluorescent pseudomonads than the Sitka spruce rhizosphere. There have been relatively few studies on microbial
diversity in the rhizosphere of trees (Fitter and Garbaye 1994)
and none on hybrid larch or Sitka spruce. Studies comparing
mycorrhizal and non-mycorrhizal yellow birch (Katznelson et
al. 1962), red alder and Douglas-fir (Neal et al. 1968), various
coniferous (Oswald and Ferchau 1968) and Eucalyptus species
(Malacjzuk and McComb 1979) have shown distinct communities of microorganisms associating with different trees.
There is evidence that these rhizosphere microorganisms can
enhance tree growth through the production of plant growth
regulators (Strelczyk and Pokojska-Burdziej 1984, Strelczyk
and Leniarka 1985), nitrogen fixation (Li and Castellano 1987,
Li and Hung 1987), solubilization of inorganic phosphates
(Leyval and Berthelin 1991) and stimulation of mycorrhization of trees (Garbaye 1994).
Conclusions
We found significant differences in the utilization of various
carbon sources by microbial communities from the rhi-
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© 1996 Heron Publishing----Victoria, Canada
Functional biodiversity of microbial communities in the rhizospheres
of hybrid larch (Larix eurolepis) and Sitka spruce (Picea sitchensis)
SUSAN J. GRAYSTON and COLIN D. CAMPBELL
Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen, AB15 8QH, U.K.
Received October 25, 1995
Summary The diversity of microorganisms associated with
trees and their different functional capabilities is thought to be
a consequence of variation in carbon compounds in the rhizosphere. We used the Biolog® system (Biolog Inc., Hayward,
CA), a redox-based test, to construct sole carbon source utilization profiles (metabolic fingerprints) of microbial communities from the rhizospheres and rhizoplanes of hybrid larch
(Larix eurolepis A. Henry) and Sitka spruce (Picea sitchensis
Bong. Carr.) taken from a farm woodland site and two secondrotation plantation forest sites. Canonical variate analysis
(CVA) of carbon utilization data differentiated among the
microbial communities from the three forest sites, with the
greatest discrimination between the farm woodland and the two
second-rotation forest sites. Carbohydrates and carboxylic acids were the substrates responsible for this discrimination.
Carbon profiles of the microbial communities from the rhizospheres of the two tree species also clustered when evaluated
by CVA, as a result of differences in utilization of carboxylic
acids and amino acids, suggesting that these tree species differ
in the exudates they produce. Isolation and enumeration of
organisms confirmed that there were qualitative and quantitative differences in the culturable populations of microorganisms at the different sites and between tree species. We
conclude that Biolog is a useful technique for evaluating the
functional diversity of microbial communities; however, to
interpret the results accurately, they must be assessed in conjunction with the actual carbon substrates available in the
particular ecosystem under study.
Keywords: Biolog, carbon utilization profiles, metabolic fingerprints, root exudates.
Introduction
Trees are grown for timber in a wide variety of soil types and
systems (e.g., coppice plantations, agroforestry and farm
woodland on former agricultural land). Because of increasing
emphasis on low-input sustainable production systems, nutrient availability is a key factor governing tree growth (Mahendrappa et al. 1986). Soil microorganisms can increase nutrient
availability through mineralization of soil organic matter and
the solubilization of soil minerals (Lee and Parkhurst 1992,
Sparling 1994). Although microbial growth in soil is carbon
limited (Wardle 1992), the presence of high concentrations of
carbon in the rhizosphere, as a result of rhizodeposition, makes
it an area of high microbial activity (Lynch and Whipps 1990).
It has been hypothesized that the diversity of microorganisms
present in different plant rhizospheres is due to variation in the
compounds exuded by plants (Curl and Truelove 1986, Bowen
and Rovira 1991, Bolton et al. 1992). Because soil microorganisms differ in their potential to perform nutrient transformations and other beneficial functions (e.g., antagonism of
pathogens), the composition of the microbial community in the
rhizosphere will have important consequences for tree growth
(Grayston et al. 1996). However, there has been little research
on the nature of tree root exudates (Leyval and Berthelin 1993)
or on the diversity of microorganisms in tree rhizospheres
(Rambelli 1973, Malacjzuk and McComb 1979, Linderman
1988).
The Biolog® system is a commercially available redoxbased sole carbon source utilization test (Biolog Inc., Hayward, CA) that is used to make metabolic fingerprints and
hence enable identification of pure cultures of microorganisms
(Biolog 1993). Recently, the Biolog system has been used to
assess differences in microbial communities from diverse
habitats (Garland and Mills 1991), soil types (Winding 1994)
and grasslands (Zak et al. 1994) and to quantify the impact of
heavy metals on microbial diversity (Campbell et al. 1995). In
all of these studies, multivariate analysis of the carbon substrate utilization profiles facilitated classification of microbial
communities based on their metabolic abilities. The technique
is, therefore, an ecologically relevant method to measure biodiversity, because it determines differences in community utilization of carbon, a key factor governing microbial growth in
soil. The community approach to measuring biodiversity may
provide more information than measuring the presence of
individuals in the population, which may not relate to the
function of the community as a whole.
The objective of this study was to determine the taxonomic
and metabolic diversity of microbial communities from the
rhizospheres and rhizoplanes of two tree species, hybrid larch
(Larix eurolepis L. Henry) and Sitka spruce (Picea sitchensis
Bong. Carr.) growing at three different forest sites, two secondrotation forest plantations and a farm woodland. We tested the
hypothesis that microbial communities from the rhizospheres
of different tree species and different sites vary in their rate of
1032
GRAYSTON AND CAMPBELL
utilization of C sources because of differences in community
composition.
Materials and methods
Sample collection
Rhizosphere soil samples were collected from three sites in the
Grampian Region in northeastern Scotland. Samples were
taken from monoculture stands of two tree species, hybrid
larch (Larix eurolepis) and Sitka spruce (Picea sitchensis) at
two second-rotation plantation forest sites, Durris (NO
772917, 2°22′32″ W 57°0′58″ N, Countesswells Series) and
Midmar (NJ 695005, 2°30′14″ W 57°8′22″ N, Countesswells
Series), and a farm woodland site at Lower Affleck (NJ
864239, 2°13′33″ W 57°18′20″ N, Thistlyhill Series). All tree
species were of the same age (7 years) and had been planted
four years previously. Trees at the forest site were planted at
2,500 stems ha −1, whereas trees at the farm forest at Lower
Affleck were planted at a density of 4,000 stems ha −1. The
ground vegetation consisted of grasses and herbs. At each site,
triplicate soil and root samples were collected to a depth of
15 cm with a soil corer (5 cm in diameter) and bulked for each
of three replicates for each tree species.
Carbon utilization profiles
Rhizosphere communities were extracted by shaking 10 g of
rhizosphere soil (soil adhering to roots) in 100 ml of 0.25strength Ringers solution (Oxoid, Unipath Ltd., Basingstoke,
U.K.) for 10 min. In a sequential process, the rhizoplane
communities were then extracted by shaking 5 g of the washed
roots in 50 ml of Ringers solution containing glass beads (20 g)
on a wrist action shaker for 10 min. Biolog GN microplates
(Biolog Inc., Hayward, CA) were used to determine the metabolic diversity of the microbial communities from these samples. The 96-well microtiter plate contained 95 different
carbon sources and a control well with no carbon source
(Biolog 1993). Each well also contained a redox indicator dye,
tetrazolium violet, and nutrients in dehydrated form, which
were rehydrated on addition of the sample (Bochner 1989). A
50-ml aliquot of a 10 −4 dilution of each rhizosphere and
rhizoplane sample was centrifuged at 750 g for 10 min to
remove any soil or root particles that might introduce extraneous carbon into the wells. The 10 −4 dilution was chosen to
minimize background color in the inoculum and was consistent for all samples to reduce confounding effects on rates of
color development caused by different inoculum densities
(Garland and Mills 1991). A 150-µl aliquot of the supernatant
from the centrifuged samples was added to each of the 96 wells
in the GN microplate. Plates were incubated at 25 °C and color
development was measured as absorbance at 590 nm every
24 h for 120 h with an optical density (OD) plate reader.
The data were evaluated by canonical variate analysis (CVA)
to differentiate samples based on the overall pattern of carbon
utilization and to identify which carbon sources influenced the
discrimination. Previous studies (Garland and Mills 1991,
Winding 1994) noted a correlation between inoculum cell
density and rate of color development that was corrected for,
when data were transformed, by dividing each well OD by the
average well color development (AWCD) for that sample (Garland and Mills 1991). The AWCD of different carbon substrate
groups (e.g., carbohydrates, carboxylic acids, amino acids,
amides, polymers and miscellaneous compounds) was calculated (as defined by Zak et al. 1994) and treatment effects
assessed by a three-way ANOVA. Canonical variate analysis
(CVA) and ANOVA were performed using Genstat 5.3 (Copyright 1992, Lawes Agricultural Trust, Rothamsted Experimental Station, U.K.).
Plate counts of microbial populations
The same rhizosphere and rhizoplane community samples
used in the carbon utilization profiles were serially diluted and
0.1 ml suspensions spread, in duplicate, on the following
selective media: Tryptone Soy agar (Oxoid, 0.1 strength)
plus cycloheximide (50 mg l −1) for enumeration of bacteria
and actinomycetes; Pseudomonas Isolation agar (Oxoid),
which is selective for populations of pseudomonads; and Rose
Bengal agar (Oxoid) for enumeration of yeasts and fungi. The
plates were incubated at 25 °C and colonies counted after
4 days for pseudomonads and fungi and after 14 days for
bacteria and actinomycetes. An ANOVA was used to determine
statistically significant treatment differences according to the
F-test, and the LSD for the 95% confidence interval (LSD0.05)
value was used in multiple comparisons. Isolates from the
Pseudomonas Isolation agar were identified using the Biolog®
system (Biolog 1993). Microlog software (Biolog Inc.) was
used to identify cultures based on their metabolic profiles.
Results
Carbon utilization profiles
Color development in the Biolog wells generally followed a
sigmoidal curve with incubation time but varied for different
groups of carbon sources (Figure 1). The order of highest
utilization after 120 h was carbohydrates > amino acids >
miscellaneous compounds > polymers > amides > carboxylic
acids. The AWCD for each group of carbon compounds
showed that there was significantly greater utilization of carbohydrates by microbial communities in rhizosphere soil compared with those on the rhizoplane (Table 1). Microbial
communities from the farm woodland site at Lower Affleck
tended to have greater rates of utilization of all six groups of C
sources than microbial communities taken from either of the
two second-rotation forest sites at Durris and Midmar (Table 1). The two tree species exhibited differences in carbon
utilization. At the Durris site, there was significantly higher
utilization of amino acids by the microbial communities from
the larch rhizosphere and rhizoplane than from the Sitka spruce
rhizosphere and rhizoplane (Table 1).
Multivariate analysis was performed on all data from each
incubation time to identify patterns of response among samples. The results presented are those after 24 h of incubation
because this incubation time resulted in the greatest discrimination among treatments. The different soils had distinct patterns of carbon utilization (Figure 2). The most distinctive
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1033
The discrimination between larch and Sitka spruce was mainly
a result of differences in utilization of some carboxylic acids
and amino acids (Table 2). Differences in utilization of carbon
sources between rhizosphere and rhizoplane samples from the
same tree species were only found at Midmar, where microbial
communities from the larch rhizoplane had significantly lower
utilization of all carbon compounds listed in Table 3 than
microbial communities from the larch rhizosphere.
Microbial populations
Figure 1. Average well color development profiles (OD 590 nm) for
rhizosphere and rhizoplane samples from Durris for each of the six
groups of carbon substrates in the Biolog GN plate ( m carbohydrates,
e carboxylic acids, h amino acids, r amides, ) polymers, n miscellaneous and j all substrates). Values are means of 12 replicate samples.
patterns were obtained with samples from the farm woodland
site at Lower Affleck, which had lower coordinate values on
canonical variate (CV)-1 (which explained 38.5% of the variance in the data) than the two second-rotation forest sites at
Midmar and Durris. The two forest sites showed some separation on the CV-2 axis (which explained 15% of the data
variance) with Midmar samples possessing a lower canonical
score than Durris samples (Figure 2). The samples from larch
at Durris were most distinct, being separated mainly on CV-1,
but with clear separation of the rhizosphere and rhizoplane on
CV-2 as well (Figure 2). The ANOVA of the AWCD of the
carbon compounds responsible for discrimination of CV indicated that microbial communities in the farm woodland had
significantly greater utilization of several carboxylic acids. In
addition, microbial communities from Durris showed significantly greater utilization of cellobiose than those from Midmar.
In addition to separation among the forest sites, there was
also clear clustering of samples between different tree species
and root sample zones (Figure 2). On CV-2, the separation of
microbial communities (e.g., Durris larch rhizosphere and
rhizoplane) was the result of differences in utilization of several carbohydrates (N-acetyl glucosamine, adonitol, cellobiose
and xylitol), carboxylic acids (D-galactonic acid lactone, itaconic acid and succinic acid) and the amide 2-amino-ethanol.
Rhizosphere and rhizoplane samples from the farm woodland
at Lower Affleck contained significantly higher populations of
all microorganisms than samples from the second-rotation
forest sites at Durris and Midmar (Figure 3). There were no
significant quantitative differences between populations of microorganisms associated with the different tree species, but
there were clear trends in associations (Figure 4). For example,
Sitka spruce had higher populations of fungi in its rhizosphere
and on its rhizoplane than larch, except for rhizoplane samples
from Midmar (Figure 4). There were more bacteria in the
rhizosphere and on the rhizoplane of larch than of Sitka spruce,
except for rhizosphere samples at Midmar. In addition to
quantitative differences in microorganisms between the two
tree species at the different sites, there were also qualitative
differences. At Lower Affleck, there were significantly greater
numbers and species of pseudomonads than at the two other
sites (Figure 5) and these pseudomonads constituted a high
proportion of the bacterial population. The Sitka spruce rhizosphere soils and rhizoplane contained significantly higher
populations of pseudomonads than those of larch at every site
except for the rhizosphere samples at Durris (Figure 5). Cultures of these pseudomonads included a mixture of fluorescent
(P. fluorescens Type G., P. corrugata, P. putida) and non-fluorescent species (P. cepacia) from the Sitka spruce rhizosphere
and rhizoplane, whereas non-fluorescent species dominated in
the larch rhizosphere.
Twenty fungal species were isolated from the three forest
sites. Nine species were present at Lower Affleck, one of which
was site specific. Sixteen species were found at the Durris site,
five of which were not isolated from the other sites. Twelve
species of fungi were present in the Midmar samples; three
were site specific. All sites appeared to have the same dominant fungal genera: Trichoderma sp., Penicillium sp., Fusarium sp., Mucor sp., Rhizopus sp. and Aspergillus sp. At all
sites, Trichoderma and Penicillium species dominated the rhizosphere of larch, and Aspergillus species dominated the rhizosphere of Sitka spruce.
Thirteen species of yeast were isolated from the three sites,
eleven from Lower Affleck, three of which were not found at
the other sites. Nine species were found at Durris, of which two
were site specific. Eight species were found at Midmar, all of
which were present at the other sites. In addition, two yeast
species were specific to larch and two were specific to Sitka
spruce.
1034
GRAYSTON AND CAMPBELL
Table 1. Average well color development in the six groups of carbon compounds in the Biolog GN plate by microbial communities from the
rhizosphere and rhizoplane of hybrid larch and Sitka spruce from three forest sites. 1
Carbon compound
Carbohydrates
Carboxylic acids
Amino acids
Amides
Polymers
Miscellaneous
1
Forest site
Hybrid larch
Sitka spruce
LSD (0.05)
Rhizoplane
Rhizosphere
Rhizoplane
Rhizosphere
Lower Affleck
Durris
Midmar
Mean (all sites)
0.715
0.654
0.244
0.537
0.739
0.857
0.567
0.721
0.789
0.485
0.326
0.533
1.012
0.548
0.583
0.715
Lower Affleck
Durris
Midmar
Mean (all sites)
0.535
0.378
0.239
0.384
0.497
0.469
0.405
0.457
0.584
0.325
0.310
0.407
0.591
0.304
0.372
0.422
Lower Affleck
Durris
Midmar
Mean (all sites)
0.797
0.464
0.232
0.498
0.666
0.675
0.414
0.585
0.769
0.370
0.343
0.494
0.701
0.378
0.426
0.502
Lower Affleck
Durris
Midmar
Mean (all sites)
0.427
0.238
0.082
0.249
0.332
0.256
0.210
0.266
0.444
0.131
0.169
0.248
0.329
0.156
0.203
0.229
Lower Affleck
Durris
Midmar
Mean (all sites)
0.546
0.416
0.209
0.390
0.523
0.364
0.349
0.412
0.497
0.325
0.260
0.361
0.592
0.309
0.293
0.398
Lower Affleck
Durris
Midmar
Mean (all sites)
1.101
0.815
0.347
0.754
1.051
1.067
0.546
0.888
1.050
0.680
0.304
0.678
1.253
0.743
0.706
0.901
0.385
0.160
0.182
0.091
0.261
0.131
0.197
0.093
0.186
0.091
0.439
0.209
Values are means of three replicate samples.
Discussion
We found quantitative and qualitative differences in the microbial populations of the rhizospheres of hybrid larch and Sitka
Figure 2. Canonical variate scores of soil samples based on sole carbon
source utilization profiles of microbial communities from either the
rhizosphere (filled symbols) or rhizoplane (open symbols) of hybrid
larch or Sitka spruce, respectively, from a farm woodland at Lower
Affleck (d, semi-circles) or second-rotation plantation forests at Durris(m, .) and Midmar (r, j). Symbols represent different samples.
spruce. These populations also varied depending on the soil
type where the trees were grown. We demonstrated that the
Biolog system can be used as a rapid screening technique to
discriminate among microbial communities from different tree
species and habitats based on metabolic profiles. The use of
differential utilization of a range of carbon compounds to
assess microbial diversity is an ecologically relevant method
for measuring diversity, because carbon is one of the main
influences on microbial growth in soil (Wardle 1992). However, the technique can only elucidate the causal relationship
in conjunction with information about what exudates are produced by trees. We found that the microbial communities from
the farm woodland system at Lower Affleck exhibited more
rapid utilization of all six groups of carbon compounds contained in the Biolog plate than the microbial communities from
the two second-rotation forest systems, indicating different
metabolic profiles between the farm woodland and second-rotation forest systems. The plate counts also confirmed that
there were qualitative and quantitative differences in the microbial communities between the forest sites.
The differences in metabolic and taxonomic diversity of the
microorganisms from Lower Affleck compared to the two
second-rotation forest sites probably reflect the previous land
use of this site that has affected the soil characteristics. Thus,
previous application of fertilizers at Lower Affleck, necessary
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1035
Table 2. Carbon compounds utilized differently by microbial communities in the rhizosphere of hybrid larch and Sitka spruce.
Carbon
compound
Community
with significantly
greater utilization
Carbohydrate
Cellobiose
Hybrid larch
Carboxylic acids
D-Galacturonic
acid
acid
D-Saccharic acid
Malonic acid
γ-Hydroxybutyric acid
Itaconic acid
Hybrid larch
Hybrid larch
Hybrid larch
Hybrid larch
Sitka spruce
Sitka spruce
Serine
γ-Aminobutyric acid
Hybrid larch
Sitka spruce
D-Glucoronic
Amino acids
in arable farming systems, likely removed any nutrient limitation to microbial growth in the soil. In addition, annual herbaceous plants exude more of their assimilated carbon into the
soil than trees (Leyval and Berthelin 1993, Grayston et al.
1996) and also exude a different spectrum of compounds
(Grayston et al. 1996), which would influence microbial community structure. The second rotation forest soils also had a
lower pH than the farm woodland system. An increase in pH
has been shown to increase root exudation in grasses (Meharg
and Killham 1990). In addition, bacteria prefer more neutral
soil conditions and therefore the low pH of the second-rotation
forest soils would select against these microorganisms.
The Biolog system assesses the metabolic diversity of the
culturable bacteria only, because fungal growth is too slow to
have an influence on the profile (Garland and Mills 1991).
Heuer et al. (1995) recently demonstrated that species of Enterobacter were mainly responsible for the color development
in Biolog wells by using 16S rRNA probes, after inoculation
with microbial communities from the phyllosphere and rhi-
Figure 3. Total populations of bacteria, actinomycetes, yeasts and
fungi in forest rhizosphere soils at Durris, Midmar and Lower Affleck.
Values are means of 12 replicate samples. Bars indicate LSD 0.05
values.
zosphere of potato plants. At Lower Affleck, a high proportion
of the bacterial population consisted of Pseudomonas species,
which likely accounts for the higher utilization of the carbon
sources in the Biolog plate by the microbial communities from
this site compared with those from the second-rotation forest
sites.
Another factor that could have influenced exudation patterns and hence microbial diversity is the presence of different
mycorrhizal symbionts (Molina et al. 1992). Native mycorrhi-
Table 3. Average well color development in the Biolog GN plates by microbial communities from a farm woodland site (Lower Affleck) and two
second-rotation forest sites (Durris and Midmar).1
Carbon compound
Forest site
Lower Affleck
Durris
Midmar
LSD (0.05)
Carbohydrates
N-Acetyl galactosamine
N-Acetyl glucosamine
Cellobiose
0.95
1.18
1.11
0.69
0.74
0.78
0.68
0.52
0.46
0.27
0.28
0.19
Carboxylic acids
D-Galactonic
acid lactone
acid
D-Galacturonic acid
γ-Hydroxybutyric acid
Malonic acid
Itaconic acid
Succinic acid
0.36
1.25
1.36
0.80
0.91
1.06
0.39
0.27
1.01
0.66
0.53
0.73
0.66
0.28
0.30
0.87
0.74
0.46
0.69
0.68
0.28
0.06
0.24
0.24
0.12
0.08
0.38
0.05
Serine
γ-Aminobutyric acid
1.14
0.65
0.80
0.38
0.68
0.37
0.20
0.10
D-Gluconic
Amino acids
1
Values are means of 12 samples.
1036
GRAYSTON AND CAMPBELL
Figure 4. Populations of (A) bacteria, (B) actinomycetes, (C)
fungi and (D) yeasts in the rhizosphere and on the rhizoplane of
Sitka spruce and hybrid larch at
Durris, Midmar and Lower Affleck. Values are means of three
replicate samples. Bars indicate
LSD0.05 values.
zal fungi present in the soil are thought to infect tree roots
(Bledsoe 1992). Therefore, the lack of previous tree growth at
Lower Affleck may have resulted in a poorer and less diverse
infection than at the second-rotation forest sites. Mycorrhizae
differ in their carbon storage patterns and their conversion of
plant sugars to metabolic intermediates (Finlay and Söderstrom 1992), and these differences could result in alterations in
the quality and quantity of carbon released from tree roots.
Leyval and Berthelin (1993) showed that beech (Fagus sylvatica L.) infected with Laccaria laccata exuded more sugars
and amino acids and a different range of organic acids than
non-mycorrhizal beech or mycorrhizal Scots pine (Pinus
sylvestris L.). Our results suggest that hybrid larch and Sitka
spruce may produce different exudates because the carbon
profiles of the microorganisms from the rhizosphere of these
two tree species vary, mainly as a result of differences in
carboxylic acid and amino acid usage. Previously, we found
that utilization of carboxylic acids was highly variable in
rhizosphere samples from different tree species growing in the
same soil type (Grayston et al. 1994). Although the carbon
compounds exuded by hybrid larch and Sitka spruce have not
been characterized, it is known that the quantity and character
of root exudates released by trees vary considerably among
species (see review by Grayston et al. 1996). A wide range of
organic acids, which are quantitatively the most important
organic component of tree root exudates (Smith 1976), are
produced by different tree species. For example, gluconic and
malonic acids, which showed differential utilization between
larch and Sitka spruce microbial communities (Table 2), are
produced by some Pinus species, but not by others (Grayston
et al. 1996). The types of amino acids exuded are also highly
variable; deciduous trees produce cystine and homoserine,
whereas these amino acids have not been isolated from
exudates of evergreens (Grayston et al. 1996).
Variation in metabolic diversity by microbial communities
from the rhizosphere of the two tree species was accompanied
BIODIVERSITY OF RHIZOSPHERE MICROBIAL COMMUNITIES
1037
zospheres of larch and Sitka spruce at different sites as a
consequence of variation in the species composition of the
different microbial populations. The identification of root
exudates from these tree species and inclusion of these carbon
sources in future utilization tests will improve the resolution of
the technique. Measurement of the production of different
types of exudates in the tree would allow us to link utilization
rates directly with exudation. The beneficial effects of rhizosphere microorganisms on tree growth highlight the need for
a greater understanding of microbial community structure and
function in the rhizosphere.
Acknowledgments
We thank D. Hirst for assistance with canonical variate analysis. C.M.
Cameron, M.S. Davidson, A. Norrie and E.J. Reid are thanked for
technical assistance. Forest Enterprises--Durris, in particular Sandy
Sandilands, are thanked for information and access to the second
rotation forest sites. This research was funded by the Scottish Office
Agriculture, Environment and Fisheries Department.
Figure 5. Populations of pseudomonads in the rhizosphere and on the
rhizoplane of Sitka spruce and hybrid larch at Durris, Midmar and
Lower Affleck. Values are means of three replicate samples. Bars
indicate LSD0.05 values.
by differences in the culturable populations present. There
appeared to be tree- and site-specific bacteria, actinomycetes
and yeasts. All the fungal species isolated were present at each
site and with both tree species, although the dominant fungal
types changed with tree species. However, all the fungal species isolated are prolific spore producers and therefore one
must be cautious when interpreting the results of these plate
count data. At Lower Affleck, a high proportion of the bacterial
population consisted of Pseudomonas species, whereas at the
second-rotation forest sites there was a greater diversity of
actinomycetes. At every site, the larch rhizosphere contained
fewer fluorescent pseudomonads than the Sitka spruce rhizosphere. There have been relatively few studies on microbial
diversity in the rhizosphere of trees (Fitter and Garbaye 1994)
and none on hybrid larch or Sitka spruce. Studies comparing
mycorrhizal and non-mycorrhizal yellow birch (Katznelson et
al. 1962), red alder and Douglas-fir (Neal et al. 1968), various
coniferous (Oswald and Ferchau 1968) and Eucalyptus species
(Malacjzuk and McComb 1979) have shown distinct communities of microorganisms associating with different trees.
There is evidence that these rhizosphere microorganisms can
enhance tree growth through the production of plant growth
regulators (Strelczyk and Pokojska-Burdziej 1984, Strelczyk
and Leniarka 1985), nitrogen fixation (Li and Castellano 1987,
Li and Hung 1987), solubilization of inorganic phosphates
(Leyval and Berthelin 1991) and stimulation of mycorrhization of trees (Garbaye 1994).
Conclusions
We found significant differences in the utilization of various
carbon sources by microbial communities from the rhi-
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