Relative abundance of ectomycorrhizas in a managed loblolly pine

924
Relative abundance of ectomycorrhizas in a
managed loblolly pine (Pinus taeda) genetics
plantation as determined through terminal
restriction fragment length polymorphism profiles
David J. Burke, Kendall J. Martin, Paul T. Rygiewicz, and Mary A. Topa
Abstract: We examined the relationship between relative abundance of ectomycorrhizas in soil cores determined using
morphotype tip counts and terminal restriction fragment length polymorphism (TRFLP) analysis. Root tips were harvested
from a total of 120 soil cores collected from six family plots in a loblolly pine (Pinus taeda L.) genetics plantation. Tips
from each soil core were morphotyped based on physical characteristics, identified through TRFLP and sequence analysis,
then pooled to reconstruct the ectomycorrhizal community within that core. The identity and relative abundance of specific
ectomycorrhizas in each reconstructed community was then determined using TRFLP analysis of the internal transcribed
spacer of the rRNA gene. Using TRFLP, we were able to detect 34 ectomycorrhizal phylotypes colonizing roots of loblolly pine. TRFLP peak area was an accurate approximation of the relative number of tips of each ectomycorrhizal type
within a soil core. Relative abundance of each ectomycorrhiza as determined by TRFLP was used to describe their distribution in the pine plantation. Although there were no differences found in ectomycorrhizal richness and evenness among
the six family plots, the two fertilized plots had generally lower levels of ectomycorrhizal richness and evenness as indicated by rank abundance curves. Our results suggest that TRFLP is a useful tool for describing the occurrence and distribution of ectomycorrhizas in environmental samples.
Key words: ectomycorrhiza, fertilization, Pinus taeda, TRFLP.
Résumé : Les auteurs ont examiné les relations entre l’abondance relative des ectomycorhizes dans des carottes de sols,
en utilisant le décompte des morphotypes des apex et le polymorphisme de la longueur des fragments de restriction terminaux (TRFLP). Ils ont examiné les apex racinaires dans un total de 120 carottes récoltées à partir des parcelles de six familles, dans une plantation génétique de pin à encens (Pinus taeda L.) . Les apex ont été classés selon les morphotypes en
se basant sur les caractéristiques physiques, identifiés par l’analyse des séquences TRFLP, et ensuite réunis pour reconstruire la communauté ectomycorhizienne dans cette carotte. Ils ont également déterminé, pour chaque communauté reconstruite, l’identité et l’abondance relative des ectomycorhizes spécifiques, en utilisant l’analyse TRFLP de l’espaceur interne
transcrit du gène rARN. À l’aide du TRFLP, il a été possible de détecter 34 phylotypes ectomycorhiziens colonisant les racines du pin à encens. La surface maximale TRFLP donne une approximation exacte du nombre relatif d’apex appartenant
à chaque type ectomycorhizien dans une carotte donnée. On a utilisé l’abondance relative de chaque ectomycorhize, tel
que déterminé par le TRFLP, pour décrire sa distribution dans la plantation de pin. Bien qu’on ait observé aucune différence dans la richesse et l’uniformité en ectomycorhizes entre les parcelles des 6 familles, les deux parcelles fertilisées
montrent généralement des degrés moindres de richesse et d’uniformité, tels qu’indiqués par les rangs dans les courbes
d’abondance. Les résultats suggèrent que le TRFLP est utile pour décrire la présence et la distribution des ectomycorhizes
dans des échantillons environnementaux.
Mots clés : ectomycorhizes, fertilisation, Pinus taeda, TRFLP.
[Traduit par la Rédaction]
Received 31 January 2006. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 3 August 2006.
D.J. Burke1,2 and M.A. Topa.2 Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA.
K.J. Martin.3 Center for Environmental Diagnostics and Bioremediation, University of West Florida, 11000 University Parkway,
Pensacola, FL 32514, USA.
P.T. Rygiewicz. USEPA National Health and Environmental Effects Research Laboratory, 200 S.W. 35th Street, Corvallis, OR 97333,
USA.
1Corresponding
author (e-mail: [email protected]).
address: The Holden Arboretum, 9500 Sperry Road, Kirtland, OH 44094, USA.
3Present address: Department of Biology, William Paterson University, Wayne, NJ 07470, USA.
2Present
Can. J. Bot. 84: 924–932 (2006)
doi:10.1139/B06-046
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2006 NRC Canada
Burke et al.
Introduction
The roots of many important tree species including members of the Pinaceae form symbiotic associations with ectomycorrhizal (ECM) fungi (Smith and Read 1997).
Mycorrhizal colonization can increase plant growth by enhancing nutrient gain, improving drought tolerance, and protecting against soil pathogens (Garbaye 1991; Newsham et
al. 1994; Smith and Read 1997; Chalot and Brun 1998). Until recently, studies of ECM diversity were limited by the
available methods, that is, the need for laborious morphotyping and enumeration of excised root tips limited the number
and size of samples that could be processed. This limitation
can be partially overcome by coupling morphological classification with molecular typing protocols, such as restriction
fragment length polymorphism (RFLP) analysis, that target
DNA with taxonomic significance (Horton and Bruns
2001). However, PCR-RFLP fingerprinting of ECM roots
requires that the DNA come primarily from a single fungal
species for proper identification. Application of this technique to DNA extracts from complex mixed communities can
result in ambiguous restriction fragment patterns. Therefore,
PCR-RFLP requires that tips be morphotyped first so DNA
extractions are done on uniform groups of tips (i.e., ideally
on single fungal types) and this restriction continues to limit
the number of samples that can be analyzed in studies of ectomycorrhizas.
Recent advances in whole community typing protocols
using terminal restriction fragment length polymorphism
(TRFLP) offer the possibility of performing detailed analyses of fungal communities in soil cores taken from ecological systems without first having to complete a
morphological analysis. TRFLP has been used to describe
soil fungal communities (Dickie et al. 2002; Klamer et al.
2002; Edel-Hermann et al. 2004; Edwards et al. 2004), but
previous studies have either employed TRFLP as a fingerprinting technique to determine the presence or absence of
ECM fungi in soil (Dickie et al. 2002; Edwards et al. 2004;
Burke et al. 2005) or have used TRFLP profiles as a screening technique to assess treatment effects on fungal community structure without specific identification of fungal types
(Klamer et al. 2002; Edel-Hermann et al. 2004).
Since TRFLP is a PCR-based technique, it is subject to
some inherent limitations that affect the quality of information obtained. The major limitations include differences in
primer affinity (Lueders and Friedrich 2003) that could result in the failure of some sequences to be sufficiently represented within the sample, and the tendency of late-stage,
enzyme-limited, PCR amplification to result in over-representation of the more minor phylotypes (Suzuki et al.
1998). Bias may also result from differences between fungal
types in the copy number of the targeted gene (Crosby and
Criddle 2003) and differences in extraction efficiency (Nagashima et al. 2003). However, despite these potential limitations, we have found that TRFLP typing of ECM
communities does provide a similar (but perhaps more conservative) estimate of ectomycorrhizal richness as compared
to more traditional morphotyping techniques (Burke et al.
2005).
Studies of bacterial communities have suggested that
PCR-based techniques can be used to estimate relative abun-
925
dance of bacterial phylotypes; the relative signal intensity of
terminal restriction fragments (TRFs) providing an accurate
quantification of the template pool in natural and experimental communities (Horz et al. 2001; Marschner et al.
2001; Lueders and Friedrich 2003). Landeweert et al.
(2003) also demonstrated that quantitative PCR used to determine the quantity of fungal hyphae in soil compared favorably with phospholipid fatty acid (PLFA) profiles. Given
the limitations of PCR methods, it seems unlikely that
TRFLP peak areas could be used as a substitute for the
number of individuals in a community. However, when
TRFLP peaks are used to identify ectomycorrhizas in natural
samples, it may also be possible to use the peak areas as an
indicator of how abundant the identified ectomycorrhizas are
relative to all other ectomycorrhizas in the sample (i.e., relative abundance). Use of peak areas as a measure of relative
abundance could provide information on the structure of ectomycorrhizas in natural systems, and how these communities and identified species types respond to environmental
change or manipulation. Nevertheless, we are unaware of
any studies that have examined how well TRFLP profiles
estimate the relative abundance of ectomycorrhizas in natural communities. In this study, we examine the relationship
between assessing relative abundance of ectomycorrhizas
based on morphotyped tip counts and on TRFLP profiles.
We then applied the TRFLP technique to estimate abundance and diversity of ECM phylotypes in six families (populations) of loblolly pine (Pinus taeda L.) grown under
ambient and fertilized soil conditions at a pine plantation.
Materials and methods
Site description and soil sampling
The study site is an 8 year old loblolly pine genetics plantation located in Scotland County, North Carolina, USA,
which has been previously described (Hockman and Allen
1990; Burke et al. 2005). A total of 120 soil cores were collected from six loblolly pine family plots representing an
Atlantic coastal plain (ACP) and Texas (TX) ecotype between 13 October and 22 November 2002. Each family plot
consists of 10 rows of 10 trees planted at 1.5 m (within
row) 2.0 m (aisle) spacing. Trees selected for sampling
were chosen randomly from each family plot while also considering locations of dead trees, if any. Samples were collected from trees in the slowest- (ACP-2 and Tx-2) and
fastest-growing (ACP-1 and TX-1) families within the two
ecotypes; rates of growth was determined using 6 year
mean heights and volumes of control trees (Retzlaff et al.
2001). Samples were also collected from two fertilized family plots (ACP-1 and TX-2) that have received annual soil
fertilizer additions to maintain optimum foliar nutrient ratios
(Hockman and Allen 1990). The trees in the fertilized treatment received the following total nutrient additions (kg/ha)
during the first 7 years: 459 N, 58 P, 78 K, 8.2 Ca, 46 Mg,
92 S, 0.5 B, 2 Cu, 5 Fe, 5 Mn, 2 Zn. Control plots received
no nutrient additions. Percent soil carbon averaged 1.8 ± 0.1
in control plots and 2.6 ± 0.2 in fertilized plots. Percent soil
moisture averaged 7.1 ± 0.1 in control plots and 8.6 ± 0.3 in
fertilized plots.
Sampling was staggered to facilitate prompt processing of
soil cores, and 24 soil cores were collected every week dur#
2006 NRC Canada
926
Can. J. Bot. Vol. 84, 2006
Fig. 1. Four of the most commonly encountered morphotypes found in an 8 year old loblolly pine (Pinus taeda) stand in Scotland County,
North Carolina. Top left, Cenococcum-like phylotype (PT1); top right, Russula-like phylotype (PT21); bottom left, Tomentella-like phylotype (PT22); bottom right, Tricholoma-like phylotype (PT10).
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Burke et al.
927
Table 1. Frequency and abundance of phylotypes detected by
TRFLP and putative identity determined through sequence analysis.
PT
No.
Phylotype sequence affinity
Frequencya
Abundanceb
1
21
22
23
27
10
6
13
31
3
8
20
25
12
7
14
17
11
29
32
4
24
9
34
16
15
26
30
33
28
2
18
19
Cenococcum geophilum
Russula sp. 1
Tomentella sp.1
Tomentella sp. 2
Basidiomycete 1
Tricholoma sp.
Tomentellopsis sp.
Piloderma sp. 1
Tylopilus sp. 1
Inocybe sp.
Xerocomus sp.
Lactarius sp. 1
Uncultured ECM
Tylopilus sp. 2
Ascomycetes
Piloderma sp. 2
Rhizopogon sp.
Laccaria sp.
Amanita sp.
Piloderma sp. 3.
Tomentella sp. 3
Tomentella sp. 4
Unknown 1
Unknown 2
Unknown 3
Tomentella sp. 5
Russula sp. 2
Russula sp. 3
Unknown 4
Unknown 5
Clavaria sp.
Basidiomycete 2
Lactarius sp. 2
52
41
35
27
25
22
22
22
19
17
16
12
12
11
9
8
5
5
5
5
4
4
4
3
3
2
2
2
<1
<1
<1
<1
<1
7.(1)
8.(1)
19.(3)
13.(2)
5.(1)
11.(2)
6.(2)
3.(1)
4.(1)
2.(1)
5.(1)
1.(1)
1.(1)
1.(1)
2.(1)
1.(1)
1.(1)
1.(1)
1.(1)
<1.
2.(1)
1.(1)
1.(1)
1.(1)
1.(1)
<1.
<1.
<1.
<1.
<1
<1.
<1.
<1
Fig. 2. Phylotype abundance curve for the ECM community found
in an 8 year old loblolly pine (Pinus taeda) stand in Scotland
County, North Carolina. The cumulative number of detected phylotypes is plotted against the cumulative number of soil cores (n =
120, x-axis bottom) and root tips from which DNA was extracted
(x-axis top). The total harvest of 120 cores represents the extraction
of DNA from approximately 50 000 root tips.
ing the 5.5-week sampling period. Cores were collected to a
depth of 20 cm with a metal coring device measuring 15 cm
in diameter (3.5 dm3). Cores were staggered around the
boles of the selected trees such that three distance classes
could be analyzed. These classes were bole (10 cm from
bole of selected tree), row (50 cm from the bole within the
planted row), and inter-row (approximately 1 m from the
tree bole). Collected samples were dry sieved at the site
with a 0.5 cm mesh screen to separate pine roots and organic material containing mycorrhizal tips from soil. Sieved
roots and organic material from each core constituted a sample and were stored at 4 8C until processed in the laboratory.
using the following protocol. Roots and adhering soil were
rinsed into a series of nested sieves and all material remaining on a 0.25-mm screen (WS Tyler Company, Cleveland,
Ohio) was emptied into a white enamel pan partially filled
with chilled reverse osmosis water. Viable ECM tips were
removed under a 2 magnification lens to Petri dishes containing cold physiological saline (NaCl concentration of
8.5 g/L). Tips were classified using a dissecting scope under
40 magnification according to general procedures outlined
in Agerer (2003). Tips retrieved from a soil core were classified by tip and patch color, patch frequency, branch pattern, tip shape, mantle texture and luster, and extent of
extramatrical hyphae. Tips were kept in cold physiological
saline throughout the morphotyping procedure. All morphotyped tips harvested from a soil core were counted and then
immediately frozen at –70 8C. Morphotyped tips were freeze
dried at –40 8C for 10 d and stored at –70 8C until DNA
extraction.
DNA extraction was performed using a 2% CTAB
(cetyltrimethyl-ammonium bromide) extraction protocol
with 0.8% mercaptoethanol and incubation at 65 8C for 1 h
(Baker and Mullin 1994). Nucleic acids were purified by
phenol–chloroform extraction and precipitated with 20%
polyethylene glycol 8000 in 2.5 mol/L NaCl. All root tips
of each morphotype found within a soil core were pooled
and extracted separately from the, respectively, pooled root
tips of all other morphotypes found within the same core.
For example, if four to five morphotypes were encountered
in a core, four to five separate DNA extractions were performed per soil core. Ten percent by mass of the extracted
nucleic acids from each morphotype within a soil core were
recombined to reconstruct the ECM fungal community
within that core (Burke et al. 2005).
Morphotyping and DNA extraction and purification
Ectomycorrhizal root tips from each soil core were sorted
into various morphotype classes within 14 d of collection
Detection of ECM through TRFLP
Recombined DNA was used as template in a PCR targeting the internal transcribed spacer (ITS) region of the rRNA
a
Indicates the percentage of soil cores (n = 120) in which the indicated
phylotype was encountered as determined by TRFLP.
b
Indicates the mean relative abundance of fungal types (percent)
averaged across all cores (n = 120) as determined by TRFLP. Standard
errors are shown in parenthesis.
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2006 NRC Canada
928
Fig. 3. Linear regression analysis of the relative abundance of the
three commonly encountered phylotypes as determined through
TRFLP integrated peak area and morphotyped tip counts. TRFLP
relative abundance was calculated as detailed in the Methods section. Tip relative abundance is the number of tips determined
through morphotyping divided by the total number of ECM tips
within the core.
Can. J. Bot. Vol. 84, 2006
Table 2. Species richness, PIE (probability of an interspecific
encounter), and the slope of rank abundance curves for sampled
family plots at the Scotland County field site. Mean ± standard
error of the mean is shown. ACP-1 and ACP-2 represent fasterand slower-growing families of the Atlantic Coastal Plain ecotype, respectively, whereas TX-1 and TX-2 represent faster- and
slower-growing families of the drought-hardy Texas ecotype.
Plot
ACP-1
ACP-2
TX-1
TX-2
ACP-1
TX-2
Treatment
Control
Control
Control
Control
Fertilized
Fertilized
n
15
15
15
15
15
15
Species
richness
4.3 (0.4)
4.7 (0.5)
4.5 (0.5)
4.2 (0.3)
3.9 (0.5)
3.2 (0.5)
PIE
0.51
0.54
0.49
0.54
0.50
0.38
(0.08)
(0.06)
(0.06)
(0.05)
(0.06)
(0.06)
Slope
–0.52
–0.63
–0.67
–0.65
–1.37
–1.85
Fig. 4. Rank abundance curves for samples collected in pine
family plots: ACP-1, ACP-2, ACP-1 fertilized, TX-1, TX-2, and
TX-2 fertilized. The slope of the linear regression line through
the points is reported in Table 2.
gene (White et al. 1990). Labeled primers NSI1 and 58A2R
were used to amplify the ITS1 region located between the
18S and 5.8S rRNA gene, and primers 58A2F and NLB4
were used to amplify the ITS2 region located between the
5.8S and 28S rRNA gene (Burke et al. 2005; Martin and
Rygiewicz 2005). Forward primers were labeled with the
dye 6FAM and reverse primers with HEX. PCR was carried
out in 50 mL reaction volumes using 2 units Faststart Taq
DNA polymerase (Roche Diagnostics GmbH, Mannheim,
Germany) on a PTC 100 thermal cycler (MJ Research, Boston, Massachusetts) using a heated lid as previously described (Burke et al. 2005). Consequently, we generated
two TRFLPs for the ECM fungal community in each core.
Endonuclease AluI was used to generate the TRFLP from
the ITS1 region, and HaeIII (Promega, Madison, Wisconsin)
was used to generate the TRFLP for the ITS2 region (Burke
et al. 2005). TRFLPs were completed through the Genomics
Technology Support Facility of Michigan State University
(East Lansing, Michigan) on an ABI 3100 (Applied Biosystems, Foster City, California).
Only TRF peak heights greater than 200 relative florescence units (on a scale of 10 000) were included in the analyses. Since two TRFLPs were generated for each soil core
ECM community using two different labeled primers, every
fungal phylotype within the community was represented by
a total of four TRFs. This method potentially provides a
larger degree of phylotype discrimination than analysis of
either region alone and can reduce the effect of primer bias
on detection of ECM phylotypes (Burke et al. 2005). TRFs
from each sample were compared against a site-specific
TRF database generated from individual morphotyped tips
and sporocarps collected (Burke et al. 2005). In this fashion,
specific ECM phylotypes could be detected within each soil
core community, and TRFs from both ITS1 and ITS2 regions served as independent confirmation of each phylotype.
Community samples that generated unique peaks not previously identified were cloned using pGEM-T Easy Vector
System (Promega, Madison, Wisconsin), and screened colonies were used as templates for sequencing using Dye Primer Cycling Kit (Applied Biosystems). Both the ITS1 and
ITS2 regions were sequenced (approximately 1000 bp)
(Burke et al. 2005). Sequences were generated at the Boyce
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Burke et al.
Thompson Institute for Plant Research (Ithaca, New York)
sequencing facility using an Applied BioSystems Prism
3100 Genetic Analyzer/DNA Sequencer. Generated sequences were compared with EMBL, GenBank, and DDBJ database entries using the FASTA program (European
Bioinformatics Institute, Cambridge, UK) to determine putative identity of the fungal phylotype.
Analysis of ECM relative abundance through TRFLP
Since very different ECM phylotypes often generated similar sized 6FAM-labeled TRFs (Burke et al. 2005), we used
the reverse-labeled HEX TRFs as the diagnostic peaks for
determining relative abundance of phylotypes in both the
ITS1 and ITS2 regions. The integrated peak florescences of
HEX-labeled peaks were used to estimate relative abundance of detected phylotypes in each ECM community (Liu
et al. 1997; Suzuki et al. 1998). Relative abundance of each
phylotype was calculated by comparing the area under the
individual peak with the total area of all peaks in the
TRFLP (Suzuki et al. 1998). The relative abundance of
each phylotype in that soil core was then determined by taking the average of its relative abundance from the ITS1 and
ITS2 region calculated separately. In those situations where
a phylotype could not be detected in one of the gene regions, the relative abundance value for that region was considered to be zero. This technique was used in an effort to
account for differences in amplification of phylotypes
caused by possible primer bias.
We used a distinctive morphotype to assess the relationship between relative abundance calculated from TRFLP to
relative abundance calculated from counted morphotyped
tips (Fig. 1). Phylotype 10 (Tricholoma-like) had a very distinctive morphotype that formed extensive white rhizomorphs within our samples. Consequently we could reliably
identify it visually, count the number of tips of this morphotype, and determine visually whether tips of this phylotype
were living or senescent. The integrated peak fluorescence
of the HEX-labeled TRFs for both the ITS1 and ITS2 region
were used to determine relative abundance of this phylotype.
We then compared relative abundance using the mean of the
ITS1 and ITS2 regions with the relative abundance of phylotype 10 as determined through visual tip counts. Phylotype
21 (Russula sp. 1) and phylotypes 22 and 23 (Tomentella
sp.1 and Tomentella sp.2) formed large tip clusters within
our samples so we also attempted to use these phylotypes to
assess morphotype- TRFLP relationships. Phylotypes 22 and
23 were very similar and could not be distinguished visually; consequently, we have combined these phylotypes for
this analysis.
Data and statistical analysis
Richness is defined as the number of ECM phylotypes
within a soil core. Relative abundance was used to calculate
the probability of an interspecific encounter (PIE) for each
soil core (Hurlbert 1971). These calculations were completed
using EcoSim (Gotelli and Entsminger 2001) and we are using
them to estimate phylotype evenness. Rank abundance curves
were used to compare differences in the distribution patterns
of ECM phylotypes within the six pine family plots included
in our study. Since our study was also meant as a preliminary
929
screening study to detect differences in ECM communities
among plants of different growth rates, additional cores were
collected from ACP-1 and TX-2 as compared with the other
family plots. Thirty soil cores were collected from ACP-1
and TX-2, whereas 15 cores were collected from the other
four family plots. For analysis of phylotype evenness, richness, and generation of rank abundance curves, 15 soil cores
were randomly selected from the 30 collected from ACP-1
and TX-2. In this fashion, each plot was represented by the
same number of soil cores (n = 15) and a total of 90 cores
were used for these analyses. Linear regression was used to
determine the relationship between phylotype abundance estimates obtained with TRFLP and morphotyping. ANOVA was
used to determine the effect of distance on the relative abundance of identified ectomycorrhizas.
Results
TRFLP analysis of ectomycorrhizas
TRFLP analysis indicated the presence of 34 identified
ECM phylotypes colonizing roots of loblolly pine at the
field site; of these, 17 were encountered in 5% or less of
the soil cores (Table 1, Fig. 1). Two Cenococcum-like phylotypes were detected (Burke et al. 2005), but due to difficulty in distinguishing between them in mixed communities,
they have been combined here (consequently, Table 1 shows
33 phylotypes). The Cenococcum-like phylotype was present
in 52% of soil cores and was the most frequently observed
phylotype. Other frequently encountered phylotypes included phylotype 21 (Russula-like), phylotype 22 and 23
(Tomentella-like) and phylotype 10 (Tricholoma-like). A
phylotype abundance curve was constructed to determine
the extent to which our sampling design allowed us to inventory the ECM fungi colonizing loblolly pine at the Scotland County site (Fig. 2). More than 95% of the phylotypes
were detected after analysis of 80 soil cores (containing approximately 40 000 ECM root tips); however, additional
TRFLP phylotypes were still being added with completion
of the final soil core.
TRFLP analysis of ectomycorrhizas relative abundance
and distribution
We observed a significant linear relationship between
TRFLP phylotype abundance and morphotyped tip abundance (Fig. 3). We found a significant linear relationship between these measures of relative abundance for the
Tricholoma-like, Tomentella-like, and Russula-like phylotypes (R2 = 0.56, 0.54, and 0.28 respectively; P < 0.0001,
<0.0001, and <0.01, respectively), indicating that the TRFLP
method compares favorably with the more traditional morphotyping technique in determining the abundance of these
ECM phylotypes.
Using relative abundance estimates from TRFLP, we
found that the most frequently detected ectomycorrhizas
were not always the most abundant (Table 1). This was the
case for both Cenococcum- and Russula-like phylotypes
(Table 1). The Cenococcum-like phylotype constituted only
7% of total phylotype abundance and phylotype 21 (Russulalike) composed only 8% of total phylotype abundance. The
most abundant phylotypes were phylotypes 10 (Tricholomalike), and 22 and 23 (Tomentella-like). The 12 most fre#
2006 NRC Canada
930
quently detected phylotypes did account for 85% of fungal
relative abundance as determined through analysis of TRFLP
integrated peak florescence (Table 1). Mean phylotype richness among the six family plots averaged 4.2 ± 0.2 phylotypes/core (median of 4.0 phylotypes/core), and PIE
averaged 0.51 ± 0.02 (median of 0.58). We did not detect significant differences in either richness or PIE among the six
family plots (Table 2). Rank abundance curves developed using TRFLP suggested that the two fertilized plots had lower
overall phylotype richness than did control plots (Fig. 4). The
slope of the linear regression line through the plotted rank
abundance points was also more negative in the two fertilized
plots as compared to control plots (Table 2).
Phylotype richness declined with distance from 4.6 ± 0.3
phylotypes/core nearest the bole to 3.9 ± 0.3 phylotypes/core
at the 1 m distance but these differences were not significant
(P > 0.10). Evenness was also not significantly affected by
distance from the bole. However, ANOVA indicated a significant effect (P < 0.10) of distance on abundance of some
phylotypes. When present, phylotype 22 (Tomentella-like)
increased in abundance with increasing distance from the
tree bole (p = 0.09). Phylotype 27 (unidentified Basidiomycete) and phylotype 3 (Inocybe-like) also significantly increased in abundance with increasing distance from the tree
(p = 0.05 and p = 0.10, respectively). In contrast, when phylotype 10 was detected (Tricholoma-like) it was significantly
more abundant in cores nearest the tree bole (p = 0.07).
Discussion
In this study, we used TRFLP profiles to describe both
the presence and relative abundance of ECM phylotypes in
a managed pine plantation. Although TRFLP has been used
to determine the presence and (or) absence of ECM phylotypes in soil cores (Dickie et al. 2002; Edwards et al. 2004;
Burke et al. 2005), we also used TRFLP peak area as a relative measure of fungal abundance in each soil core, an approach not previously taken in studies of ectomycorrhizas.
PCR-based approaches can provide an accurate estimate of
fungal hyphae in soil (Landeweert et al. 2003) or of the distribution of bacterial phylotypes in natural and experimental
communities (Horz et al. 2001; Marschner et al. 2001;
Lueders and Friedrich 2003). Although it may not be possible to use TRFLP peak areas as a substitute for ectomycorrhizal tip counts, our results suggest that TRFLP can provide
an accurate estimate of the relative abundance and distribution of ectomycorrhizal tips in natural samples. TRFLP peak
areas can then be used to describe the distribution of specific ectomycorrhizas in natural communities.
Since all of the tips from soil cores included in this study
were morphotyped prior to molecular analysis, we were able
to measure the accuracy of TRFLP relative abundance estimates using ECM phylotypes readily identifiable within our
soil cores. We found good correspondence between relative
abundance as estimated through TRFLP and tip counts.
However, TRFLP was a better estimate of root tips for phylotypes 10 and 22–23 than for phylotype 21. We previously
found that primer bias may partly limit the ability to detect
some phylotypes in experimental communities (Burke et al.
2005) and this could partly explain the weaker relationship
between abundance measures for phylotype 21. In addition,
Can. J. Bot. Vol. 84, 2006
PCR-TRFLP measures are also likely to be sensitive to the
quantity of fungal biomass produced since this will affect
the quantity of target available for amplification. This may
have the potential disadvantage of over representing those
phylotypes that naturally produce large quantities of hyphal
biomass even if tip numbers are comparable, but has the advantage of producing a more accurate estimate of the viable
population of ECM since senescing or dormant ECM are not
likely to amplify well. This could be particularly helpful in
accurately estimating the abundance of Cenococcum-like
fungal types where it can be difficult to distinguish between
viable and non-viable ECM tips.
Using TRFLP, we found that the general distribution pattern and identity of ECM fungi colonizing loblolly pine at
the Scotland County genetics plantation is similar to the pattern found for other coniferous forest systems. We observed
a large number of ECM phylotypes in this study but most of
the phylotypes encountered were rare, with half of the phylotypes occurring in less than 5% of the soil cores. ECM
fungal communities are known to be patchy in space and
time, and most ECM species are rarely found even with
more extensive sampling, which is an expected result in
community studies of ECM fungi (Visser 1995; Horton and
Bruns 2001; Taylor 2002). Although 95% of our phylotypes
were found within the first 80 cores, our species area curve
indicates that additional species types could be encountered
with more extensive sampling; a result not unexpected in
community studies of ECM fungi (Horton and Bruns 2001;
Taylor 2002). The Tomentella-like phylotypes (Thelephorales) were the most abundant ECM fungi detected in our
soil cores, whereas the Cenococcum-like phylotype was the
most frequently encountered type but its abundance was
comparatively low. The genus Tomentella is considered one
of the most common ECM fungi on conifers (Horton and
Bruns 2001) while low levels of Cenococcum are typically
encountered in other ECM community studies (Visser 1995;
Rygiewicz et al. 2000; Horton and Bruns 2001; Valentine et
al. 2004).
The distribution of some ectomycorrhizas was influenced
by distance from the tree bole. Distance from the tree has
been shown to affect sporocarp production (Mason et al.
1982; Last et al. 1983) especially in disturbed sites. Some
ECM fungi may colonize only certain parts of the root system as a function of root age, class, or development. Gibson
and Deacon (1988) observed that the late-stage Lactarius
pubescens only colonized short roots originating from older
parts of the root system whereas the early-stage Hebeloma
sp. colonized tips from any portion of the root system
(Smith and Read 1997). The genetics plantation is historically similar to many managed plantations in the southeastern USA, where the previous stand was cleared, the soil was
disked prior to tree planting and mowing was used to minimize competition for soil resources. This can create differences in soil compaction between sites close to the tree and
farther away where machine tire compaction may be most
pronounced. ECM distributions in the genetics plantation
could partly reflect such differences in soil compaction but
could also reflect adaptation of ECM fungi to spatially explicit soil conditions or root growth (Gibson and Deacon
1988; Coleman et al. 1989; Nilsen et al. 1998).
Fertilization can reduce richness and alter ECM distribu#
2006 NRC Canada
Burke et al.
tion patterns in natural systems (Treseder and Allen 2000;
Peter et al. 2001; Lilleskov et al. 2002; Edwards et al.
2004). Although our sampling design was not adequate for
detecting differences among the sampled family plots, rank
abundance plots suggest that ectomycorrhizal communities
in fertilized family plots were less diverse than in control
family plots. The two fertilized family plots contained fewer
ectomycorrhizas as compared to respective control plots, and
were more likely to be dominated by a few ectomycorrhizal
types as revealed by the slope of the curves. In general, the
two fertilized plots were dominated by two Tomentella-like
phylotypes, whereas the Tricholoma- and Russula-like phylotypes were more abundant in the control plots (data not
shown). Changes of this nature under fertilization are similar
to those observed by other investigators including previous
studies conducted on loblolly pine (Visser 1995; Lilleskov
et al. 2002; Edwards et al. 2004). In conclusion, we found
that TRFLP can be used to estimate the relative abundance
of ectomycorrhizas in soil cores. This technique can be
used to describe the distribution of ectomycorrhizas in natural systems.
Acknowledgements
This work was funded by National Science Foundation
grant No. IBN-0212892 Ecology and Evolutionary Physiology Program. The authors thank Matthew Garner, Nathaniel
Hubert, Paul King, Alberto Stolfi, and Dr. Steve McKeand
for technical assistance. We also thank Dr. Darlene Southworth for reviewing an earlier version of this manuscript.
The document was subjected to the peer and administrative
reviews of the U.S. Environmental Protection Agency at the
National Health and Environmental Effects Research Laboratory, Western Ecology Division, and was approved for
publication. Mention of trade names or commercial products
in this paper does not constitute endorsement or recommendation of use.
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