Content uploaded by Stephen Pecot
Author content
All content in this area was uploaded by Stephen Pecot on Feb 12, 2014
Content may be subject to copyright.
ORIGINAL PAPER
Nitrogen decreases and precipitation increases
ectomycorrhizal extramatrical mycelia production
in a longleaf pine forest
Stephanie E. Sims & Joseph J. Hendricks &
Robert J. Mitchell & Kevin A. Kuehn & Stephen D. Pecot
Received: 31 October 2006 / Accepted: 26 December 2006 / Published online: 27 January 2007
#
Springer-Verlag 2007
Abstract The rates and controls of ectomycorrhizal fungal
production were assessed in a 22-year-old longleaf pine (Pinus
palustris Mill.) plantation using a complete factorial design
that included two foliar scorching (control and 95% plus
needle scorch) and two nitrogen (N) fertilization (control and
5gNm
−2
year
−1
) treatments during an annual assessment.
Ectomycorrhizal fungi production comprised of extramatrical
mycelia, Hartig nets and mantles on fine root tips, and
sporocarps was estimated to be 49 g m
−2
year
−1
in the control
treatment plots. Extramatrical mycelia accounted for approx-
imately 95% of the total mycorrhizal production estimate.
Mycorrhizal production rates did not vary significantly
among sample periods throughout the annual assessment
(p=0.1366). In addition, reduction in foliar leaf area via
experimental scorching treatments did not influence mycor-
rhizal production (p=0.9374), suggesting that stored carbon
(C) may decouple the linkage between current photosynthate
production and ectomycorrhizal fungi dynamics in this forest
type. Nitrogen fertilization had a negative effect, whereas
precipitation had a positive effect on mycorrhizal fungi
production (p=0.0292; r
2
=0.42). These results support the
widely speculated but poorly documented supposition that
mycorrhizal fungi are a large and dynamic component of C
flow and nutrient cycling dynamics in forest ecosystems.
Keywords Mycorrhizal fungi
.
Extramatrical mycelia
.
Extraradical hypha
.
Biomass
.
Production
Introduction
Mycorrhizal fungi mediate carbon (C) and nutrient exchange
between plants and soil in forest ecosystems (Treseder and
Allen 2000;Hartnettetal.2004; Godbold et al. 2005;
Hobbie 2006). Although previous studies have suggested
that mycorrhizal fungi are a large and dynamic component of
C flow and nutrient cycles, the majority of these studies have
been conducted on fungal species growing either axenically
or in association with seedlings in artificial (e.g., growth
chambers and glasshouses) environments for relatively short
(e.g., weeks to months) periods (Arnebrant 1994; Treseder
and Allen 2000; Bidartondo et al. 2001; Staddon et al. 2002,
2003). As a result, the rates and environmental controls of
mycorrhizal processes in situ are poorly understood
(Wallander et al. 2001; Högberg and Högberg 2002;
Treseder 2004;Hobbie2006). Longer-term, field-based,
ecosystem-scale studies of the rates and controls of
mycorrhizal production are critical for assessing the role
of these fungi in the C flow and nutrient cycling dynamics of
forest ecosystems (Norby and Jackson 2000; Treseder and
Allen 2000, 2002; Godbold et al. 2005).
Mycorrhiza (2007) 17:299–309
DOI 10.1007/s00572-007-0105-x
S. E. Sims (*)
:
R. J. Mitchell
:
S. D. Pecot
Joseph W. Jones Ecological Research Center at Ichauway,
Rt. 2,
P.O. Box 2324, Newton, GA 39870, USA
e-mail: ssims@jonesctr.org
R. J. Mitchell
e-mail: rmitchel@jonesctr.org
S. D. Pecot
e-mail: specot@jonesctr.org
J. J. Hendricks
Department of Biology, University of West Georgia,
Carrollton, GA 30118, USA
e-mail: jhendric@westga.edu
K. A. Kuehn
Department of Biological Sciences,
The University of Southern Mississippi,
Hattiesburg, MS 39406, USA
e-mail: kevin.kuehn@usm.edu
Many ecologically and econo mically important tree
species in boreal, temperate, and tropical forests form
ectomycorrhizal associations (Fogel 1980;Allen1991;
Hobbie 2006). The fungal component of such associations
consists of the following: (1) a Hartig net of fungal mycelia
surrounding the cortical cells of many fine root tips, (2) a
mantle of fungal mycelia surrounding the surface of these
root tips, (3) extramatrical mycelia that radiate from the
mantle into the surrounding soil environment, and (4)
sporocarp formation (Allen 1991; Wilcox 1991). Compre-
hensive field-based assessments of ectomycorrhizal pro-
duction have been hindered by the inability to measure the
growth of extramatrical mycelia (Wallander et al. 2001;
Högberg and Högberg 2002; Treseder 2004). Con sequently,
mycorrhizal production asses sments in field settings have
been typically limited to ectomycorrhizal development on
root tips and sporocarp production, despite laboratory-
based studies that have documented the quantitative
importance of extramatrical mycelia (Colpaert et al. 1992;
Arnebrant 1994; Treseder and Allen 2000; Wallander et al.
2001; Staddon et al. 2002).
One promising approach for measuring extramatrical
mycelia production of ectomycorrhizal fungi in situ was
pioneered by Wallander et al. (2001). In the field, the accrual
of fungal-specific biomarkers that break down rapidly after
cell death (e.g., ergosterol and signature phospholipid fatty
acids; Antibus and Sinsabaugh 1993; Wallander et al. 2001;
Gessner and Newell 2002) was assessed using ingrowth
mesh bags containing acid-washed sand. Paired sets of
ingrowth bags were placed within “closed” (i.e., in polyvinyl
chloride [PVC] pipes that prevented the in-growth of roots
and ectomycorrhizal fungi) and “open” (i.e., areas that could
be colonized by roots and ectomycorrhizal fungi) cores and
harvested periodically throughout the year. The biomarker
accrual in the closed cores reflected only the growth of
saprotrophic fungi, whereas the biomarker accrual in the
open cores represented growth of both ectomycorrhizal and
saprotrophic fungi. Rates of ectomycorrhizal fungi produc-
tion were estimated as the difference in biomarker accrual
between the open and closed cores (Wallander et al. 2001).
Recently, the utility of the Wallander et al. ( 2001)
ingrowth core approach for measuring rates of extramatrical
mycelial production was assessed in a longleaf pine (Pinus
palustris Mill.) plantation using acid-washed sand and
native soil as ingrowth matrixes (Hendricks et al. 2006a).
Extramatrical mycelial production of ectomycorrhizal fungi
was significantly influenced by the ingro wth matrix, with
production rates in the native soil more than 300% higher
than corresponding rates in acid-washed sand. Thus,
modification of the Wallander et al. (2001) approach to
use native soil as the ingrowth matrix may provide
estimates of extramatrical mycelial production that better
reflect the indigenous rates (Hendricks et al. 2006a). In
turn, extramatrical mycelial production assessmen ts via the
modified Wallander et al. (
2001) approach may be coupled
with more traditional assessments of ectomycorrhizal
development on root tips and sporocarp formation to
provide a more comprehensive assessment of ectomycor-
rhizal production rates in forest ecosystems.
This study evaluated t he rates and ecophysiological
controls on in situ ectomycorrhizal fungi production in a
22-year-old longleaf pine plantation. The primary objectives
of this study were to (1) assess the various components of
ectomycorrhizal fungal production, including the growth of
extramatrical mycelia, Hartig nets and mantles on root tips,
and sporocarps, (2) compare the temporal production patterns
of the various fungal constituents, (3) evaluate how reduced C
source strength (i.e., reduced foliar leaf area via scorching
and, consequently, decreased current photosynthate produc-
tion) influences ectomycorrhizal production, and (4) evaluate
how increased root C sink strength (i.e., increased soil N
availability via fertilization and, consequently, increased fine
root and fungal enzyme concentrations and maintenance
respiration rates) influences ectomycorrhizal production
during an annual assessment.
Materials and methods
Study site
This study was conducted in a 50-ha longleaf pine plantation
located at the Joseph W. Jones Ecological Research Center in
Baker County, GA (31°15′ latitude, 84°30′ longitude). The
plantation was established in 1980 using an approximate 2×
3 m spacing between trees. The closed canopy of the
plantation resulted in very little understory vegetation, and a
4% glyphosate herbicide solution was applied to understory
plants 1 year before plot establishment (and thereafter as
necessary) to ensure that longleaf pine was the only plant
species growing in the study plots. Tree heights at the time of
study initiation ranged from 10.7 to 13.7 m. The soil in the
plantation has been classified as a loamy, siliceous, thermic
Arenic Paleudult with weakly developed horizons because of
past anthropogenic site use, mixing by fauna, low organic
matter content, and lack of silt and clay (Goebel et al. 1997).
The climate for this region has been characterized as humid
subtropical with an average annual precipitation of 131 cm
distributed evenly throughout the year and mean annual low
and high temperatures of 9 and 28°C, respectively (Goebel et
al. 1997).
Treatments
In the summer of 2001, treatment plots were established in
the plantation using two foliar scorching (control and 95%
300 Mycorrhiza (2007) 17:299–309
plus needle scorch) and two N fertilization (control and 5 g
Nm
−2
year
−1
) treatments in a complete factorial design
yielding four treatment combinations replicated eight times
each for a total of 32 plots (see Guo et al. 2004). Each plot
was 20×20 m. To minimize potential edge effects, the
sample collections were confined to the central 15×15 m
subplot of each treatment plot, and the treatment plots were
separated by at least a 20-m buffer zone.
In June and July of 2002, after the initiation of first year
needle production, foliage scorch treatments were con-
ducted to assess the effects of current photosynthate
production, or C source strength, on ectomycorrhizal
growth. Approximately 95% of the foliage (i.e., all needles
except those on the apical branch of each tree) was
removed in 16 randomly selected treatment plots. A 12-m
hydraulic lift was used to access the canopy, and a handhel d
torch connected to a 1,900-l propane tank was used to
scorch the foliage for approximately 10 s per needle flush.
Previous foliar scorch treatments in the plantation revealed
that this method was sufficient to kill foliage without killing
branches and terminal buds (Carter et al. 2004). Although
this method and degree of defol iation may seem drastic,
this treatment has a direct ecological analogue in that
longleaf pine occasionally receive and survive 100% crown
scorching during prescribed burning events. The hydraulic
lift and propane tank were also driven across the non-
scorched treatment plots to min imize potential differences
in ectomycorrhizal product ion because of compaction and
soil disturbance associated with the scorching process.
The N fertilizati on tre atmen t was init iat ed in Janu ary
2001 to assess the effects of increasing soil N availabili ty
and assimilation, or C sink strength, on ectomycorrhizal
production. Consistent with the factorial design, eight
foliar scorch and eight nonfoliar scorch treatment plots
were randomly select ed and fertilized using ammonium
nitrate at a rate of 5 g N m
−2
year
−1
,anapproximate
twofold increase above N mineralization rates measured in
comparable longleaf pine ecosystems (Wilson et al. 1999,
2002). Nitrogen additions were conduct ed in a manner that
tracked natural temporal patterns of N mineralization; the
proportion of the 5 g N m
−2
year
−1
added each month to
the N fertilization treatment plots was based on the
percentage of annual N mineralization occurring during
that particula r month in compa rabl e stand s (Wilson et al.
1999, 2002).
Production assessments
Ectomycorrhizal production rates were assessed from
February 2002 to February 2003. To obtain comprehensive,
ecosystem-scale estimates, extramatrical mycelia, Hartig
net, mantle, and sporocarp production were quantified
during the annual assessment.
The production of extramatrical mycelia as well as the
production of Hartig nets and mantles on fine root tips were
measured using a modification of the Wallander et al.
(2001) ingrowth core approach (as described in Hendricks
et al. 2006a). At the beginning of each sample interval, five
sets of “closed” (i.e., 10-cm-diameter × 30-cm-deep cores
placed within PVC pipes to prevent the ingrowth of roots
and mycorrhizal fungi ) and “open” (i.e., 10-cm-diameter ×
30-cm-deep ingrowth cores exposed to the bulk soil that
could be colonized by roots and mycorrhizal fungi) cores
were established in each plot. The ingrowth soil was
collected from adjacent sites that received the same N
amendments, sieved to remove coarse organic matter, and
homogenized before filling the cores. The cores were
established by filling the holes, tamping the soil to the
approximate bulk density of the adjacent soil, and placing a
PVC pin flag directly in the center to mark the core
location. Thus, the open-core ingrowth soil was directly
adjacent to the bulk soil (as opposed to being contained in
nylon mesh bags used by Wallander et al. 2001). After
either a 1- to 2-mo nth ingrowth period to coincide with
associated assessments of fine root dynamics, the cores
were collected using a slightly smaller diameter corer (8 cm
diameter × 30 cm deep) with the PVC pin flag serving as
the center point (Hendrick s et al. 2006a). The samples were
composited by core type in each plot and placed in a cooler
for transport to the laboratory.
Field samples were processed within 1 h of collection. In
the laboratory, each sample was homogenized , and soil
samples were collected for ergosterol concentration (5–7g
preserved in 5 ml of KO H methanol, 0.8% KOH in high-
pressure liquid chromatography [HPLC] grade methanol,
stored at −20°C; Gessner and Newell 2002) and moisture
(5–7 g dried at 70°C to a constant mass; Jones 1984)
analysis to express ergosterol concentrations on a dry-mass
basis. After subsampling for ergosterol and soil moisture,
the remaining soil for each open core sample was wet
sieved (#10 mesh) to collect fine root samples to assess
ectomycorrhizal fungal producti on in Hartig nets and
mantles. Hartig net and mantle production were assessed
jointly by extracting ergosterol from the fine root tissues
that colonized the open ingrowth cores during the sample
period. Isol ated fine root tissues were rinsed with cold
water, blotted dry, and preserved (5 ml KOH methanol,
0.8% KOH in HPLC grade methanol, stored at −20°C) for
ergosterol analys is.
Ergosterol in the preserved soil and fine root samples
was extracted in alcoholic KOH (0.8% KOH in HPLC
grade methanol, total extraction volume of 10 ml) for
30 min at 80°C in tightly capped thick-walled digestion
tubes. The resultant crude extract was cleaned by solid
phase extraction (Gessner and Schmitt 1996), and ergoster-
ol was purified and quantified by HPLC. A LichroSpher
Mycorrhiza (2007) 17:299–309 301
100 RP-18 column (0.46×25 cm) maintained in a Shimadzu
column oven (CTO-10AS) at 40°C and connected to a
Shimadzu autosampler (SIL-10AD) and Shimadzu liquid
chromatograph system (Pumps LC-10AT, Controller SCL-
10A) was used for separation and analysis. The mobile
phase was HPLC grade methanol at a flow rate of 1.5 ml
min
−1
. Ergosterol was detected at 282 nm using a Shimadzu
(SPD-10A) UV/VIS detector (retention time = approx.
8 min) and was identified and quantified based on
comparison with ergosterol standards (Fluka Chemical).
Laboratory trials using this extraction procedure on auto-
claved soil samples spiked with 25 μg of ergosterol yielded
a 71.3±2.6% recovery of ergosterol. Consequently, esti-
mates for the field samples were adjusted for unrecovered
ergosterol. Ectomycorrhizal extramatrical mycelial produc-
tion was calculated as the difference in ergosterol accrual
between the paired open and closed cores.
Fungal sporocarp production was assessed in coordina-
tion with extramatrical mycelia, Hartig net, and mantle
production assessments. At the end of each sample period,
sporocarps were collected from five 1×1 m subplots in
each of the 32 study plots. The sporocarps were dried at
70°C to a constant mass, weighed, and identified. Sporo-
carps that could not be conclusively identified visually were
grouped based on similarity in appearance. Representative
samples for each identified species and unknown group
were homogenized using a Wig-L-Bug Model 6 Amalgam-
ator (Reflex Analytical Corporation, Ridgewood, NJ). A 2-
to 4-mg subsample of each sample was analyzed for
13
C
and
15
N natural abundance at the U.C. Davis Stable Isotope
Facility to potentially assess the trophic status (i.e.,
mycorrhizal vs saprotrophic) based on the significant
difference in
13
C and
15
N natural abundanc e concentrations
between mycorrhizal and saprotrophic species (Taylor et al.
1997; Gebauer and Taylor 1999; Hobbie et al. 1999;
Högberg et al. 1999; Kohzu et al. 1999). In addition, a 3-
to 5-g subsample was ashed (500°C for 4 h; Jones 1984)to
express sporocarp mass es on an ash-free, dry-mass basis.
The estimates of extramatrical mycelia, Hartig net and
mantle, and ectomycorrhizal sporocarp production were
summed for each sample interval. To express the produc-
tion estimates in the same units (g/m
2
), ergosterol produc-
tion estimates associated with the extramatrical mycelia and
root tips (i.e., Hartig nets and mantles) were converted to
fungal biomass values using a conversion factor of 5 μg
ergosterol per milligram fungal biomass based on the
average of published estimates for ectomycorr hizal speci es
(5.04±3.80 μg ergosterol per milligram fungal biomass; n=
36 cited in Salmanowicz and Nylund 1988; Martin et al.
1990; Antibus and Sinsabaugh 1993; Sung et al. 1995;
Wallander et al. 1997;Colpaertetal.1999). Fungal
biomass estimates expressed in milligrams per gram of soil
were then converted to a ground surface area basis (to a 30-
cm depth) using a soil bulk density value of 1.15 g/cm
3
.In
turn, the extramatrical mycelia, Hartig net and mantle, and
ectomycorrhizal sporocarp production estimates were
summed across the sample intervals to yield an annual
production estimate.
Edaphic resource availabil ity assessmen ts
Nitrogen mineralization was measured during the annual
assessment using a closed core incubation technique
(Raison et al. 1987; Blair 1997). Ten soil cores (2 cm
diameter) were collected from the 0–10 cm horizon in each
plot at the beginning of an incubation cycle, sieved (#10
mesh) in the laboratory, and subsampled for estimation of
initial pools of inorganic N NH
þ
4
and NO
3
and moisture
content. In addition, five subsamples were taken from each
sample, placed in gas-impermeable PVC pipes (2 cm
diameter) that were capped and buried (to 10 cm depth) in
their original plot within a 24-h period. After an incubation
period ranging from 28 to 35 days, samp les were retrieved,
composited by plot, and subsampled for inorganic N and
moisture analyses.
Inorganic N in the initial and incubated soil samples was
extracted with 2 M KCl (10 g:25 ml) by vigorous agitation
on a mechanical shaker for 15 min, followed by centrifu-
gation (2,500 rpm) for an additional 15 min. The
supernatant for each sample was then carefully drawn off
and analyzed colorimetrically for NH
þ
4
and NO
3
concen-
trations using a Lachat Flow Injection Analyzer (Kenney
and Nelson 1982; Lachat Instruments 1992, 1997). Net
ammonification and nitrification were then calculated by
subtracting the initial concentrations from the final pools of
extractable NH
þ
4
NandNO
3
N, respectively. Net
mineralization was calculated as the sum of net ammoni-
fication and nitrification during the incubation period
expressed on a soil dry mass basis.
Soil moisture availability was assessed for each sample
interval as well. In association with the N mineralization
assessments, gravimetric soil moisture (g/g) was measured
at the beginning and end of each sample interval. In
addition, precipitation during the sample interval was
measured at a weather station located approximately 2 km
from the longleaf pine plantation.
Data analysis
Statistical analyses were conducted using mixed-models
analysis of variance and regression analyses in SAS® (SAS
Institute, Cary, NC). All variables were normally distribut-
ed, so data transformations were not necessary. Mixed-
models analysis of variance was used to test for main and
interaction effects of scorching and fertilization on ectomy-
corrhizal production through time (Littell et al. 1996).
302 Mycorrhiza (2007) 17:299–309
Where significant interactions were present, contrasts were
performed to detect specific differences. In addition, simple
and multiple regression analyses were used to assess
relationships between mycorrhizal fungi production and
edaphic resource availability indices.
Results
Production rates
Ectomycorrhizal production including extramatrical myce-
lia, Hartig nets and mant les, and sporocarps was estimated
to be 49 g m
−2
year
−1
in the control plots of the longleaf
pine plantation (Fig. 1). Extramatrical mycelia comprised
95% of the total annual production estimate. This can be
considered a conservati ve production estimate, as extrama-
trical mycelial turnover may have occurred during the
sample intervals. In contrast to extramatrical mycelia, the
Hartig nets and mantles on root tips and mycorrhizal
sporocarps constituted only 4.5 and 0.5%, respectively, of
the total annual production (Fig. 1).
Estimates of mycorrhizal sporocarp production were also
probably conservative because of the difficulty of positively
identifying the trophic status of some speci mens. Based on
visual identification, 84% (by mass) of the sporocarps were
confirmed to be ectomycorrhizal, belonging to the genera
Amanita, Cantharellus , Cortinarius, Geastrum, Laccaria,
Pisolithus, Rhizopogon, Scleroderma, Thelephora,and
Tricholoma. Three percent of the sporocarps were con-
firmed to be saprophyti c, belong ing to the genera Agaricus,
Clathrus, Peziza, Polyporus, and Ramaria, and 13% could
not be conclusively categorized by trophic status. Natural
abundance analyses of
13
Cand
15
N also failed to
conclusively reveal the trophic status of the “unknown”
sporocarp specimen (data not shown). Consequently, the
sporocarp masses used in the production estimate were
limited to those conclusively identified as mycorrhizal.
Although some mycorrhizal sporocarps were likely omitted
from the production estimate, the error attributed to this
source was considered small because: (1) the unknown
specimens as a whole represented only a small percentage
of the total sporocarp mass (13%), and (2) the sporocarps as
a whole represented on ly a small percentage of the total
ectomycorrhizal production estimate (<1%).
Temporal variation
Ectomycorrhizal production rates did not vary significantly
(p=0.1366) during the annual assessment (Fig. 2). Although
the differences were not significant, extramatrical mycelia
did exhibit maximum production rates during the late June–
early August sampling interval and minimum rates during
the mid December–mid February sampling interval. The
annual minimum rate of extramatrical mycelial production
Month
February
M
a
r
c
h
A
p
ri
l
M
a
y
J
u
ne
J
ul
y
A
ugu
s
t
S
e
p
t
e
m
b
e
r
O
c
t
o
b
e
r
N
o
ve
m
b
e
r
D
e
c
e
m
b
e
r
J
a
nua
ry
F
e
b
rua
ry
Cumulative production (g/m
2
)
0
10
20
30
40
50
60
Total
Extramatrical mycelia
Hartig net/mantle
Sporocarp
Fig. 1 Cumulative mycorrhizal fungi production in the control plots
over the course of the annual assessment. Values represent means +
SD for the sample interval
Control
Production (g m
-2
30 d
-1
)
0
2
4
6
8
10
12
14
16
Total
Extramatrical mycelia
Hartig net/mantle
Sporocarp
Fertilized
Month
F
e
b
rua
ry
M
a
rc
h
A
p
ri
l
M
a
y
J
un
e
J
u
l
y
A
ugus
t
S
e
p
t
e
m
b
e
r
O
c
t
o
b
e
r
N
o
ve
m
b
e
r
D
e
c
e
m
b
e
r
J
a
nua
ry
F
e
b
rua
ry
Production (g m
-2
30 d
-1
)
0
2
4
6
8
10
12
14
16
Total
Extramatrical mycelia
Hartig net/mantle
Sporocarp
Fig. 2 Temporal pattern of mycorrhizal fungi production standardized
to 30-day production intervals in the control and fertilized treatment
plots
Mycorrhiza (2007) 17:299–309 303
coincided with the maximum period of Hartig net and
mantle production on fine roots. Mycorrhizal sporocarp
production was consistently low throughout the study period.
Carbon source and sink controls
The mixed-models analysis of variance test for fixed effects
did not reveal significant differences in total mycorrhizal
fungi production because of scorching (p=0.9374; 47±4 vs
46±7 g m
−2
year
−1
for scorched and nonscorched treatments,
respectively) or fertilization (p=0.7865; 45±6 vs 48±4 g
m
−2
year
−1
for fertilized and nonfertilized treatments which
exhibited mineralization rates of 2.0±0.2 and 0.9±0.1 g
m
−2
year
−1
, respectively). However, there was a significant
fertilization × time interaction effect (p=0.0414). Subse-
quent contrast analyses revealed that fertilization had a
significant effect on ectomycorrhizal production during the
fifth s ampling interval (early August– mid September),
which followed months of relatively large N additions in
fertilized treatment plots (Fig. 2).
A linear regression analysis indicated that the relation-
ship between N availability and total ectomycorrhizal fungi
production was not significant (p=0.2210). However, a
multiple regression analysis using both N availability and
precipitation during the interval as the independent varia-
bles revealed a significant relationship (p=0.0292; r
2
=0.42)
with total ectomycorrhizal production during each sample
interval (Fig. 3). The multiple regression equation (y=4.48−
4.60[nitrogen availability] + 0.13[precipitation]) indicates
that nitrogen had a negative impact, whereas precipitation
had a positive impact on ectomycorrhizal produc tion.
Comparison of the absolute values of the standardized
parameter estimates for N (−0.22) and precipitation (0.57)
indicated that precipitation had a greater effect than
nitrogen availability on total mycorrhizal fungi production.
Discussion
Mycorrhizal production dynamics are currently among the
most poorly understood aspects of forest ecosy stem
ecology primarily because of the long-standing inability to
assess the patterns and controls of extramatrical mycelia
production in field settings (Treseder and Allen 2000;
Wallander et al. 2001, 2004; Högb erg and Högberg 2002;
Hobbie 2006). The results of this study may provide
valuable insight for several reasons. First, this study
employed a modified version of the Wallander et al.
(2001) ingrowth core approach (i.e., native soil matrix) to
obtain more field-realistic estimates of extramatrical myce-
lia production, in association with assessments of Hartig
net, mantle, an d sporocarp production. Second, t hese
production assessments were conducted over the course of
1 year in an established (i.e., 22-year-old) longleaf pine
forest. Lastly, manipulations of C source strength and C
sink strength to assess the ecophysiological controls on
ectomycorrhizal production in the plantation were con-
ducted in a manner that simulated natural disturbanc es and
processes at the ecosystem scale.
Production rates
Ectomycorrhizal fungi production was estimated to be 49 g
m
−2
year
−1
in the longlea f pine plantation (Fig. 1). Hartig
net, mantle, and sporocarps cumulatively constituted only
5% of the total production estimate, whereas extramatrical
mycelia constituted 95% of the total (Fig. 1). Although few
field-based ecosystem studies have assessed the propor-
tional allocation of ectomycorrhizal production to extrama-
trical mycelia (Hobbie 2006), the estimate of 95% reported
here is comparable to 60–85% reported by Colpaert et al.
(1992) for a variety of ectomycorrhizal species in a
laboratory-based study and approximately 80% reported
by Wallander et al. (2001) for a field-based study in a
Norway spruce fores t ecosystem.
The annual ectomycorrhizal production estimate
reported for this longleaf pine forest was approximately
four times higher than the estimate reported by Wallander et
al. (2001) for the Norway spruce forest (12.5 g m
−2
year
−1
based on ergosterol analysis). In addition, crude estimates
of the ratio of ectomycorrhizal fungi production to fine root
production were higher in the longleaf pine forest (10.8%
Fig. 3 Mult iple regressio n relationship depicting the interactive
effects of nitrogen availability and precipitation on mycorrhizal fungi
production
304 Mycorrhiza (2007) 17:299–309
based on an annual fine root production estimate of 448 g
m
−2
year
−1
derived for this site in 1999 using the
minirhizotron approach; Carter et al. 2004) compared to
the Norway spruce forest (3.5% based on an average fine
root production estimate of 351 g m
−2
year
−1
derived for
this site from 1995 to 1996 using a root window
observation approach; Stober et al. 2000). The differences
in absolute and relative ectomycorrhizal production esti-
mates between these studies may be attributed to the
inherent differences in climate, edaphic resource availabil-
ity, and net primary productivity between the two sites.
However, the differences in ectomycorrhizal production
estimates may also be attributed to the underlying ingrowth
core methodology. Although both studies employed the
ingrowth core approach, Wallander et al. (2001) used acid-
washed sand as the ingrowth matrix, whereas we used
native soil. In a previous study conducted in this same
longleaf pine plantation site, Hendricks et al. ( 2006a)
observed that ectomycorrhizal extramatrical mycelial pro-
duction using native soil was 300% higher than
corresponding production rates in acid-washed sand. This
difference is consistent with the difference in ectomycor-
rhizal production observed between this and the Wallander
et al. (2001) study.
Temporal patterns
Ectomycorrhizal production did no t exhibit significant
temporal variation during the annual assessment (Fig. 2).
The lack of significant seasonal differences in the longleaf
pine plantation contrasts with the patterns reported for
boreal coniferous forests, where minimum and maximum
ectomycorrhizal production rates generally occurred in the
spring and fall, respectively (Wallander et al. 1997, 2001).
Wallander et al. (2001) noted anecdotally that rates of
ectomycorrhizal fungi production corresponded with fine
root production rates reported by Stober et al. (2000),
which were measured at the same study site but in a
different year. The observation of relatively continuous
ectomycorrhizal fungi pro duction in the longleaf pine
plantation is consistent with the fine root production
patterns recently reported for this plantation (Carter et al.
2004) and other longleaf pine forests (West et al. 2004,
Hendricks et al. 2006 b), which typically have relatively
mild winters and precipitation evenly distributed through-
out the year (Mitchell et al. 1999; Kirkman et al. 2001). The
relationship between fine root and ectomycorrhizal fungi
production warrants further research.
Carbon source controls
Mycorrhizal fungi depend on C from the host plant for
production and maintenance respiration. Although it is clear
that the host may allocate a significant proportion of
photosynthate to mycorrhizal fungi, the mechanisms that
control this C flux to these fungal microsymbionts are
poorly understood (Bidartondo et al. 2001 ; Högberg et al.
2001; Staddon et al. 2002, 2003).
Several studies have demonstrated that mycorrhizal
fungi depend on recently fixed C, or current photosynthate,
for production and maintenance activities (Söderström and
Read 1987; Cairney et al. 1989; Cairney and Alexander
1992; Högberg et al. 2001; Hobbie et al. 2002; Högberg
and Högberg 2002; Johnson et al. 2002; Treseder et al.
2004). Reductions in leaf area and current photosynthate
production via defoliation, herbivory, or pruning have been
shown to reduce C allocation to fine roots, and presumably
their mycorrhizal fungal symbionts, in mature trees (Wargo
et al. 1972; Parker and Patton
1975; Webb 1981; Eissenstat
and Duncan 1992; Kielland et al. 1997; Ruess et al. 1998).
Högberg et al. (2001) reported that stem girdling reduced
soil CO
2
efflux rates by 37% within 5 days in Scots pine
(P. sylvestris) forests. In addition, C isotope tracer assess-
ments have indicated that mycorrhizal mycelia production
and respiration are sustained using recently (e.g., ≤24 h)
fixed C in a mature deciduous forest (Keel et al. 2006) and
seedlings (Cairney et al. 1989; Cairney and Alexander
1992; Johnson et al. 2002; Wu et al. 2002; Staddon et al.
2003). Collectively, these studies suggest that disruption of
current photosynthate prod uction via anthropogenic or
natural disturbances may have particularly adverse effects
on mycorrhizal production.
In contrast, the finding that foliar scorching did not
significantly reduce mycorrhizal production in this study
suggests that stored C reserves may be used to support fine
root and ectomycorrhizal fungi dynamics after disruption of
current photosynthate production. It should be noted that
stored C reserves and fluxes were not assessed in this study
and that the C used to support mycorrhizal production on
the roots of scorched trees may have been derived from
other sources, most notably via mycorrhizal network
connections to adjacent nonscorched trees (Southworth et
al. 2005). However, it is unlikely that compensatory C
supply via mycorrhizal networks was a significant C source
in this system. Fine root and mycorrhizal dynamics were
assessed in the interior subplot (15×15 m) of a large
treatment plot (20×20 m), and C supply via a common
mycorrhizal network likely decreases with distance from
the original source (Southworth et al. 2005). In addition, the
lack of a significant difference (p=0.9374) between
mycorrhizal production in the scorched and nonscorched
treatments was attributed to the similarity of the estimates
(i.e., 47±4 vs 46±7 g m
−2
year
−1
for scorched and
nonscorched treatments, respectively) rather than an error
associated with the estimates, and it is unl ikely that
mycorrhizal networks may totally compensate for the large
Mycorrhiza (2007) 17:299–309 305
reduction in C supply strength of the scorched trees. In
addition, the lack of a significant foliage-scorching treat-
ment effect on ectomycorrhizal product ion is consistent
with prior findings that foliage scorching did not signifi-
cantly affect the production and mortality of fine roots
(Carter et al. 2004) or the nonstructural carbohydrate
concentration of fine roots (Guo et al. 2004), although this
treatment did significantly reduce stored C reserves (e.g.,
coarse root C concentrations; Guo et al. 2004) in this
longleaf pine plantation. This suggests that C reserves in
coarse roots may be used to compensate for reductions in
current photosynthate production and maintain the relative-
ly high metabolic activity of fine roots and associated
mycorrhizal fungi.
Theapparentimportanceof stored C reserves for
maintaining fine root and mycorrhizal fungi dynamics in
longleaf pine forests is consistent with the findings of other
studies conducted in systems subject to regular f oliar
disturbances. Kosola et al. (2001) observed that severe
insect defoliation of Eugeneii hybrid poplars did not affect
the nonstructural carbohydrate concentration or mortality
rate of fine roots Edwards and Ross-Todd (1979) reported
that stem girdling in a mixed deciduous forest did not
significantly reduce fine root biomass or soil CO
2
efflux
rates for up to 2 years after treatment initiation. Research by
Langley et al. (2002), us ing
13
C tracer techniques,
demonstrated that fine roots and associated ectomycorrhizal
symbionts were maintained by stored C for more than
2.5 years after a prescribed burn in a scrub oak (Quercus
spp.) ecosystem. In addition, foliage removal in grazing-
tolerant grasses via burning or mowing also had no effect
on fungal colonization or extramatrical mycelia develop-
ment (Wallace 1987; Allen et al. 1989; Eom et al. 1999).
Although a direct linkage between current photosynthate
production and fine root dynamics has been reported
(Högberg et al. 2001; Johnson et al. 2002; Staddon et al.
2003; Treseder et al. 2004; Keel et al. 2006), other studies
suggest that stored C may decouple the linkage between
current photosynthate production and fine root dynamics in
ecosystems subject to frequent foliar disturbances (e.g., fire,
grazing, insect herbivory, etc.) (Eom et al. 1999; Kosola et
al. 2001; Langley et al. 2002; Carter et al. 2004; Guo et al.
2004). Our results support the hypothesis recently proposed
by Guo et al. (2004), which maintains that plant species
may differ in their belowground C allocation relationships
based on their natural history and resilience to disturbance.
Carbon sink controls
Plants use mycorrhizal fungi for nutrient acquisition, and
thus the nutrient status of the host plant and soil has long
been considered a primary factor influencing C allocation
rates to mycorrhizal production (Allen 1991; Arnebrant
1994; Treseder and Allen 2000, 2002). Although it has
been widely hypothesized that the host plant will allocate
proportionately more C to associated mycorrhizal sym-
bionts in nitrogen-poor environments (R ead 1991; Smith
and Read 1997; Wallenda and Kottke 1998; Treseder and
Allen 2000, 2002), this hypothesis has not been rigorously
tested in field settings because of the difficulty of
measuring extramatrical mycelia production (Wallander et
al. 2001; H ögberg and Högberg 2002; Nilsson and
Wallander 2003).
In this study, N fertilization reduced ectomycorrhizal
production rates (Figs. 2 and 3). This pattern is consistent
with recent observations reported by Nilsson and Wallander
(2003), which indicated that N fertilization reduced extra-
matrical mycelial production by approximately 50% in a
Norway spruce forest. In addition, in a recent meta-analysis
of 31 studies, Treseder (2004) noted that mycorrhizal fungi
colonization of fine root tips decreased 15% under N
fertilization. Furthermore, the significant decline in ecto-
mycorrhizal sporocarp production in forests subjected to
atmospheric N deposition has been well documented
(Wallenda and Kottke
1998; Cairney and Meharg 1999;
Wallander et al. 1999; Treseder and Allen 2000; Lilleskov
et al. 2002).
A multiple regression analysis indicated that N avail-
ability and precipitation together accounted for 42% of the
variation in ectomycorrhizal production rates during the
annual assessment (Fig. 3). Whereas N availability had a
negative effect, precipitation had a positive effect on
ectomycorrhizal production. The strong and contrasting
effects of N and water availability on ectomycorrhizal
production have important implications for C flow and
nutrient cycling dynamic assessments in forests, as these
resources may be altered signifi cantly and independently by
anthropogenic and natural disturbances (Norby and Jackson
2000; Treseder and Allen 2000; Aber and Melillo 2001;
Staddon et al. 2002).
Implications for C flow and nutrient cycling assessments
This assessment of the rates and ecophys iological controls
of ectomycorrhizal fungi production in the longleaf pine
plantation support the widely speculate d but poorly
substantiated supposition that mycorrhizal fungi are a large
and dynamic component of C flow and nutrient cycles in
forest ecosystems (Hobbie 2006). Extramatrical mycelia,
which have been traditionally ignored in field assessments,
constituted the vast majority of total mycorrhizal fungi
production. In addition, the manipulation of C source and
sink strengths elucidated the importance of stored C, N, and
water availability in the regulation of ectomycorrhizal
production. Ecosystem-scale studies that assess the linkage
between C assimilation and belowground allocation with an
306 Mycorrhiza (2007) 17:299–309
emphasis on extramatrical mycelial production, respiration,
mortality, and decomposition may provide valuable insight
into the role of ectomycorrhizal fungi in C flow and
nutrient cycle dynamics and are thus critical in assessing
and predicting t he responses of forest ecosystems to
anthropogenic and natural disturbances.
Acknowledgments Many people have supported this project, and it
is with deep appreciation that we acknowledge their contributions.
Angela Colley, Kate Earnest, Audrey Johnson, Nancy Newberry,
Russell Nix, Marc Sims, Sara Snow, Dwan Williams, and Justin
Williams assisted in the execution of this study. Dr. David Porter
assisted in sporocarp identification. Dr. E. Barry Moser provided
valuable statistical consultation services. Drs. Leos G. Kral and Carl J.
Quertermus provided incisive and helpful criticisms on previous drafts
of this manuscript. Support for this project was provided by the
Graduate School at Eastern Michigan University, National Research
Initiative of the USDA Cooperative State Research, Education and
Extension Service (Grants 00-35101-9283 and 2006-35101-16557),
and Robert W. Woodruff Foundation.
References
Aber JD, Melillo JM (2001) Terrestrial ecosystems. Saunders College,
Philadelphia, PA
Allen MF (1991) The ecology of mycorrhizae. Cambridge Univ.
Press, Cambridge, UK
Allen MF, Richards JH, Busso CA (1989) Influence of clipping and
soil water status on vesicular–arbuscular mycorrhizae of two
semi-arid tussock grasses. Biol Fertil Soils 8:285–289
Antibus RK, Sinsabaugh RL (1993) The extraction and quantification
of ergosterol from ectomycorrhizal fungi and roots. Mycorrhiza
3:137–144
Arnebrant K (1994) Nitrogen amendments reduce the growth of
extramatrical ectomycorrhizal mycelium. Mycorrhiza 5:7–15
Bidartondo MI, Ek H, Wallander H, Söderström B (2001) Do nutrient
additions alter carbon sink strength of ectomycorrhizal fungi?
New Phytol 151:543–550
Blair JM (1997) Fire, N availability, and plant response in grass-
lands: a test of the transient maxima hypothesis. Ecology
78:2359–2368
Cairney JWG, Alexander IJ (1992) A study of ageing of spruce [Picea
sitchensis (Bong.) Carr.] ectomy corrhizas. II. carbohydrate
allocation in ageing Picea sitchensis/Tylospora fibrillose (Burt.)
Donk ectomycorrhizas. New Phytol 122:153–158
Cairney JWG, Meharg AA (1999) Influences of anthropogenic
pollution on mycorrhizal fungal communities. Environ Pollut
106:169–182
Cairney JWG, Ashford AE, Allaway WG (1989) Distribution of
photosynthetically fixed carbon within root systems of Eucalyp-
tus pilularis plants ectomycorrhizal with Pisolithus tinctorius.
New Phytol 112:495–500
Carter DC, Hendricks JJ, Mitchell RJ, Pecot SD (2004) Fine root
carbon allocation and fates in longleaf pine forests. For Sci
50:177–187
Colpaert JV, van Assche JA, Luijtens K (1992) The growth of
extramatrical mycelium of ectomycorrhizal fungi and the growth
response of Pinus sylvestris L. New Phytol 120:127–135
Colpaert JV, Van Tichelen KK, Van Assche JA, Van Laere A (1999)
Short-term phosphorus uptake rates in mycorrhizal and non-
mycorrhizal roots of intact Pinus sylvestris seedlings. New Phytol
143:589–597
Edwards NT, Ross-Todd BM (1979) The effects of stem girdling on
biogeochemical cycles within a mixed deciduous forest in eastern
Tennessee. Oecologia 40:247–257
Eissenstat DM, Duncan LK (1992) Root growth and carbohydrate
responses in bearing citrus tress following partial canopy
removal. Tree Physiol 10:245–257
Eom AH, Hartnett DC, Wilson GWT, Figge DAH (1999) The effect of
fire, mowing and fertilizer amendment on arbuscular mycorrhizas
in tallgrass prairie. Am Midl Nat 142:55–70
Fogel R (1980) Mycorrhizae and nutrient cycling in natural forest
ecosystems. New Phytol 86:199–212
Gebauer G, Taylor AFS (1999)
15
N natural abundance in fruit bodies
of different functional groups of fungi in relation to substrate
utilization. New Phytol 142:93–101
Gessner MO, Newell SY (2002) Biomass, growth rate, and production
of filamentous fungi in plant litter. In: Hurst CJ, Crawford RL,
Knudsen GR, McInerny MJ, Stetzenbach LD (eds) Manual of
environmental microbiology. ASM, Washington, DC, pp 390–
408
Gessner MO, Schmitt AL (1996) Use of solid-phase extraction to
determine ergosterol concentrations in plant tissue colonized by
fungi. Appl Environ Microbiol 62:415–419
Godbold D, Hoosbeek M, Lukac M, Cotrufo M, Janssens I,
Ceulemans R, Polle A, Velthorst E, Scarascia-Mugnozza G,
Angelis P, Miglietta F, Peressotti A (2005) Mycorrhizal hyphal
turnover as a dominant process for carbon input into soil
organic matter. Plant Soil 281:15–24
Goebel PC, Palik BJ, Kirkman LK, West L (1997) Field guide: land-
scape ecosystem types of Ichauway. Technical report no. 97-1.
Joseph W. Jones Ecological Research Ce nter at Ichauway,
Newton, GA
Guo D, Mitchell RJ, Hendricks JJ (2004) Fine root branch orders
respond differentially to carbon source–sink manipulations in a
longleaf pine forest. Oecologia 140:450–457
Hartnett DC, Potgieter AF, Wilson GWT (2004) Fire effects on
mycorrhizal symbiosis and root system architecture in southern
African savanna grasses. Afr J Ecol 42:328–337
Hendricks JJ, Mitchell RJ, Kuehn KA, Pecot SD, Sims SE
(2006a) Measuring external mycelia production of ectomycor-
rhizal fungi in the field: the soil matrix matters. New Phytol
171:179–186
Hendricks JJ, Hendrick RL, Wilson CA, Mitchell RJ, Pecot SD, Guo
D (2006b) Assessing the patterns and controls of fine root
dynamics: an empirical test and methodological review. J Ecol
94:40–57
Hobbie EA (2006) Carbo n a llocation to ectomycorrhizal fungi
correlates with belowground allocation in cult ure studies.
Ecology 87:563–569
Hobbie EA, Macko SA, Shugart HH (1999) Insights into nitrogen and
carbon dynamics of ectomycorrhizal and saprotrophic fungi from
isotopic evidence. Oecologia 118:353–360
Hobbie EA, Weber NS, Trappe JM, van Klinken GJ (2002) Using
radiocarbon to determine the mycorrhizal status of fungi. New
Phytol 156:129–136
Högberg MN, Högberg P (2002) Extramatrical ectomycorrhizal
mycelium contributes one-third of microbial biomass and
produces, together with associated roots, half the dissolved
organic carbon in a forest soil. New Phytol 154:791–795
Högberg P, Plamboeck AH, Taylor AFS, Fransson PMA (1999)
Natural
13
C abundance reveals trophic status of fungi and host-
origin of carbon in mycorrhizal fungi in mixed forests. Proc Natl
Acad Sci USA 96:8534–8539
Högberg P, Nordgren A, Buchmann N, Taylor AFS, Ekblad A,
Högberg MN, Nyberg G, Ottosson-Lofvenius M, Read DJ (2001)
Mycorrhiza (2007) 17:299–309 307
Large-scale forest girdling shows that current photosynthesis
drives soil respiration. Science 411:789–792
Johnson D, Leake JR, Read DJ (2002) Transfer of recent photosyn-
thate into mycorrhizal mycelium of an upland grassland: short-
term respiratory losses and accumulation of
14
C. Soil Biol
Biochem 34:1521–1524
Jones JB Jr (1984) A laboratory guide of exercises for conducting soil
tests and plant analyses. Benton Laboratories, Athens, GA
Keel SG, Siegwolf RTW, Körner C (2006) Canopy CO
2
enrichment
permits tracing the fate of recently assimilated carbon in a mature
deciduous forest. New Phytol 172:319–329
Kenney DR, Nelson DW (1982) Nitrogen: inorganic forms. In: Page
AL, Miller RH, Keeney DR (eds) Methods of soil analysis. Part
2. Chemical and microbial properties. American Society of
Agronomy, Madison, WI, pp 672–684
Kielland K, Bryant JP, Ruess RW (1997) Moose herbivory and carbon
turnover of early successional stands in interior Alaska. Oikos
80:25–30
Kirkman LK, Mitchell RJ, Helton RC, Drew MB (2001) Productivity
and species richness across an environmental gradient in a fire-
dependent ecosystem. Am J Bot 88:2119–2128
Kohzu A, Yoshioka T, Ando T, Takahashi M, Koba K, Wada E (1999)
Natural
13
C and
15
N abundance of field-collected fungi and their
ecological implications. New Phytol 144:323–330
Kosola KR, Dickman DI, Paul EA, Parry D (2001) Repeated insect
defoliation effects on growth, nitrogen acquisition, carbohy-
drates, and root demography of poplars. Oecologia 129:65–74
Lachat Instruments Inc (1992) QuickChem method no. 12-107-04-1-b.
Lachat Instruments, Milwaukee, WI
Lachat Instruments (1997) QuickChem method no. 12-107-06-1-b.
Lachat Instruments, Milwaukee, WI
Langley JA, Drake BG, Hungate BA (2002) Extensive belowground
carbon storage supports roots and mycorrhizae in regenerating
scrub oaks. Oecologia 131:542–548
Lilleskov EA, Hobbie EA, Fahey TJ (2002) Ectomycorrhizal fungal
taxa differing in response to nitrogen deposition also differ in
pure culture organic nitrogen use and natural abundance of
nitrogen isotopes. New Phytol 154:219–231
Littell RC, Milliken GA, Stroup WW, Wolfinger RD (1996) SAS
system for mixed models. SAS Institute, Cary, NC
Martin F, Delaruelle C, Hilbert JL (1990) An improved ergosterol
assay to estimate fungal biomass in ectomycorrhizas. Mycol Res
94:1059–1064
Mitchell RJ, Kirkman LK, Pecot SD, Wilson CA, Palik BJ, Boring LR
(1999) Patterns and controls of ecosystem function in longleaf
pine–wiregrass savannas. I. aboveground net primary productiv-
ity. Can J For Res 29:743–751
Nilsson LO, Wallander H (2003) Production of external mycelium
by ectomycorrhizal fungi in a Norway spruce forest was
reduced in response to nitrogen fertilization. New Phytol 158:
409–416
Norby RJ, Jackson RB (2000) Root dynamics and global change:
seeking an ecosystem perspective. New Phytol 147:3–12
Parker J, Patton RL (1975) Effects of drought and defoliation on some
metabolites in roots of black oak seedlings. Can J For Res 5:457–
463
Raison R, Connell M, Khanna P (1987) Methodology for studying
fluxes of soil mineral-N in situ. Soil Biol Biochem 19:521–
530
Read DJ (1991) Mycorrhizas in ecosystems. Experientia 47:376–391
Ruess RW, Hendrick RL, Bryant JP (1998) Regulation of fine root
dynamics by mammalian browsers in early successional Alaskan
taiga forest. Ecology 79:2706–2720
Salmanowicz B, Nylund JE (1988) High performance liquid chroma-
tography determination of ergosterol as a measure of ectomycor-
rhiza infection in Scots pine. Eur J For Pathol 18:291–298
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn.
Academic, San Diego, CA
Söderström B, Read DJ (1987) Respiratory activity of intact and
excised ectomycorrhizal mycelial systems growing in unsterilized
soil. Soil Biol Biochem 19:231–236
Southworth D, He XH, Swenson W, Bledsoe CS, Horwath WR (2005)
Application of network theory to potential mycorrhizal networks.
Mycorrhiza 15:589–595
Staddon PL, Heinemeyer A, Fitter AH (2002) Mycorrhizas and global
environmental change: research at different scales. Plant Soil
244:253–261
Staddon PL, Ramsey CB, Ostle N, Ineson P, Fitter AH (2003) Rapid
turnover of hyphae of mycorrhizal fungi determined by AMS
microanalysis of
14
C. Science 300:1138–1140
Stober C, George E, Persson H (2000) Root growth and response to
nitrogen. In: Schulze ED (ed) Carbon and nitrogen cycling in
European forest ecosystems. Springer, Berlin Heidelberg New
York, pp 99–121
Sung SJS, White LM, Marx DH, Otrosina WJ (1995) Seasonal
ectomycorrhizal fungal biomass development on loblolly pine
(Pinus taeda L.) seedlings. Mycorrhiza 5:439–447
Taylor AFS, Hogbom L, Högberg M, Lyon AJE, Nasholm T,
Högberg P (1997) Natural
15
N abundance in fruit bodies of
ectomycorrhizal fungi from boreal forests. New Phytol 136:713–
720
Treseder KK (2004) A meta-analysis of mycorrhizal responses to
nitrogen, phosphorus, and atmospheric CO
2
in field studies. New
Phytol 164:347–355
Treseder KK, Allen MF (2000) Mycorrhizal fungi have a potential
role in soil carbon storage under elevated CO
2
and nitrogen
deposition. New Phytol 147:189–200
Treseder KK, Allen MF (2002) Direct nitrogen and phosphorus
limitation of arbuscular mycorrhizal fungi: a model and field test.
New Phytol 155:507–515
Treseder KK, Masiello CA, Lansing JL, Allen MF (2004) Species-
specific measurements of ectomycorrhizal turnover under N-fer-
tilization: combining isotopic and genetic approaches. Oecologia
138:419–425
Wallace LL (1987) Mycorrhizae in grasslands: interactions of
ungulates, fungi and drought. New Phytol 105:619–632
Wallander H, Massicotte H, Nylund JE (1997) Seasonal variation in
ergosterol, chitin and protein in ectomycorrhizal roots collected
in a Swedish pine forest. Soil Biol Biochem 29:45–53
Wallander H, Arnebrant K, Dahlberg A (1999) Relationships between
fungal uptake of ammonium, fungal growth and nitrogen avail-
ability in ectomycorrhizal Pinus sylvestris seedlings. Mycorrhiza
8:215–223
Wa llander H, Nilsson LO, Hagerberg D , Baath E (2001)
Estimation of the biomass and seasonal growth of external
mycelium of ectomycorrhizal fungi in the fi eld. New Phytol
151:753–760
Wallander H, Goransson H, Rosengren U (2004) Production, standing
biomass and natural abundance of
15
N and
13
C in ectomycor-
rhizal mycelia collected at different soil depths in two forest
types. Oecologia 139:89–97
Wallenda T, Kottke I (1998) Nitrogen deposition and ectomycorrhizas.
New Phytol 139:169–187
Wargo PM, Parker J, Houston DR (1972) Starch content in roots of
defoliated sugar maple. For Sci 18:203–204
Webb WL (1981) Relation of starch content to conifer mortality and
growth loss after defoliation by the Douglas-fir tussock moth. For
Sci 27:224–232
West JB, Espeleta JF, Donovan LA (2004) Fine root production and
turnover across a complex edaphic gradient of a Pinus palustris–
Aristida stricta savanna ecosystem. For Ecol Manag 189:397–
406
308 Mycorrhiza (2007) 17:299–309
Wilcox HE (1991) Morphology and development of ecto- and
ectoendomycorrhizae. In: Schenck NC (ed) Methods and
principles of mycorrhizal research. American Phytopathological
Society, St. Paul, MN, pp 103–113
Wilson CA, Mitchell RJ, Hendricks JJ, Boring LR (1999) Patterns
and controls of ecosystem function in longleaf pine–wiregrass
savannas. II. nitrogen dynamics. Can J For Res 29:752–760
Wilson CA, Mitchell RJ, Boring LR, Hendricks JJ (2002) Soil
nitrogen dynamics in a fire-maintained forest ecosystem: results
over a 3-year burn interval. Soil Biol Biochem 34:679–689
Wu B, Nara K, Hogetsu T (2002) Spatiotemporal transfer of
carbon-14-labelled photosynthate from ectomy corrhizal Pinus
densiflora seedlings to extraradical mycelia. Mycorrhiza 12:83–
88
Mycorrhiza (2007) 17:299–309 309
