Ectomycorrhizal responses to organic and inorganic nitrogen sources when associating with two host species.
ABSTRACT While it is established that increasing atmospheric inorganic nitrogen (N) deposition reduces ectomycorrhizal fungal biomass and shifts the relative abundances of fungal species, little is known about effects of organic N deposition. The effects of organic and inorganic N deposition on ectomycorrhizal fungi may differ because responses to inorganic N deposition may reflect C-limitation. To compare the effects of organic and inorganic N additions on ectomycorrhizal fungi, and to assess whether host species may influence the response of ectomycorrhizal fungi to N additions, we conducted an N addition experiment at a field site in the New Jersey pine barrens. Seedlings of two host species, Quercus velutina (black oak) and Pinus rigida (pitch pine), were planted at the base of randomly-selected mature pitch pine trees. Nitrogen was added as glutamic acid, ammonium, or nitrate at a rate equivalent to 227.5 kg ha(-1) y(-1) for eight weeks, to achieve a total application of 35 kg ha(-1) during the 10-week study period. Organic and inorganic N additions differed in their effects on total ectomycorrhizal root tip abundance across hosts, and these effects differed for individual morphotypes between oak and pine seedlings. Mycorrhizal root tip abundance across hosts was 90 % higher on seedlings receiving organic N compared to seedlings in the control treatment, while abundances were similar among seedlings receiving the inorganic N treatments and seedlings in the control. On oak, 33-83 % of the most-common morphotypes exhibited increased root tip abundances in response to the three forms of N, relative to the control. On pine, 33-66 % of the most-common morphotypes exhibited decreased root tip abundance in response to inorganic N, while responses to organic N were mixed. Plant chemistry and regression analyses suggested that, on oak seedlings, mycorrhizal colonization increased in response to N limitation. In contrast, pine root and shoot N and C contents did not vary in response to any form of N added, and mycorrhizal root tip abundance was not associated with seedling N or C status, indicating that pine received sufficient N. These results suggest that in situ organic and inorganic N additions differentially affect ectomycorrhizal root tip abundance and that ectomycorrhizal fungal responses to N addition may be mediated by host tree species.
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ABSTRACT: Observed modifications of ectomycorrhizal (ECM) communities have been connected to the increased N depositions of the 20th century. Because of their narrow niche width, small disturbances of soil conditions can produce greater effects on the fungal species than on their host trees. This study investigated the ECM community in a black spruce (Picea mariana (Mill.) BSP) stand subjected to long-term additions of 9 and 30 kg N·ha–1·year–1 of ammonium nitrate, representing 3 and 10 times the atmospheric N deposition at the site, respectively. Root tip vitality and ECM presence were detected on samples collected from the organic horizon and ECM were classified into morphotypes according to their morphological and anatomical characters. In the control, 80.6% of the root tips were vital, 76.5% of them showing ECM colonization. Higher root tip vitality and mycorrhization were observed in the treated plots. Forty-one morphotypes were identified, most of them detected at the higher N inputs. Results diverging from the expectations of a reduction in ECM presence and diversity could be related to a higher growth rate of the trees following fertilization. The repeated application of small N doses could have been a better imitation of natural inputs from atmospheric deposition and could have provided more reliable responses of ECM to treatment.Canadian Journal of Forest Research 04/2012; 42(7):1204-1212. · 1.66 Impact Factor
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Ectomycorrhizal responses to organic and inorganic nitrogen
sources when associating with two host species
Meghan L. AVOLIO1, Amy R. TUININGA*, J. D. LEWIS, Michael MARCHESE
Louis Calder Center and Department of Biological Sciences, Fordham University, 53 Whippoorwill Road, Box 887, Armonk, NY 10504, USA
a r t i c l e i n f o
Received 25 February 2009
Received in revised form
8 May 2009
Accepted 12 May 2009
Published online 22 May 2009
John W.G. Cariney
a b s t r a c t
While it is established that increasing atmospheric inorganic nitrogen (N) deposition re-
duces ectomycorrhizal fungal biomass and shifts the relative abundances of fungal spe-
cies, little is known about effects of organic N deposition. The effects of organic and
inorganic N deposition on ectomycorrhizal fungi may differ because responses to inorganic
N deposition may reflect C-limitation. To compare the effects of organic and inorganic N
additions on ectomycorrhizal fungi, and to assess whether host species may influence
the response of ectomycorrhizal fungi to N additions, we conducted an N addition exper-
iment at a field site in the New Jersey pine barrens. Seedlings of two host species, Quercus
velutina (black oak) and Pinus rigida (pitch pine), were planted at the base of randomly-
selected mature pitch pine trees. Nitrogen was added as glutamic acid, ammonium, or ni-
trate at a rate equivalent to 227.5 kg ha?1y?1for eight weeks, to achieve a total application
of 35 kg ha?1during the 10-week study period. Organic and inorganic N additions differed
in their effects on total ectomycorrhizal root tip abundance across hosts, and these effects
differed for individual morphotypes between oak and pine seedlings. Mycorrhizal root tip
abundance across hosts was 90 % higher on seedlings receiving organic N compared to
seedlings in the control treatment, while abundances were similar among seedlings receiv-
ing the inorganic N treatments and seedlings in the control. On oak, 33–83 % of the most-
common morphotypes exhibited increased root tip abundances in response to the three
forms of N, relative to the control. On pine, 33–66 % of the most-common morphotypes ex-
hibited decreased root tip abundance in response to inorganic N, while responses to organic
N were mixed. Plant chemistry and regression analyses suggested that, on oak seedlings,
mycorrhizal colonization increased in response to N limitation. In contrast, pine root
and shoot N and C contents did not vary in response to any form of N added, and mycor-
rhizal root tip abundance was not associated with seedling N or C status, indicating that
pine received sufficient N. These results suggest that in situ organic and inorganic N addi-
tions differentially affect ectomycorrhizal root tip abundance and that ectomycorrhizal
fungal responses to N addition may be mediated by host tree species.
ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ1 914 273 3078; fax: þ1 914 273 2167.
E-mail addresses: email@example.com (M. L. Avolio), firstname.lastname@example.org (A. R. Tuininga)
1Present address: Department of Ecology and Evolutionary Biology, Yale University, P.O. Box 208106, New Haven, CT 06520-8106, USA.
Fax: þ1 203 432 2374.
0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
journal homepage: www.elsevier.com/locate/mycres
mycological research 113 (2009) 897–907
Author's personal copy
Atmospheric nitrogen (N) deposition has increased in many
N-limited temperate ecosystems since the 1940s (Vitousek et
al. 1997). Across ecosystems and among mycorrhizal types
(e.g. arbuscular and ectomycorrhizal), increased inorganic N
deposition reduces the abundance of mycorrhizal fungi mea-
suredaspercentcolonizationof roottips(Treseder2004). Inor-
ganic N deposition also alters fungal community structure by
inducing shifts in the relative abundances of fungal species
(Dighton et al. 2004; Newton & Pigott 1991; Peter et al. 2001;
Wallenda & Kottke 1998; Wiklund et al. 1995) and changes in
the composition of the fungal community (Avis et al. 2003;
Lilleskov et al. 2002a). While the effects of inorganic N deposi-
tion on mycorrhizal fungal communities are well established
(Brandrud 1995; Brandrud & Timmermann 1998; Lilleskov et
al. 2001; Taylor et al. 2000), relatively little is known about
the effects of organic N deposition on these communities.
More research on the potential effects of organic N deposition
is critical because, on average, 30 % of all N deposition is in an
organic form (Neff et al. 2002) and it is unclear whether fungal
responses to organic N deposition parallel responses to inor-
ganic N deposition.
The relative effects of organic and inorganic N deposition
on mycorrhizal communities are difficult to predict in part be-
cause the mechanisms regulating mycorrhizal responses to
suggestthat a reduction in carbon (C) allocation to roots by the
host plant may be a key driver (Buscot et al. 2000; Chalot et al.
2002; Hampp et al. 1999; Ho ¨gberg et al. 2007). Increased N sup-
ply to the plant induces a shift from starch and sugar forma-
tion to energy generation for amino acid and protein
production (Buscot et al. 2000; Hampp et al. 1999). This shift
may result in fewer carbohydrates in the roots, causing the
fungi to starve. If fungal C-limitation is a driver, then ectomy-
corrhizal fungal responses to organic N deposition should be
different than responses to inorganic N deposition, because
organic N can also serve as a C source for the fungi. Alterna-
tively, it has been suggested that fungal responses to inor-
ganic N deposition may be regulated by the soil C:N ratio
(Ho ¨gberg et al. 2007). Increased N availability may reduce the
C:N ratio below a critical level, causing the fungi to become
C limited. If either of these mechanisms affects mycorrhizal
fungal responses to N deposition, responses to organic and in-
organic N deposition should differ because organic N deposi-
tion should maintain a greater soil C:N ratio and because
organic N forms can serve as a C source for fungi.
At least two other factors, though, may influence mycor-
rhizal responses to organic and inorganic N deposition. First,
there may be inherent differences among fungal species in
their responses to N deposition. It has been shown that
some ectomycorrhizal fungal species are less sensitive than
other species to inorganic N deposition (Lilleskov et al.
2001). While many species show a negative response to in-
creased inorganic N deposition, some species show a neutral
or even positive response. Second, the host tree species with
which the fungi are associated may differentially affect
fungal responses. Newton & Pigott (1991) observed that the
response of two species of ectomycorrhizal fungi to inorganic
N additions differed depending on whether they were
associated with an oak or a birch seedling. Few other studies
have addressed the role that host species may play in deter-
mining how mycorrhizal fungi respond to either organic or
inorganic N deposition. As a result, despite the potential im-
portance of host species in regulating fungal responses to N
deposition, little is known about whether the host species in-
fluences the relative response of fungi to organic and inor-
ganic N deposition.
A more fundamental limitation to predicting mycorrhizal
fungal responses to organic N deposition is that, even though
most N in forest soils is in organic forms (Tamm 1991), organic
N is usually overlooked in field studies of mycorrhizal fungi.
Culture studies indicate that some ectomycorrhizal fungi
can grow on organic N forms, including amino acids (Abuzina-
dah & Read 1988; Guidot et al. 2005; Wipf et al. 2002) and pro-
teins (Dickie et al. 1998; Eaton & Ayres 2002; Lilleskov et al.
2002b), as their sole N source. Greenhouse and culture studies
indicate that some ectomycorrhizal fungi can transfer N to
host trees from a range of organicN sources. These sources in-
clude amino acids and proteins (Abuzinadah & Read 1986,
1989; Finlay et al. 1992; Melin & Nilsson 1953; Plassard et al.
2000; Wallenda & Read 1999), pollen (Perez-Moreno & Read
2001), and dead soil arthropods (Klironomos & Hart 2001). Pre-
sumably ectomycorrhizal fungi are using a variety of organic
N forms in situ (Read & Perez-Moreno 2003). However, aside
from Wallenda et al. (2000), who demonstrated that ectomy-
corrhizal fungi took up glutamic acid and transferred the N
to associated host trees in situ, to our knowledge no other
studies have examined mycorrhizal fungal responses to or-
ganic N sources in the field.
In this study, we conducted a field experiment to examine
whether increased organic and inorganic N supply differen-
whether host species plays a role in regulating fungal re-
sponses to organic and inorganic N additions. We hypothe-
sized that: (i) ectomycorrhizal fungi differentially respond to
organic and inorganic N additions in situ, and (ii) ectomycor-
rhizal fungal responses to organic and inorganic N differ
depending on the host seedling species. To test these hypoth-
eses, a field study was conducted to examine ectomycorrhizal
fungal abundance and community composition on two seed-
ling species (black oak, Quercus velutina Lam., and pitch pine,
Pinus rigida Mill.) given organic N (glutamic acid) or inorganic
N (ammonium or nitrate) additions.
Materials and methods
The study took place in the Brendan T. Byrne State Forest
within the pine barrens of New Jersey (39?51.2770N, 74?
30.6940W). The oligotrophic, mor soils are acidic, sandy and
porous, and hold little water (Boyd 1991). They are covered
by thin (5–10 cm) organic horizons (Forman 1998; Tuininga &
Dighton 2004; Wang 1984). Wildfires are a common phenome-
non of the pine barrens (Collins & Anderson 1994; Little 1979),
affecting soil structure and nutrient dynamics. The pine bar-
rens receive low levels of N deposition (w4.2 kg ha?1y?1of
N; Dighton et al. 2004; Wang 1984), and the majority of soil
898 M. L. Avolio et al.
Author's personal copy
nutrients are in organic matter (Wang 1984). There are unde-
tectable levels of soil extractable nitrate in the study area, as
nitrate rapidly leaches out of the very sandy soils (Gray un-
published data; Wang 1984). At our study site, an upland
pine-oak stand, pitch pine trees dominated the tree canopy.
Black oak trees were also a common component of the over-
story, and there is a thick understory of ericaceous shrubs
(Collins & Anderson 1994; Matlack et al. 1993; McCormick
1998). Most of the plant species at this site form mycorrhizal
associations with symbiotic fungi (Dighton et al. 2004;
Tuininga 2000; Tuininga & Dighton 2004). Currently there are
120 described morphotypes of ectomycorrhizas (the morpho-
type includes the fungus and the root) in the New Jersey pine
barrens (Tuininga 2000). In these pine barrens, many ectomy-
corrhizal fungi have been identified as indicators of N deposi-
tion, having reduced abundances along a gradient of
increasing N deposition (Dighton et al. 2004). For the months
this study took place, September through the first week in No-
vember 2004, the pine barrens received greater than average
rainfall, 23.3 cm mo?1(Gray, personal communication). The
long-term average rainfall for the months of September and
October in the pine barrens is 18.4 cm mo?1(Gray, personal
communication, National Weather Service Station 28-6124).
Two bait seedling species, pitch pine and black oak, were used
to assess whether organic and inorganic N additions differen-
tially affect ectomycorrhizal fungi and whether host seedling
species affect the response of ectomycorrhizal fungi to N
addition. Studies have shown that ectomycorrhizal fungal
communities on bait seedlings reflect the overall ectomycor-
rhizal fungal community (Jonsson et al. 1999a; Walker et al.
Pitch pine seeds collected from the field site were germinated
one year after collection. Seeds were soaked in sterile deion-
ized water (diH2O) overnight, then surface sterilized by soak-
ing in 30 % H2O2(Mallinkrodt Baker, Inc., Phillipsburg, NJ,
USA) for 20 min, followed by three rinses in sterile diH2O for
10 min each. Seeds were spread on moist Kimwipes (Kim-
berly-Clark, Dallas, TX, USA) in sterile Petri dishes and incu-
bated (Model I-35LL, Percival Scientific Inc., Perry, IA, USA)
for no longer than 6 weeks in air temperatures maintained
at 25 and 20?C coinciding with a daily cycle of 8 h of light
and 16 h of dark using fluorescent light bulbs on racks located
about 30 cm above the incubating seeds. Seedlings were then
planted in plastic planter flats filled with a 1:2 v/v mixture of
unsterilized peat and sterilized, sieved, sandy pine barrens
soil collected from the A horizon. Seedlings were grown in
flats in the greenhouse at the Louis Calder Center (Armonk,
NY, USA), watered as needed, and exposed to natural light,
until they were planted in the field when they were about
three months old.
Black oak acorns (Sheffield Seed Company, Locke, NY, USA)
were soaked in sterile diH2O overnight, surface sterilized by
soaking in 10 % hypochlorite for 20 min, and washed three
times in sterile diH2O for 15 min each time. Seeds were then
germinated, using the same temperature and light regime as
for the pine seedlings, in 1037 cm3pots containing a 1:1 v/v
mixture of unsterilized peat and sterilized, sieved, sandy soil
collected from the A horizon of the field site. Once the seeds
had germinated, seedlings were moved to the greenhouse,
watered as necessary, and grown in natural light until they
were planted in the field when they were about four months
The experimental design was a 4 ? 2 factorial, with four N
treatments and two host seedling species; there were 8 repli-
cates within each treatment, and 64 seedlings total. Each host
seedling was planted at the base of a mature pitch pine tree.
From an approximately 5 ha stand, 32 mature pitch pines
were selected at random and a meter square plot was ran-
domly located at the base of each tree. On August 26th, 2004,
two seedlings, one pitch pine and one black oak, were planted
half a meter apart in the center of each plot (i.e. 0.5 m from the
mature pitch pine). Due to the heterogeneous nature of the
soils, each seedling was treated as a discrete replicate. Nitro-
gen was added in the form of ammonium (ammonium chlo-
ride), glutamic acid (glutamic acid sodium salt hydrate), or
nitrate (sodium nitrate) over the course of the experiment. Ni-
trogen was added at a rate equivalent to 227.5 kg ha?1y?1for
eight weeks, to achieve a total application of 35 kg ha?1during
the 10-week study period, simulating a high level of deposi-
tion in the United States. Starting 12 d after planting, 0.125 L
of a 0.25 M solution of the appropriate N form was added
weekly using a handheld spray bottle to apply the treatment
evenly over the entire plot. Control plots received 0.125 L of
diH2O added as for the N treatments. Plots were randomly
assigned to an N treatment or the control, with eight replicate
plots for each of the N treatments and the control. Glutamic
acid was chosen as the organic N form because it is commonly
found in sandy loamy soils typical of the study site (Sowden &
Ivarson 1966). The seedlings were harvested the first week of
November, 10 d after the last N addition.
At the harvest, seedlings were carefully removed from the
soil. Roots were separated from shoots and stored in wet pa-
pertowelsat4?C fornolongerthan twoweeks.The entiremy-
corrhizal community on seedling roots was assessed using
standard morphological characters, including color, shape,
branching pattern, and mantle structure (Agerer 1987–2002;
Goodman et al. 1996–2000; Ingleby et al. 1990). These data
were used to determine morphotype richness, abundance,
and diversity, the latter of which was calculated using Simp-
son’s index (Begon et al. 2006).
Growth and seedling chemistry
At the start and the end of the experiment, height of all seed-
lingswas measuredand the number of leavesoneach oakwas
counted. After the harvest, seedlings were separated into
Ectomycorrhizal responses to organic and inorganic nitrogen sources899
Author's personal copy
roots, stems, and leaves. Stems and leaves were immediately
driedat 70?C forone weekwhilerootsweredriedforone week
above). After drying, tissues were weighed to determine dry
mass production. Tissues were ground using either a mortar
and pestle or a Wiley mill (40 mesh) and subsampled to deter-
mine % C and N using a Perkin-Elmer 2400 Series II CHNS/O
analyzer (Perkin-Elmer Life and Analytical Sciences, Inc.,
Wellesley, MA, USA).
?C after mycorrhizal community measurements (see
To investigate the response of the soil mycorrhizal commu-
nity on mature pitch pine trees to N addition, one soil core
was collected within each plot using a soil corer (5 cm diam.
up to 7 cm deep; AMS Inc., American Falls, ID, USA). Soil cores
for analysis of the soil mycorrhizal community were collected
adjacent to the soil cores collected for soil chemistry (see be-
low), and during the same sampling events. The organic hori-
zon of each core was stored in a plastic bag at 4?C for no
longer than three months. The entire mycorrhizal community
within each core was assessed as described above.
To determine whether the N additions affected soil N pools,
two soil cores were collected within each plot using the
same soil corer as above. The first soil core was taken 7 d be-
fore the N additions were initiated and the second was taken
10 d after the N additions were terminated, coinciding with
the seedling harvest. These cores were stored in airtight plas-
tic bags at 4?C for no longer than 48 h. The cores were sieved
through a 4 mm sieve and subsamples of soil from each core
were used to determine ammonium and nitrate concentra-
tions, water content, pH, total N, and total C. Ammonium
and nitrate were extracted from 10 g of soil that was stirred
for 45 min in 50 mL of 1 M KCl (Robertson et al. 1999). Samples
were then vacuum-filtered and immediately frozen for up to
three months at ?20
a TrAAcs 800 auto-analyzer (BranþLuebbe, SPX, Charlotte,
NC, USA) according to the manufacturer’s recommendations.
Ammonium was measured using Berthelot reaction chemis-
try (Searle 1984) and nitrate was measured using hydrazine
sulfate (Kamphake et al. 1967). Gravimetric water content
was determined as described by Brady & Weil (2002). Soil pH
was determined by mixing 2 g of fresh, sieved soil with 5 mL
diH2O for 1 h, after which the pH was measured using a digital
pH meter (Beckman Coulter F295, Fullerton, CA, USA). Total
soil C and N (mg g?1) were determined on 1.5–2.5 mg of soil
that was dried and ground (with a mortar and pestle) prior
to analysis using a Perkin-Elmer 2400 Series II CHNS/O ana-
lyzer (Perkin-Elmer Life and Analytical Sciences, Inc., Welles-
ley, MA, USA).
?C, until they were analyzed using
All statistical analyses, except Chi-square tests and principal
components analyses (PCA), were conducted using SAS soft-
ware (version 9.1.3, 2003, SAS Institute, Cary, NC, USA). All
treatment effects in all tests were considered significant at
a ? 0.1, to take into account variability of environmental fac-
tors in the field. The Shapiro–Wilk statistic was used to assess
normality of the data and Levene’s test was used to examine
the homogeneity of variance of the data. Data were trans-
formed, when necessary, using either a square root (soil core
morphotype data), a log10(gravimetric soil moisture data) or
rank (seedling C and N concentration data, seedling growth
data, seedling C and N content data) transformation in order
tohave normally distributed
The effects of the N treatments over time on gravimetric
soil moisture content, soil pH, soil extractable ammonium
and nitrate, soil C:N, soil core mycorrhizal community proper-
ties, and oak leaf count were tested using repeated-measures
analysis of variance (ANOVA), where N treatment was the be-
tween-subject factor and time and time ? N treatment were
the within-subject factors. All other seedling and mycorrhizal
community data were tested using two-way ANOVAs with
host tree and N treatment as factors. The seedlings within
each plot were treated as independent replicates due to the
distance between seedlings. In cases of significance, Tukey’s
Honestly Significant Difference test was used for pairwise
comparisons of means. Dunnett’s test was used to compare
number of mycorrhizal root tips in the inorganic and organic
N treatments to the control. Separate PCA of the abundances
of ectomycorrhizal morphotypes comprising communities
on the seedlings and on mature tree roots in the soil cores
were performed using PC-Ord (v4, 1999, Gleneden Beach, OR,
USA). For each of the six most-common morphotypes on
a host species, Chi-square tests with Yates correction were
used to examine whether the observed number of root tips
in each N treatment was greater or less than the number of
tips in the control. Only two of the six morphotypes, Ceno
and Copper, were found on both host species. For these two
morphotypes, we additionally used 2 ? 2 Chi-square contin-
gency tables with Yates correction to test whether the re-
sponse to a given N form, relative to the control, differed
between host species. For the Chi-square analyses, we used
the average number of root tips per seedling in a treatment,
rather than the total number of root tips, to compensate for
unequal number of host seedlings in a treatment caused by
seedling death. Multiple regression analysis was performed
separately for each host species using the STEPWISE proce-
dure in SAS to model the relationships betweenectomycorrhi-
zal root tip abundance and seedling dry mass, and C and N
shoot and root concentrations.
data with homogeneous
A total of 15 morphotypes was identified on pine seedlings,
with a mean (?1 S.E.) of 4.93 ? 0.36 morphotypes seedling?1.
A totalof15 morphotypeswasalsoidentifiedonoak seedlings,
with a mean of 3.71 (?0.35) morphotypes seedling?1. Morpho-
type richness was significantly lower on oak seedlings than on
900M. L. Avolio et al.
Author's personal copy
pine (p ¼ 0.0225), but was not observed to differ among N
treatments (p ¼ 0.7120). In contrast, total ectomycorrhizal
root tip abundance did respond to N addition, with seedlings
receiving glutamic acid having 90 % more ectomycorrhizal
root tips than seedlings in the control (p ¼ 0.0360; Fig. 1A). To-
tal abundance of ectomycorrhizal root tips significantly dif-
fered among N treatments (p ¼ 0.0310; Fig. 1B), but not
between host seedling species (p ¼ 0.5222). Treatment effects
on abundance were tested using the total number of ectomy-
corrhizal root tips per seedling rather than the percent of root
tips that were ectomycorrhizal because colonization was
greater than 80 % for all seedlings. Diversity, as measured by
Simpson’s index, was not observed to differ among N treat-
ments (p ¼ 0.7518) or between host seedling species
(p ¼ 0.2228). The effect of N treatment on ectomycorrhizal
abundance, diversity, or richness for all morphotypes com-
bined was not observed to differ between host species (data
In contrast, individual morphotype responsesto N addition
varied by host species. The direction of the responses of the
six most-common morphotypes on oak to the N treatments
generally was positive compared to negative responses of in-
dividual morphotypes on pine (Table 1). On oak, both nitrate
and glutamic acid additions were associated with significant
increases, relative to the control, in the abundances of five
of the six most-common morphotypes, while ammonium ad-
dition was associated with increases in abundance of two of
the six most-common morphotypes. On pine seedlings, how-
ever, eight of the ten significant responses to N addition were
negative. Ammonium and nitrate addition were associated
with reductions in abundance of four and two, respectively,
of thesix most-common morphotypes. With glutamicacidad-
dition, two morphotypes increased in abundance and two de-
creased. The differences between host species in the patterns
of response of individual morphotypes to nitrate, ammonium
and glutamic acid were qualitatively similar to the responses
of total mycorrhizal root tip abundance (Fig. 1B). However,
for total mycorrhizal root tip abundance, variation within
each treatment combination was large enough that the effect
of N addition did not significantly differ between host species.
Similarly, PCA analysis did not reveal any clear separation of
fungal groups by N treatment within a host seedling species
or across host species (analyses not shown).
Host species effects on response of two morphotypes to
Two fungal morphotypes, Ceno and Copper, were associated
with both host seedling species. Both were clearly distinct in
all morphological characteristics (see Appendix) across host
species and were more negatively affected by N addition
when associated with pine seedlings than when associated
with oak (Table 1). Tests of independence, from the 2 ? 2
Chi-square contingency table analyses, demonstrated that
these two morphotypes had different responses to one form
of N added, nitrate. When nitrate was added, the response
of Ceno differed between host species, but the response of
Copper was independent of host species. Nitrate addition on
oak was associated with 98 % more Ceno root tips relative to
the control, but on pine was associated with 75 % fewer
Ceno root tips relative to the control. Though the two morpho-
types differed between hosts in their response to nitrate, they
had similar responses to glutamic acid and to ammonium.
Glutamic acid addition was associated with five-fold more
Copper root tips and 79 % more Ceno root tips on oak relative
to the control, but on pine Copper and Ceno had 88 and 48 %
fewer tips, respectively, relative to the control. Neither mor-
photype responded to the ammonium treatments on both
host tree species.
Growth and chemistry
Oak seedlings were significantly larger than pine, on average,
for all measured growth variables (data not shown). Across
species, growth and biomass allocation did not significantly
vary among treatments (Table 2). However, leaf senescence
Fig 1 – Number of ectomycorrhizal tips per seedling
(mean ± 1 S.E.) by nitrogen treatment. There were more
tips in the organic N treatment compared the control
(A; Dunnett’s test, p [ 0.0360) while there was no difference
between the inorganic treatment and the control (Dunnett’s
test, p [ 0.7890). The number of mycorrhizal tips was
affected by the N treatments (p [ 0.0310; B), there was no
interaction between host seedlings (p [ 0.5222). Different
letters indicate means that are significantly different at
p < 0.1 based on Tukey’s HSD test for pairwise comparisons
(For oak seedlings n [ 8 for all treatments, for pine seed-
lings n [ 6 for nitrate and control, n [ 7 for glutamic acid,
and n [ 8 for ammonium).
Ectomycorrhizal responses to organic and inorganic nitrogen sources901
Author's personal copy
(p ¼ 0.0509). Oak seedlings in the control shed the fewest
leaves, 1.38 ? 0.73 leaves seedling?1, while those in the nitrate
treatment shed the most leaves, 3.38 ? 0.25 leaves seedling?1.
Across host species, shoot C:N ratio and N concentration,
whole plant dry mass and C:N ratio significantly varied among
N treatments (Table 2). Seedlings receiving ammonium had
significantly greater shoot N concentrations, and significantly
lower shoot and whole plant C:N ratios compared to those re-
ceiving nitrate. In addition, seedlings receiving ammonium
had significantly lower whole plant dry mass compared to
those receiving nitrate or glutamic acid. Shoot dry mass and
C concentration,root C:N ratio and N concentration,and root:-
shoot ratio were not observed to vary among N treatments
(Table 2). Similarly, shoot C and N content were not observed
to vary among N treatments (data not shown). The responseof
whole plant C content to N additionfollowed the same pattern
as the response of whole plant dry mass (data not shown).
There were significant interactions between host seedling
species and N treatment for root dry mass and C concentra-
tion (Table 2). For each interaction there were significant dif-
ferences between the N additions for the oak seedlings but
not for the pine. Oak seedlings receiving glutamic acid and ni-
trate had roughly two-fold greater root dry mass than seed-
lings receiving ammonium, while seedlings in the control
were not observed to vary from seedlings in the other N treat-
ments. Oak seedlings receiving glutamic acid had greater root
C concentration than oak seedlings in the ammonium and
control treatments. The interactive effects of host species
and N addition on root C and N content generally paralleled
the interactive effects on root dry mass (data not shown).
Table 1 – The average number of root tips from individual morphotypes on the oak and pine seedlings separated by
nitrogen treatment followed by a dash (-) and then the Chi-square statistic for each comparison. Chi-square tests were
performed for each morphotype by N treatment, using the average number of tips found in the control as the expected
value. Chi-square values that are significant at p ? 0.1 are bolded. An up- or a down-arrow signals the direction of the
difference between the observed number of tips versus the control. Morphotype abbreviations are explained in Appendix
Brn – c.p.
Brn/wh – b.
Orange – c.
Wht – m.p.
50.25 – 27.6 [
8.00 – 210 [
67.13 – 31.7 [
3.25 – 0.40
6.50 – 18.1 [
40.75 – 5.10 [
24.63 – 0.00
11.25 – 441 [
27.13 – 1.55
1.88 – 0.00
0.25 – 0.2
41.75 – 5.98 [
46.63 – 20.4 [
42.63 – 7016 [
60.63 – 20.3 [
9.38 – 26.1 [
0.00 – 2.45
89.38 – 130 [
Brn – Russ
Brn/Wh – d.b.
Brn/wh – d.b.-br.
1.00 – 0.28
10.83 – 13.8 Y
9.16 – 0.78
5.50 – 11.8Y
5.00 – 3.81Y
54.83 – 3.26Y
1.50 – 0.04
30.63 – 0.06
9.25 – 0.74
14.88 – 2.08
0.38 – 9.97Y
47.13 – 6.97Y
0.86 – 0.38
50.00 – 8.89 [
18.14 – 1.80
11.42 – 4.74 Y
1.42 – 8.15 Y
85.57 – 3.04 [
Table 2 – Results of effects of nitrogen treatment on seedlings, separated by species when there was a significant
interaction between host seedling and nitrogen treatment, for dry mass (g), C:N ratios, C and N concentration (mg gL1),
separated by shoots (Sht), roots (Rt), and whole plant (WP), presented as means (±1 S.E.). Effects significant at p ? 0.1 are in
bold, * indicates there was a significant interaction between host tree species and N treatment, different letters indicate
means are significantly different at p ? 0.1 based on Tukey’s HSD by treatment, and where applicable by treatment and
Sht Dry Mass
Sht N Conc.
Sht C Conc.
Rt Dry Mass*
Rt N Conc.
Rt C Conc.*
Rt:Sht Dry Mass
WP Dry Mass
902M. L. Avolio et al.
Author's personal copy
Multiple regression analyses were conducted to examine
relationships between total ectomycorrhizal root tip abun-
dance and seedling mass and chemistry for each host species.
On oak seedlings, the number of ectomycorrhizal root tips in-
creased with seedling dry mass (Fig. 2A), but decreased with
shoot N concentration (Fig. 2B), which together explained
(p < 0.0001). Similarly, on pine seedlings, number of root tips
increased with seedling dry mass (Fig. 2C), which explained
50 % of the variation in the number of root tips (p < 0.0001).
thenumber of root tips
Extractable ammonium increased by 12 %, on average, over the
79 %, and 83 % in plots receiving ammonium, nitrate, or diH2O,
respectively. Soil total C and total N concentrations did not sig-
nificantly vary among treatments (p ¼ 0.3834 and p ¼ 0.2164 re-
spectively). As with soil N concentrations, soil pH did not
significantly vary among treatments (p ¼ 0.1352; Table 3), but
both significantly declined over time (p < 0.0001 and
p ¼ 0.0108 respectively). Across treatments, mean (?1 S.E.) soil
N concentration decreased from 5.73 ? 0.43 mg N g?1soil in
August to 3.01 ? 0.25 mg N g?1soil in November, while [Hþ]
decreased from 1.92 ? 10?4? 1.9 ? 10?5(mol/L) in August to
1.3 ? 10?4? 1.5 ? 10?5(mol/L) in November. Gravimetric water
content did not significantly vary among treatments over time
(p¼ 0.1611).Extractablenitrate wasonlydetectable infive sam-
(ppm; 1 ppm is the detection limit of the methods described
above), which likely reflects the sandy soils of the pine barrens,
from which nitrate readily leaches out.
types of ectomycorrhizas were identified in the soil cores
taken adjacent to mature pitch pine trees. Mean richness
declined 18.3 % over the study period (p ¼ 0.0372), from
4.09 ? 0.231 morphotypes soil core?1in August to 3.34 ? 0.252
morphotypes soil core?1in November. The magnitude of the
decline did notsignificantly
(p ¼ 0.7751). The abundance (p ¼ 0.7098) and diversity
(p ¼ 0.5129) of ectomycorrhizal morphotypes in the soil cores
did not significantly vary among treatments over time. PCA
analysis did not reveal any clear separation by N treatment of
morphotypes associated with mature roots (data not shown).
Organic N deposition accounts for about a third of all N depo-
sition (Neff et al. 2002), and most of the N in forest soils is or-
ganic (Tamm 1991), yet most field research on the effects of N
deposition on ectomycorrhizal fungal communities has
Fig 2 – Regressions between abundance of mycorrhizal root tips and host seedling chemistry, from the multiple regression
analysis. On oaks seedlings (n [ 32) mycorrhizal abundance is affected by (A) whole seedling dry mass (r2[ 0.383,
p [ 0.0010) and (B) seedling shoot N concentration (r2[ 0.085, p [ 0.0402). On pine seedlings (n [ 27) mycorrhizal abun-
dance is affected by (C) whole seedling dry mass only (r2[ 0.5002, p < 0.0001).
Ectomycorrhizal responses to organic and inorganic nitrogen sources 903
Author's personal copy
focused on inorganic forms of N. Consistent with our hypoth-
eses, organic and inorganic N additions differentially affected
the ectomycorrhizal fungal community. Ectomycorrhizal root
tips were nearly twice as abundant on seedlings receiving glu-
tamic acid compared to seedlings receiving deionized water,
while seedlings receiving inorganic N did not differ from those
receiving deionized water. Although N addition significantly
altered total mycorrhizal abundance, it was not associated
with significant changes in the composition of the ectomycor-
rhizal fungal community, either on the seedlings or in the soil
cores taken from under mature pitch pine. These results are
consistent with the results of other studies on the effects of
disturbances on ectomycorrhizal fungal communities, in
that disturbances generally have to be very large (Dahlberg
et al. 2001) or continue for many years (at least one to two de-
cades) before changes in species composition occur (Avis et al.
2003; Ka ˚re ´n & Nylund 1997; Peter et al. 2001; Tuininga 2000;
Visser 1995). Shorter-term disturbances typically are associ-
ated with changes in relative abundance of species rather
than changes in composition (Jonsson et al. 1999b; Tuininga
2000; Tuininga & Dighton 2004), as was seen in our study.
The increased abundance of mycorrhizal root tips on field-
grown seedlings in response to amino acid additions is a new
finding, but parallels the results of studies conducted under
controlled conditions. In a greenhouse experiment, Holopai-
nen & Heinonen-Tanski (1993) demonstrated that the num-
bers of mycorrhizal root tips increased with additions of
urea formaldehyde to Pinus sylvestris seedlings grown in
pots. Other greenhouse studies have observed an increase in
the number of mycorrhizal root tips when grown with other
organic N sources such as squirrel fecal pellets (Lilleskov &
Bruns 2003) and humus (Slankis 1974). Taken together, these
results suggest that organic N addition may increase ectomy-
corrhizal fungal abundance under at least some conditions in
the field, in contrast to inorganic N addition, which has been
observed to reduce the abundance of many ectomycorrhizal
fungal species (Brandrud 1995; Brandrud & Timmermann
1998; Lilleskov et al. 2001; Taylor et al. 2000).
their number of root tips to N addition differed between host
species. Oak mycorrhizas that responded to N addition
responded with increased abundance, whereas nearly all
pine mycorrhizal responses to N additions were negative.
More specifically, for the two morphotypes found on both
host species, the responses to N addition were affected by the
host species with which they were associated. Both morpho-
types, Ceno and Copper, were more negatively affected by
responses to inorganic N additions differed between host spe-
cies (Baum & Makeschin 2000; Newton & Pigott 1991). There is
atively affected by N additions than hardwood stands (Magill
et al. 2004), and ectomycorrhizas are more resilient to N depo-
sition in hardwood than in conifer stands (Taylor et al. 2000).
The differential effects of oak and pine on the response
patterns of morphotypes to N addition may reflect differential
responses of host plant species to the form of N added. Root
dry mass of pine seedlings did not respond to N treatments,
suggesting that the pine seedlings were not N limited, which
may account for the lack of a relationship between mycorrhi-
zal root tip abundance and shoot N concentration on pine
negative relationship between mycorrhizal root tip abun-
dance and shoot N concentration, and there were significant
differences in root dry mass among N treatments, suggesting
that mycorrhizal root tip abundance on oak seedlings was af-
fected by seedling responses to N addition. Ammonium-
treated plants, which had the greatest shoot N concentration,
had the least root dry mass, and just 33 % of the six most-com-
mon morphotypes exhibited increased root tip abundance rel-
ative to the control. In contrast, nitrate-treated plants had the
least shoot N concentration and the most root dry mass, and
exhibited an overall pattern of increased number of root tips
by most of the associated mycorrhizal fungi, suggesting the
nitrate-treated seedlings were N limited. The results of gluta-
mic acid addition were less straightforward, perhaps due to
the nature of this form of N. Shoot N concentration in the glu-
tamic acid treatment did not differ from seedlings in the other
treatments. Therefore, it appears that the plants that were
treated with glutamic acid were not N limited, but most of
the associated morphotypes exhibited increased number of
mycorrhizal tips relative to the control. This could result
from glutamic acid serving as an alternate source of C, sup-
porting more mycorrhizal fungal growth.
Table 3 – Soil chemistry and soil moisture from before to after the nitrogen additions in the field experiment, presented as
means (±1 S.E.)
Soil N (mg N g soil?1)
Before nitrogen additions
After nitrogen additions
Soil pH ([Hþ])
Before nitrogen additions
After nitrogen additions
Soil Moisture (g H2O g soil?1)
Before nitrogen additions
After nitrogen additions
Soil Extractable NH4
(mg NH4-N g soil?1)
Before nitrogen additions
After nitrogen additions
2.26 ? 10?4(4.8 ? 10?5)
1.61 ? 10?4(2.5 ? 10?5)
1.93 ? 10?4(3.9 ? 10?5)
8.96 ? 10?5(1.8 ? 10?5)
1.99 ? 10?4(3.9 ? 10?5)
3.80 ? 10?5(3.8 ? 10?5)
1.50 ? 10?4(1.9 ? 10?5)
2.41 ? 10?5(2.4 ? 10?5)
904M. L. Avolio et al.
Author's personal copy
Amino acids can be used as C sources by some ectomycor-
rhizal fungi (Chalot et al. 1994; Chalot & Brun 1998). This alter-
nate source of C may partially account for the increases in
morphotype abundances relative to the control observed for
five of six morphotypes on oak with glutamic acid additions.
Even two of six pine morphotypes responded positively to glu-
tamic acid addition, while all of the responses of these six my-
corrhizas to nitrate and ammonium addition were neutral or
negative. Thus, our results provide some support for the hy-
pothesis that C-limitation may be a key driver regulating the
response of ectomycorrhizal fungi to N addition (Buscot et al.
2000; Chalot et al. 2002; Hampp et al. 1999; Ho ¨gberg et al.
2007), as our results suggest that at least some morphotypes
are not negatively affected by N addition when they have their
own C supply separate from that supplied by the host.
An alternative hypothesis that has been proposed to ac-
count for ectomycorrhizal fungal responses to inorganic N de-
position is that the association is regulated by the soil C:N
ratio (Ho ¨gberg et al. 2007). As with the study by Ho ¨gberg et
al.(2007), our results were not consistent with this hypothesis.
Although fungal abundance significantly varied among N
treatments, soil C and N concentrations did not significantly
vary among N treatments. Further, our results indicate that
the response of two ectomycorrhizal fungal morphotypes to
N addition was not uniform across host species, suggesting
that the host seedling had a greater effect than soil character-
istics on the ectomycorrhizal fungal community. Accordingly,
our results suggest that changes in fungal communities in re-
sponse to N addition are a function of interactions between
the host plant and the ectomycorrhizal fungal community.
In summary, organic N addition resulted in significantly
more ectomycorrhizal root tips relative to the control, while
the number of ectomycorrhizal root tips in the inorganic N
treatments did not differ from the control. Further, relative
to the control, all the significant responses of the six most-
common morphotypes to N additions on oak seedlings were
positive, while on pine seedlings eight of ten of the significant
responses of morphotypes to N additions were negative. For
two morphotypes associated with both host species, Ceno
and Copper, we found neither form of N added nor associated
host species determined the response of the morphotypes rel-
ative to the control. Taken together, our results demonstrate
that there is no one overriding factor that determines the re-
sponse of mycorrhizal morphotypes to N additions, but is in-
stead a combination of factors. Thus, the effects of organic
N deposition on ectomycorrhizal abundance cannot be pre-
dicted from studies on inorganic N, and fungal responses to
N form when associated with a particular host species may
not predict responses when associated with other host spe-
cies. In the future, individual mechanisms will need to be
tested further under field conditions.
This research was funded in part by the New Jersey Depart-
ment of Environmental Protection and by the Cooperative
State Research, Education and Extension Service, U.S. Depart-
ment of Agriculture under Award 2003-35107-13775. We thank
the New Jersey Forest Service for access to the field site. We
also thank Rebecca Huskins, Dr. Abby Sirulnik, and Alissa
Perrone for help in the field and laboratory. Lastly, we thank
Drs. Erik Lilleskov, Robert Ross, Andy Taylor, John Wehr,
and two anonymous reviewers whose comments greatly
improved this manuscript. This is Contribution #242 from
the Louis Calder Center and Biological Station, Fordham
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.mycres.2009.05.001.
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