Tree Physiology 20, 599–606
© 2000 Heron Publishing—Victoria, Canada
Effects of continuous optimal fertilization on belowground
ectomycorrhizal community structure in a Norway spruce forest
PETRA M. A. FRANSSON, ANDY F. S. TAYLOR and ROGER D. FINLAY
Department of Forest Mycology and Pathology, SLU, Box 7026, S-75007 Uppsala, Sweden
Received June 21, 1999
Summary Studies of effects of fertilizer treatment on ectomycorrhizal fungal community structure have predominantly
been based on large, single additions of nitrogen. Studies involving chronic additions of nutrients in combination with irrigation are much less common. We used morphotyping to study
effects of balanced additions of a nutrient solution on ectomycorrhizal fungal community structure in a 36-year-old stand
of Picea abies (L.) Karst. Despite high variability among individual samples, principal components analysis revealed a clear
shift in community structure in response to fertilization. Irrigated plots receiving only water did not differ significantly
from untreated control plots. Mycorrhizal root tips colonized
by Cenococcum geophilum Fr. were significantly more common in fertilized plots than in control plots. Possible responses
by other ectomycorrhizal species were masked by high variability. Over sixty morphotypes were distinguished, but there
was no measurable effect of either fertilizer or irrigation treatment on morphotype richness or total number of root tips.
Keywords: biodiversity, fungi, mycorrhiza, Picea abies.
Introduction
In Northern Europe, forest fertilization is a well-established
practice for increasing tree yield (Lundmark 1986, Tamm
1991). In addition to increased tree biomass production, fertilizer additions can have significant effects on other biota within
the forest ecosystem (Kellner 1993, Arnebrant et al. 1996,
Boxman et al. 1998, Nohrstedt 1998). In particular, ectomycorrhizal (ECM) fungi, which are obligate root symbionts
of most boreal forest trees, are sensitive to changes in soil nutrient status (see Wallenda and Kottke 1998). The mycelia of
ECM fungi extend from tree roots into the soil and are directly
involved in mobilization and uptake of soil nutrients, a proportion of which is then passed on to the host tree (Smith and
Read 1997). Changes in soil nutrients, particularly nitrogen,
can markedly affect the production of extramatrical mycelium
that can, in theory, alter nutrient availability to the host
(Wallander and Nylund 1991, 1992).
Numerous investigations of the effects of forest fertilization
on ECM fungi have been conducted based on sporocarp abundance as a measure of community response to the applied
treatments (Kuyper and de Vries 1990, Rühling and Tyler
1991, Wiklund et al. 1994, Brandrud 1995). However, recent
investigations indicate that sporocarp inventories of ECM
fungi correlate poorly with community structure on mycorrhizal root tips (Gardes and Bruns 1996, Kårén and Nylund
1996, Dahlberg et al. 1996). This is partly because a proportion of the ECM community on root tips is commonly formed
by species that either do not produce sporocarps or form inconspicuous reproductive structures (Taylor and Alexander
1989, Dahlberg et al. 1996, Kårén and Nylund 1997, Brandrud
and Timmermann 1998). Few investigations of ECM fungal
communities have directly examined ectomycorrhizal root
tips (Kårén and Nylund 1996, Jonsson et al. 1999b).
The last 15 years has witnessed a revolution in our ability to
distinguish the ECM fungi colonizing roots. Morphological
characterization (Agerer 1986–1998) and molecular identification by PCR-RFLP analysis of the internal transcribed
spacer (ITS) region (Gardes and Bruns 1993) are the preferred
methods. Morphological characterization (here referred to as
morphotyping) has evolved from a coarse recognition tool
with a few ill-defined morphotypes or groups of morphotypes
distinguished (e.g., Ahlström et al. 1988, Ohtonen et al. 1990,
Arnebrant and Söderström 1992), to a reproducible technique
that can differentiate among mycorrhizal fungi on root tips at
the species or genus level (Brand 1991, Lilleskov and Fahey
1996, Goldack et al. 1998, Wöllecke et al. 1998). The main
disadvantages of morphotyping are that, to date, the number of
published descriptions of ectomycorrhizas formed by known
species of fungi is limited and it is not always possible to distinguish among some groups of closely related fungi (Müller
and Agerer 1990). However in some groups of fungi, such as
the genus Cortinarius, the latter disadvantage also applies to
the most commonly used molecular identification method of
PCR-RFLP analysis of the ITS-region of the fungal rDNA
(Kårén et al. 1997). The advantage of morphotyping over molecular identification is that it is relatively inexpensive and
large numbers of root tips can be assessed.
Studies of the response of mycorrhizal fungi to forest fertilization have generally involved single or multiple applications
of solid N-fertilizer (Alexander and Fairley 1983, Kuyper and
de Vries 1990, Rühling and Tyler 1991, Arnebrant and Söderström 1992). Some field studies have included continuous applications of liquid fertilizer in the irrigation water (Arnebrant
600
FRANSSON, TAYLOR AND FINLAY
and Söderström 1992, Wiklund et al. 1994). Such chronic additions (low amounts applied over an extended period of time)
should give a more even dose and reduce nutrient leaching,
which has been observed after both a single dose application
(150 kg N ha –1) of solid fertilizer (Westling and Hultberg
1990/91) and multiple applications (30–120 kg N ha –1 year –1)
of fertilizer (Berdén et al. 1997). Chronic additions should also
reduce the risk of possible toxic effects on soil biota (Tamm
1991).
At the Flakaliden research site in northern Sweden, a nutrient optimization experiment involving chronic additions of
balanced nutrients to a young Norway spruce (Picea abies (L.)
Karst.) stand has been running since 1987 (Linder 1995). The
purpose of the Flakaliden experiment is to demonstrate the potential yield of Norway spruce, by optimizing the nutritional
status of the stand while simultaneously minimizing losses of
nutrients to groundwater by leaching. We investigated the impact of these balanced nutrient additions on the ECM fungal
community by detailed morphotyping of the ECM root tips.
No measurable effect of the balanced nutrient addition (referred to as liquid fertilizer treatment) was found on morphotype richness or on the total number of root tips sampled in the
plots. However, there was a shift in community structure associated with the liquid fertilizer treatment, compared with the
control and irrigation treatments.
Materials and methods
Study site
The study site at Flakaliden, northern Sweden (64°07′ N;
19°27′ E; altitude 310 m a.s.l.), was planted with Norway
spruce after clear felling in 1963. A balanced nutrient solution
(macro- and micro-) is supplied daily throughout the growing
season (June to mid-August). The nutrient solution was initially based on the recommendations of Ingestad (1967, 1979),
and subsequently adjusted annually on the basis of the nutrient
status of the trees and the soil. During the first ten years of the
experiment, the liquid fertilization treatment included a total
of 825 kg N ha –1 added as both ammonium and nitrate. This
treatment, subsequently referred to as the fertilizer treatment,
was compared with control plots receiving no treatment and an
irrigation treatment receiving water without added nutrients.
features (Agerer 1986–1998). The total abundance (total number of mycorrhizal root tips per core) and relative abundance
(number of each morphotype per total no. of living mycorrhizal root tips) of each mycorrhizal type were recorded.
Non-mycorrhizal root tips were also recorded. Unidentified
morphotypes were given numbers as they occurred in the examination.
To investigate the vertical distribution of mycorrhizal types
between soil horizons, an additional 45 cores were sampled
from one control and one fertilizer plot. Mycorrhizal and
non-mycorrhizal root tips from the organic and mineral layers
were processed separately as described above.
Statistical analysis
Treatment effects on morphotype richness, total root tip numbers and individual morphotype abundance (both relative and
total) were analyzed by one-way analysis of variance
(ANOVA).
To investigate treatment effects on overall community
structure, a principal components analysis (PCA) was carried
out on the total numbers of mycorrhizal root tips per
morphotype, the total numbers of morphotypes with a relative
abundance above 5% (23 morphotypes) and on identified
morphotypes only (18 morphotypes). Community diversity
was evaluated by several methods. Rank-abundance graphs
were constructed with log abundance (log10 no. tips colonized)
plotted against ranked morphotype abundance (Tokeshi
1993). Within- and between-treatment sample similarities
were calculated by the Bray-Curtis similarity index (Equation
1) (Krebs 1989), based on the numbers of mycorrhizal tips in
each of the morphotype categories for each replicate plot.
Overall community diversity was measured by the Shannon-Wiener diversity index (Equation 2) (Krebs 1989), and
the Berger-Parker index (Equation 3) was used to assess evenness (Magurran 1988). For the Bray-Curtis and Berger-Parker
indices, complement values were used to achieve a positive relationship between similarity/evenness and the value of the indices.
Bray-Curtis similarity index:
n
 n

1 −  ∑ [ X ij − X ik ] / ∑ [ X ij + X ik ] ,
 i=1

i=1
(1)
Sampling and identification of ectomycorrhizal fungi
Samples were taken from three replicate plots within each
treatment. Within each replicate plot, five soil cores (2.8-cm
diameter) were taken from the organic soil layer beneath each
of six randomly chosen trees. Thirty soil cores were thus taken
from each of nine plots, giving a total of 270 soil cores. Soil
samples were placed in separate plastic bags and maintained at
4 °C, until examined. The depth of the organic layer in each
core was measured before soaking the sample in water for
30 min. Living root tips were then extracted by wet sieving
through a combination of 1.0 mm and 500 µm sieves.
Root tips were examined and classified into mycorrhizal
morphotypes on the basis of macroscopic and microscopic
where Xij and Xik = number of individuals in morphotype i in
samples j and k, respectively, and n = number of morphotypes
in each sample.
Shannon-Wiener index:
n
− ∑ pi ln pi ,
(2)
i=1
where pi = proportion of individuals found in the ith
morphotype.
TREE PHYSIOLOGY VOLUME 20, 2000
FERTILIZATION AND ECTOMYCORRHIZAL COMMUNITY STRUCTURE
Berger-Parker evenness measure:
1 − ( N max/ N ),
(3)
where Nmax = number of individuals in the most abundant
morphotype, and N = total number of individuals recorded.
Effects of soil fraction, organic versus mineral, and treatment on morphotype distribution were tested with a model
based on the binomial distribution and the Glimmix macro for
SAS (SAS Institute Inc., Cary, NC).
Results
Fertilizer effects and fungal community composition
The PCA indicated an altered mycorrhizal community structure in response to fertilization treatment (Figure 1), and the
results were similar for all three categories of morphotypes
tested. The community structure in control and irrigation treatment plots was similar, with the plots forming a single tight
group suggesting little effect of irrigation. The first axis
(PCA1) of the PCA accounted for nearly 60% of the variation,
whereas an additional 17% of the variation was explained by
axis 2 (PCA2). Despite the fertilizer-induced alteration in
mycorrhizal community structure, there were no measurable
treatment effects on morphotype richness (P = 0.369) or total
number of root tips in samples (P = 0.589).
High variability within the data effectively masked possible
individual responses to treatments by many of the morphotypes. Only one morphotype showed a significant response to
the treatments; Cenococcum geophilum Fr. mycorrhizas were
more abundant on fertilized plots than on control plots (P =
0.017). Several morphotypes showed a tendency to become
more common with fertilizer treatment. Tylospora fibrillosa
(Burt.) Donk and Amphinema byssoides (Pers.:Fr) Erikss.
both showed an increased abundance in the fertilized plots.
Tomentelloid mycorrhizas were found on all three fertilized
plots, but only on one each of the other two treatment plots. In
Figure 1. First and second axes of a Principal Components Analysis of
the ectomycorrhizal fungal communities in Norway spruce plots receiving either irrigation (IR), nutrient solution (F) or no treatment (C).
601
contrast, Piloderma byssinum (Karst.) Jül. and P. croceum
Erikss. & Hjortst. showed a tendency to become less common
with fertilization. Piloderma morphotypes were classified into
three groups (P. byssinum, P. croceum and P. sp. 1).
Piloderma sp. 1 had a distinctive appearance with a large
amount of emanating hyphae and no distinct rhizomorphs as
often seen in P. byssinum and P. croceum. Cenococcum
geophilum, P. byssinum, P. croceum and T. fibrillosa occurred
in all plots. Cortinarius spp. were found in all control plots,
but not in all irrigated and fertilized plots. Cortinarius spp.,
Inocybe spp., Lactarius spp., Russula spp. and tomentelloid
types each consisted of several different types and are therefore listed as groups in Table 1.
All of the investigated plots showed a high morphotype
richness and high variability in morphotype composition. The
number of morphotypes found in the different plots varied between 13 and 22. The total number of morphotypes found was
68; 18 were identified with previously described morphotypes
but 50 morphotypes did not correspond with any previously
published descriptions. Unidentified morphotypes were assigned numbers as they occurred during processing of the
roots but some morphotypes were difficult to distinguish and it
is possible that the number of unidentified morphotypes was
overestimated. Relative abundances of Norway spruce root
tips colonized by each ectomycorrhizal morphotype are listed
for each plot in Table 1. An average of 67% of the community
in each plot consisted of root tips colonized by taxa producing
resupinate fruit bodies (e.g., Amphinema, Piloderma and
Tylospora) or species that do not produce sporocarps (Cenococcum). The frequency of root colonization was high. In six
plots, 100% of the examined root tips were colonized by
mycorrhizal fungi, whereas in the other three plots over 93%
of the root tips were colonized.
Diversity
Rank abundance curves (Figure 2) for the treatments revealed
a close similarity in species abundance distributions between
the control and fertilized plots. However, some morphotypes
with intermediate abundance were missing from the irrigated
plots. The Berger-Parker evenness index indicated that the
ECM communities in the fertilized plots had a tendency (P =
0.073) to become less even compared with the other two treatments (Table 2). Analysis of variance of the Shannon-Wiener
indices showed no significant differences between treatments
with respect to diversity. The degree of similarity in community composition among the three replicate plots within each
treatment, as measured by the Bray-Curtis index, did not differ. However, the degree of similarity between control plots
and irrigation plots was significantly greater (P < 0.0001) than
that between control and fertilized plots or irrigation and fertilized plots. This finding reinforces the shift in community
structure in the fertilized plots suggested by the PCA.
Vertical distribution of morphotypes
The binomial model used to test whether different morphotypes were distributed unequally between the organic and
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
602
FRANSSON, TAYLOR AND FINLAY
Table 1. Relative abundance of ectomycorrhizal morphotypes, expressed as the percentage of root tips examined, which were sampled from Norway spruce plots receiving no treatment, irrigation or nutrient solution. Abbreviation: SE = standard error.
Replicate plot no.
Control
1
Amphinema sp.
Cenococcum
Cortinarius spp.
Dermocybe sp.
Hydnellum sp.
(cf) Inocybe spp.
Lactarius spp.
L. (cf) rufus
L. deterrimus
Piceirhiza gelatinosa
P. rosa-nigrescens
Piloderma sp. 1
P. byssinum
P. croceum
Russula spp.
Tomentelloid
Tuber sp.
Tylospora sp.
Unknown 1-50
Non-mycorrhizal tips
Totals (no. tips)
Extent of colonization (%)
No. types
No. tips/100 cm –3 ± SE
Irrigation
2
3.2
11.9
8.7
3
5.5
13.8
6.6
1
0.5
10.6
4.0
1.1
21.0
11.4
4.0
0.6
Fertilization
2
3
1
2
17.2
3.1
15.7
6.3
39.5
3
17.7
30.0
1.1
1.6
15.2
0.6
1.8
7.1
1.3
0.6
3.5
2.0
0.4
10.5
29.4
25.4
3.9
2.0
21.0
27.6
3.9
2.8
15.8
13.8
6.8
6.3
9.1
28.4
0.9
26.2
22.5
0.2
4.4
7.6
25.5
24.2
9.1
0.2
2.0
12.3
4.0
6.0
1.3
2.4
2.2
1.3
1.3
6.3
11.9
20.7
4.1
3.1
6.6
2.5
0.8
0.6
0.5
0.6
2.8
1.1
4.7
44.4
13.5
14.4
41.5
10.8
20.0
16.2
14.3
13.1
6.0
5.1
0.2
6.4
126
181
549
176
325
451
301
463
513
100
100
94.0
94.9
100
100
100
100
93.6
13
15
20
18
13
14
15
22
20
34.7 ± 9.9 74.8 ± 27.0 140.3 ± 49.2 32.3 ± 8.7 53.7 ± 15.7 87.3 ± 18.9 44.8 ± 13.3 99.4 ± 31.5 96.8 ± 23.4
mineral soil layers (vertical distribution) is only applicable to
morphotypes occurring in more than 10 cores out of 45. In the
present study only three morphotypes fulfilled this criterion
(C. geophilum, T. fibrillosa and Piloderma sp.). The model
showed that significantly more C. geophilum mycorrhizal root
tips were associated with the organic layer than with the mineral layer (P = 0.04). In contrast, significantly more T. fibrillosa mycorrhizal root tips were associated with the mineral
layer than with the organic layer (P = 0.02). Piloderma did not
show any significant association with soil fraction. Although
the data for Lactarius spp. did not fit the model, all root tips
colonized by Lactarius spp. were found in the mineral layer.
No significant effect of treatment on morphotype distribution
was detected for any of the morphotypes tested.
Discussion
Figure 2. Rank-abundance graphs for ectomycorrhizal fungal communities in Norway spruce plots receiving either irrigation (IR), nutrient solution (F) or no treatment (C).
Boreal forests have developed under conditions of N limitation (Tamm 1991). The actual N pool is often large but the
greater part of the nutrient is relatively inaccessible, being
bound in organic matter that accumulates at the soil surface
(Persson et al. 2000). It is in this organic layer that the majority
of mycorrhizal roots of boreal forest trees are found (Meyer
1973, Ogawa 1977, 1985). On the basis of this apparent association between organic matter and ectomycorrhizal roots,
Frank suggested over 100 years ago that ECM fungi might
play an important role in the acquisition of N from organic
sources (see Read 1987). Recent experiments support this
view (see Leake and Read 1997, Näsholm et al. 1998).
The application of inorganic fertilizers, which include N, to
boreal systems reduces the importance of organic matter as the
main N source. Under these modified conditions, it has been
suggested that ECM fungi that are specialized in the utilization of organic N sources will be outcompeted by more generalist mineral N fungi (Read et al. 1989).
TREE PHYSIOLOGY VOLUME 20, 2000
FERTILIZATION AND ECTOMYCORRHIZAL COMMUNITY STRUCTURE
603
Table 2. Diversity indices for ectomycorrhizal communities in Norway spruce plots receiving either no treatment, irrigation or nutrient solution.
Diversity index
Berger-Parker 1
Shannon-Wiener
1
Control
Irrigation
Fertilization
1
2
3
1
2
3
1
2
3
0.71
1.96
0.72
2.14
0.83
2.44
0.70
2.14
0.74
2.01
0.74
2.06
0.60
2.11
0.70
2.31
0.52
1.91
Complement values.
In fertilization studies of forests, N has commonly been
added as ammonium nitrate (e.g., Rühling and Tyler 1991,
Högberg et al. 1992, Emmett et al. 1995, Brandrud 1995) or
ammonium sulfate (e.g., Alexander and Fairley 1983, Termorshuizen 1993, Wiklund et al. 1994). During the 1970s, ammonium nitrate was commonly used in Swedish forestry, but at
present the most common form is calcium nitrate (Nohrstedt
1998).
Analysis of published investigations of fertilization effects
on ECM fungi suggests that the observed effects may depend
critically on the type of fertilizer used, the method of application—large single doses or continuous application of smaller
doses, and on the mycorrhizal structures enumerated. We used
continuous, balanced additions of liquid fertilizer and found a
clear effect of the treatment on community structure, but no
overall effect on total number of morphotypes or total number
of root tips colonized by mycorrhizal fungi. Studies based on
mycorrhizal root tips involving larger, less frequent additions
of N (Kårén and Nylund 1997, Jonsson et al. 1999a) and
N-free fertilization (Kårén and Nylund 1996) have also failed
to demonstrate a reduction in the number of taxa recorded. Additions of water can alter the community composition of ECM
fungi recorded as sporocarps (Wiklund et al. 1994), but no
data appear to be available on effects of irrigation on
mycorrhizal root tips. In our study, irrigation alone did not significantly change the community structure compared with the
control treatment, suggesting that it is the addition of nutrients
rather than irrigation that affects community composition.
Bergh et al. (1999) reported that there were also no measurable
effects of irrigation alone on tree growth at the Flakaliden field
site.
Reductions in the percentage of colonized tips are often
seen after fertilizing with single, high-N additions (Wallenda
and Kottke 1998). We found no change in colonization frequency in response to continuous application of fertilizer; all
plots had a high degree of colonization. Other studies involving three annual applications of N (Termorshuizen 1993) and
weekly additions of N (Brandrud and Timmermann 1998) also
found no effect on colonization.
In our study, the mycorrhizal community was dominated by
species that do not form conspicuous sporocarps. Piloderma
croceum colonized almost 30% of the roots in some plots, and
C. geophilum colonized up to 40% of the roots in one plot.
T. fibrillosa was also common, especially in one of the fertilized plots (45%). High relative abundances of P. croceum
(19%), T. fibrillosa (9%) and C. geophilum (18%) were also
found by Dahlberg et al. (1996) on mycorrhizal root tips in an
old-growth spruce forest in southern Sweden.
The high diversity of the ECM community found in the
present study appears to be a common feature of boreal forests
(Dahlberg et al. 1996, Väre et al. 1996, Taylor et al. 1997,
Högberg et al. 1999). High diversity of soil biota, including
ECM fungi, has been interpreted by some authors (e.g.,
Schimel 1995) as evidence that a high degree of functional redundancy may exist. However, the interpretation of shifts in
ECM community structure is complicated by the fact that the
degree of intraspecific variation in physiology may be high
(Wagner et al. 1988, Gay et al. 1993). Cairney (1999) points
out that it is not yet possible to draw general conclusions about
links between community structure and function because relatively few investigations of ECM fungal physiology have been
conducted with more than five isolates of the same species.
Changes in the relative abundance of different species
might lead to changes in function if accompanied by large
changes in morphology and metabolic activities. Relevant factors to consider are the amount and distribution of
extramatrical mycelium, the capacity to utilize more or less
complex organic substrates, and carbon-use efficiency.
Changes in the concentrations of inorganic nutrients can influence growth and production of extramatrical mycelium. Reduced development has been observed where phosphorus
concentrations are elevated, and increased growth has been
observed in relation to P starvation (Piché and Fortin 1982,
Jones et al. 1990, Wallander and Nylund 1992). Wallander
and Nylund (1992) found reductions in fungal biomass associated with mycorrhizal seedlings in response to excess N and
the effect was most pronounced on the extramatrical mycelium. These effects are probably related to patterns of assimilate allocation within the symbiosis (Wallander 1995).
Changed nutrient availabilty may alter species composition
as well as resulting in changed activity within a species (Taylor et al. 2000). Bending and Read (1995) found that the differing abilities of Suillus bovinus (Fr.) Roussel and Thelephora
terrestris Ehrenb. to obtain nutrients from litter was consistent
with their differing patterns of mycelial growth and enzymatic
capabilities. Fertilization, which changes the amount of extramatrical mycelium or the enzymatic activity of the mycelium,
is thus likely to alter the capacity of ectomycorrhizal fungi to
utilize various organic nutrients. High concentrations of ammonium may repress protease production (Chalot et al. 1995)
and alter the ability of the fungal community to utilize organic
forms of nitrogen.
It has been suggested that ECM fungal species that produce
a large amount of mycelial biomass consume more energy
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
604
FRANSSON, TAYLOR AND FINLAY
from the host than those that produce less (Colpaert et al.
1992). Godbold et al. (1997) found an increase in mycorrhizal
morphotypes with a large amount of extramatrical mycelium
in response to elevated CO2 concentration. However, little is
known about differences in carbon-use efficiency among
ectomycorrhizal fungi, and the significance of changes in the
ECM community structure for the carbon economy of the
whole forest is still unclear. Increased tree growth in response
to fertilization may change carbon allocation patterns. Bergh
et al. (1999) reported a doubling of height and diameter growth
in trees on fertilized plots compared to trees on control plots at
Flakaliden after only three years of treatment. Taken over 10
years of treatment, fertilization increased stem volume production by a factor of four compared with control plots. Detailed data on belowground patterns of carbon allocation at
Flakaliden are not yet available.
Both temporal and spatial variations seem to be high for
ECM fungi, but no systematic investigation of this variation
has been made. In this study the large differences between replicate plots indicate high spatial variability. Concerning the
vertical distribution of mycorrhizal fungi down the soil profile, some species or morphotypes have been observed to occur more frequently in either the organic or the mineral layer
(Reddell and Malajczuk 1984, Jonsson et al. 1999a). Reddell
and Malajczuk (1984) found brown and white mycorrhizal
types in a Eucalyptus marginata forest were mainly associated
with the upper organic soil profile, whereas black types dominated the mineral layer. The black types were probably
C. geophilum, because the authors found sclerotia of that species in the soil. At Flakaliden, we found the reverse: a significantly higher abundance of Cenococcum in the organic layer
than in the upper mineral soil, in agreement with the findings
of Jonsson et al. (1999a). In the present study, T. fibrillosa was
more common in mineral soil, and Lactarius spp. were found
only in the mineral fraction. Knowledge about vertical distribution of ECM fungal species is important when evaluating
pertubations caused by silvicultural practices such as fertilization, biomass harvesting and addition of ash (Mahmood et al.
1999). Further knowledge about the vertical distribution of
different mycorrhizal fungi is also an important prerequisite to
understanding the possible roles of these species in podsolization and weathering interactions (Jongmans et al 1997).
The function of ECM fungi may be related to vertical distribution, because different species differ in their ability to utilize
various nitrogen forms, which in turn are distributed differently in the soil horizon. The ECM fungal species may also
differ in uptake of other mineral nutrients and water, the latter
being scarce in the upper layer of soil during dry periods.
Three main points emerged from this study. First, balanced
nutrient additions to a Norway spruce boreal forest can significantly affect the associated mycorrhizal fungi and alter community structure. Second, the effect on community structure
does not necessarily entail a reduction in species richness.
Finally, the changed community structure and the current lack
of knowledge concerning physiological variation in ECM
fungi suggests that there is an urgent need for further research
into how changes within ECM communities may be reflected
in the functioning of the ecosystem as a whole.
Acknowledgment
This work was supported by the Swedish National Energy Administration.
References
Agerer, R. 1986–1998. Colour atlas of ectomycorrhizae. EinhornVerlag, Germany.
Alexander, I.J. and R.I. Fairley. 1983. Effects of N fertilisation on
populations of fine roots and mycorrhizas in spruce humus. Plant
Soil 71:49–53.
Ahlström, K., H. Persson and I. Börjesson. 1988. Fertilization in a
mature Scots pine (Pinus sylvestris L.) stand—effects on fine
roots. Plant Soil 106:179–190.
Arnebrant, K. and B. Söderström. 1992. Effects of different fertiliser
treatments on ectomycorrhizal colonisation potential in two Scots
pine forests in Sweden. For. Ecol. Manage. 53:77–89.
Arnebrant, K., E. Bååth, B. Söderström and H.-Ö. Norhstedt. 1996.
Soil microbial activity in eleven Swedish coniferous forests in relation to site fertility and nitrogen fertilization. Scand. J. For. Res.
11:1–6.
Bending, G.D. and D.J. Read. 1995. The structure and function of the
vegetative mycelium of ectomycorrhizal plants V. Foraging behaviour and translocation of nutrients from exploited litter. New
Phytol. 130:401–409.
Berdén, M., I. Nilsson and P. Nyman. 1997. Ion leaching before and
after clear-cutting in a Norway spruce stand—effects of long-term
application of ammonium nitrate and superphosphate. Water Air
Soil Pollut. 93:1–26.
Boxman, A.W., K. Blanck, T.E. Brandrud, B.A. Emmett,
P. Gundersen, R.F. Hogervorst, O.J. Kjønaas, H. Persson and
V. Timmermann. 1998. Vegetation and soil biota response to experimentally-changed nitrogen inputs in coniferous forest ecosystems of the NITREX project. For. Ecol. Manage. 101:65–79.
Bergh, J., S. Linder, T. Lundmark and B. Elfving. 1999. The effects of
water and nutrient availability on the productivity of Norway
spruce in northern and southern Sweden. For. Ecol. Manage.
119:51–62.
Brand, F. 1991. Ektomykorrhizen an Fagus sylvatica—Charakterisierung und identifizierung, Ökologische kennzeichnung und unsterile kultivierung. Libri Bot. 2:1–229.
Brandrud, T.E. 1995. The effects of experimental nitrogen addition
on the ectomycorrhizal fungus flora in an oligotrophic spruce forest at Gårdsjön, Sweden. For. Ecol. Manage. 71:111–122.
Brandrud, T.E and V. Timmermann. 1998. Ectomycorrhizal fungi in
the NITREX site at Gårdsjön, Sweden; below and above-ground
responses to experimentally-changed nitrogen inputs 1990–1995.
For. Ecol. Manage. 101:207–214.
Cairney, J.W.G. 1999. Intraspecific physiological variation: implications for understanding functional diversity in ectomycorrhizal
fungi. Mycorrhiza 9:125–135.
Chalot, M., M.M. Kytöviita, A. Brun, R.D. Finlay, and B. Söderström. 1995. Factors affecting amino acid uptake by the
ectomycorrhizal fungus Paxillus involutus. Mycol. Res. 99:
1131–1138.
Colpaert, J.V., J.A. van Assche and K. Luijtens. 1992. The growth of
the extra-matrical mycelium of ectomycorrhizal fungi and the
growth response of Pinus sylvestris. New Phytol. 120:127–135.
TREE PHYSIOLOGY VOLUME 20, 2000
FERTILIZATION AND ECTOMYCORRHIZAL COMMUNITY STRUCTURE
Dahlberg, A., L. Jonsson and J-E. Nylund. 1996. Species diversity
and distribution of biomass above and below ground among
ectomycorrhizal fungi in an old-growth Norway spruce forest in
southern Sweden. Can. J. Bot. 75:1323–1335.
Emmett, B.A., S.A. Brittain, S. Hughes, J. Gorres, V. Kennedy,
D. Norris, R. Rafarel, B. Reynolds and P.A. Stevens. 1995. Nitrogen additions (NaNO3 and NH4NO3) at Aber forest, Wales: I. Response of throughfall and soil water chemistry. For. Ecol. Manage.
71:45–59.
Gardes, M. and T. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113–118.
Gardes, M. and T.D. Bruns. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and belowground views. Can. J. Bot. 74:1572–1583.
Gay, C., R. Marmeisse, P. Fouillet, M. Buntertreau and J.C. Debaud.
1993. Genotype/nutrition interactions in the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi. New Phytol. 123:
335–343.
Godbold, D.L., G.M. Berntson and F.A. Bazzaz. 1997. Growth and
mycorrhizal colonization of three North American tree species under elevated atmospheric CO2. New Phytol. 137:433–440.
Goldack, J., B. Münzenberger and R.F. Hüttl. 1998. Ectomycorrhizal
community on reclamation sites in the Lusatian lignite mining region. In Proc. Second International Conference on Mycorrhiza,
Uppsala, Sweden, p 71.
Högberg, P., C.-O. Tamm and M. Högberg. 1992. Variations in 15N
abundance in a forest fertilization trial: critical loads of nitrogen,
nitrogen saturation, contamination and effects of revitalization fertilization. Plant Soil 142:211–219.
Högberg, P., A.H. Plamboeck, A.F.S. Taylor and P.M.A. Fransson.
1999. Natural 13C abundances reveals trophic status of fungi and
host-origin of carbon in mycorrhizal fungi in mixed forests. Proc.
Nat. Acad. Sci. 96:8534–8539.
Ingestad, T. 1967. Methods of uniform optimum fertilization of forest
tree plants. In Proc. XIVth IUFRO Congress, Münich, Section
22:265–269.
Ingestad, T. 1979. Mineral nutrient requirements in Pinus sylvestris
and Picea abies seedlings. Physiol. Plant 45:373–380.
Jones, M.D., D.M. Durall, and P.B. Tinker. 1990. Phosphorus relationship and production of extramatrical hyphae by two types of
willow ectomycorrhizas at different soil phosphorus levels. New
Phytol. 115:259–267.
Jongmans, A.G., N. van Breemen, U. Lundström, P.A.W. van Hees,
R.D. Finlay, M. Srinivasan, T. Unestam, R. Giesler, P.-A. Melkerud and M. Olsson. 1997. Rock-eating fungi. Nature 389:
682–683.
Jonsson, L., A. Dahlberg and T.-E. Brandrud. 1999a. Spatiotemporal
distribution of an ectomycorrhizal community in an oligotrophic
Swedish Picea abies forest subject to experimental nitrogen addition: above- and below-ground views. For. Ecol. Manage. 129:
1–14.
Jonsson, L., A. Dahlberg, M.-C. Nilsson, O. Zackrisson and O. Kårén.
1999b. Ectomycorrhizal fungal communities in late-successional
Swedish boreal forests and composition following wildfire. Mol.
Ecol. 8:205–215.
Kellner, O. 1993. Effects on associated flora of sylvicultural nitrogen
fertilization repeated at long intervals. J. Appl. Ecol. 30:563–574.
Krebs, C.J. 1989. Ecological methodology. Harper and Row Publishers. New York, 620 p.
Kuyper, T.W. and B.W.L. de Vries. 1990. Effects of fertilisation on
the mycoflora of a pine forest. Wageningen Agricultural Univ.
Papers 90(6):102–111.
605
Kårén, O. and J-E. Nylund. 1996. Effects of N-free fertilization on
ectomycorrhiza community structure in Norway spruce stands in
Southern Sweden. Plant Soil 181:295–305.
Kårén, O. and J.-E. Nylund. 1997. Effects of ammonium sulphate on
the community structure and biomass of ectomycorrhizal fungi in a
Norway spruce stand in southwestern Sweden. Can. J. Bot.
75:1628–1642.
Kårén, O., N. Högberg, A. Dahlberg, L. Jonsson and J-E. Nylund.
1997. Inter- and intraspecific variation in the ITS region of rDNA
of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytol. 136:313–325.
Leake, J.R. and D.J. Read. 1997. Mycorrhizal fungi in terrestrial habitats. In The Mycota, Vol. 4: Environmental and Microbial Relationships. Eds. D.T. Wicklow and
B. Söderström.
Springer-Verlag, Berlin, pp 281–301.
Lilleskov, E.A and T.J. Fahey. 1996. Patterns of ectomycorrhizal diversity over an atmospheric nitrogen deposition gradient near
Kenai, Alaska. In Proceedings of the First International Conference on Mycorrhizae. Eds. T.M. Szaro and T.D. Bruns, p 76.
Linder, S. 1995. Foliar analysis for detecting and correcting nutrient
imbalances in Norway spruce. Ecol. Bull. 44:178–190.
Lundmark, J.E. 1986. Skogsmarkens ekologi, ståndortsanpassat skogsbruk. Skogsstyrelsen, 158 p.
Magurran, A.E. 1988. Ecological diversity and its measurements.
Croom Helm, London, pp 41, 79.
Mahmood, S., R.D. Finlay and S. Erland. 1999. Effects of repeated
harvesting of forest residues on the ectomycorrhizal community in
a Swedish spruce forest. New Phytol. 142:577–585.
Meyer, F.H. 1973. Distribution of mycorrhizae in native and
man-made forests. In Ectomycorrhizae. Eds. G.C.Marks and T.T.
Kozlowski. Academic Press, London, pp 79–105.
Müller, W. and R. Agerer. 1990. Studies on ectomycorrhizae
XXIX—Three mycorrhizae of the Leccinum scabrum-group. Nova
Hedwigia 51:381–410.
Nohrstedt, H-Ö. 1998. Residual effects of N fertilization on soil–water chemistry and ground vegetation in a Swedish Scots pine forest.
Environ. Pollut. 102 (Suppl. 1):77–83.
Näsholm, T., A. Ekblad, A.Nordin, R. Geisler, M. Högberg and
P. Högberg. 1998. Boreal forest plants take up organic nitrogen.
Nature 392:914–916.
Ogawa, M. 1977. Ecology of higher fungi in Tsuga diversiflora and
Betula ermani–Abies mariesii forests of subalpine zone. Trans.
Mycol. Soc. Jpn. 18:1–19.
Ogawa, M. 1985. Ecological characters of ectomycorrhizal fungi and
their mycorrhizae—an introduction to the ecology of higher fungi.
Jpn. Agric. Res. Quart. 18:305–314.
Ohtonen, R., A.M. Markkola, H. Heinonen-Tanski and H. Fritze.
1990. Soil biological parameters as indicators of changes in Scots
pine forests (Pinus sylvestris L.) caused by air pollution. In Acidification in Finland. Eds. P. Kauppi, P. Anttila, and K. Kenttamies.
Springer-Verlag, Berlin, Heidelberg, pp 373–393.
Persson, T., H. van Oene, T. Harrison, G. Bauer, P. Bottner, E. Dambrine, G. Matteucci, P. Karlsson and A. Rudebeck. 2000. Experimental sites in the NIPHYS/CANIF project. In Carbon and
Nitrogen Cycling in European Forest Ecosystems. Ecol. Stud. In
press.
Piché, Y. and J.A. Fortin. 1982. Development of mycorrhizae, extramatrical mycelium and sclerotia on Pinus strobus seedlings. New
Phytol. 91:211–220.
Read, D.J. 1987. In support of Frank’s organic nitrogen theory.
Angew. Bot. 61:25–37.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
606
FRANSSON, TAYLOR AND FINLAY
Read, D.J., J.R. Leake and A.R. Langdale. 1989. The nitrogen nutrition of mycorrhizal fungi and their host plants. In Nitrogen, Phosphorus and Sulphur Utilization by fungi. Eds. L. Boddy,
R. Merchant and D.J. Read. Cambridge University Press, pp
181–204.
Reddell, P. and N. Malajczuk. 1984. Formation of mycorrhizae by
Jarrah (Eucalyptus marginata Donn ex Smith) in litter and soil.
Aust. J. Bot. 32:511–520.
Rühling, Å. and G. Tyler. 1991. Effects of simulated nitrogen deposition to the forest floor on the macrofungal flora of a beech forest.
Ambio 20:261–263.
Schimel, J. 1995. Ecosystem consequences of microbial diversity and
community structure. Ecol. Stud. 113:239–254.
Smith, S.E. and D.J. Read. 1997. Mycorrhizal symbiosis, 2nd Edn.
Academic Press, New York, 605 p.
Tamm, C.-O. 1991. Nitrogen in terrestrial ecosystems. Ecol. Stud.
81:115.
Taylor, A.F.S. and I. Alexander. 1989. Demography and population
dynamics of ectomycorrhizas of Sitka spruce fertilised with N.
Agric. Econ. Environ. 28:493–496.
Taylor, A.F.S., L. Högbom, M. Högberg, A.J.E. Lyon, T. Näsholm
and P. Högberg. 1997. Natural 15N abundance in fruit bodies of
ectomycorrhizal fungi from boreal forests. New Phytol.
136:713–720.
Taylor, A.F.S., F. Martin and D.J. Read. 2000. Fungal diversity in
ectomycorrhizal communities of Norway spruce (Picea abies (L.)
Karst.) and beech (Fagus sylvatica L.) along north–south transects
in Europe. In Carbon and Nitrogen Cycling in European Forest
Ecosystems. Ecol. Stud. In press.
Termorshuizen, A.J. 1993. The influence of nitrogen fertilisers on
ectomycorrhizas and their carpophores in young stands of Pinus
sylvestris. For. Ecol. Manage. 57:179–189.
Tokeshi, M. 1993. Species abundance patterns and community structure. Adv. Ecol. Res. 24:112–186.
Väre, H., E. Ohenoja and R. Ohtonen. 1996. Macrofungi of oligotrophic Scots pine forests in northern Finland. Karstenia 36:1–18.
Wagner, F., G. Gay and J.C. Debaud. 1988. Genetic variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic
mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum.
Appl. Microbiol. Biotech. 28:566–576.
Wallander, H. 1995. A new hypothesis to explain allocation of drymatter between mycorrhizal fungi and pine seedlings in relation to
nutrient supply. Plant Soil 168/169:243–248.
Wallander, H. and J.-E. Nylund. 1991. Effects of excess nitrogen on
carbohydrate concentration and mycorrhizal development of Pinus
sylvestris L. seedlings. New Phytol. 119:405–411.
Wallander, H. and J.-E. Nylund. 1992. Effects of excess nitrogen and
phosphorus starvation on the extramatrical mycelium of mycorrhizas of Pinus sylvestris L. seedlings. New Phytol. 120:
495–503.
Wallenda, T. and I. Kottke. 1998. Nitrogen deposition and ectomycorrhizas. New Phytol. 139:169–187.
Westling, O. and H. Hultberg. 1990/91. Liming and fertilization of
acid forest soil: short-term effects on runoff from small catchments. Water Air Pollut. 54:391–407.
Wiklund, K., L-O. Nilsson and S. Jacobsson. 1994. Effects of irrigation, fertilisation, and artificial drought on basidioma production in
a Norway spruce stand. Can. J. Bot. 73:200–208.
Wöllecke, J., B. Münzenberger and R.F. Hüttl. 1998. Effects of enlarged N-deposition on the mycorrhizal diversity of a pine stand in
northeastern Germany. In Proc. Second International Conference
on Mycorrhiza, Uppsala, Sweden, p 186.
TREE PHYSIOLOGY VOLUME 20, 2000