ectomycorrhizal status of norway spruce seedlings from bare-root forest nurseries
TRANSCRIPT
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Forest Ecology and Management 236 (2006) 375–384
Ectomycorrhizal status of Norway spruce seedlings from
bare-root forest nurseries
Maria Rudawska a,*, Tomasz Leski a, Lidia K. Trocha a, Roman Gornowicz b
a Institute of Dendrology, Polish Academy of Sciences, 5 Parkowa St., 62-035 Kornik, Polandb Agriculture University, Faculty of Forestry, 28 Wojska Polskiego St., 60-637 Poznan, Poland
Received 8 October 2005; received in revised form 4 July 2006; accepted 21 September 2006
Abstract
We examined the ectomycorrhizal communities associated with Norway spruce seedlings grown in 16 bare-root nurseries in northwest Poland. One
through four-year-old seedlings were examined and compared. We found 11 morphotypes in total with an average of 2.7 morphotypes per nursery.
Among these 11 morphotypes, RFLP-analysis detected 17 distinct RFLP-patterns. By comparison with the available reference material, 12 of these
could be identified to genus or species. These were Amphinema byssoides, Hebeloma crustuliniforme, Hebeloma longicaudum, Paxillus involutus,
Thelephora terrestris, Cenococcum geophilum, Phialophora finlandia, Tuber sp., Wilcoxina mikolae, Wilcoxina sp. 1, Wilcoxina sp. 2 and Tricharina
ochroleuca. The five unidentified morphotypes were: basidiomycetes ITE.5 and four ascomycetes designated as IDPAN.1, IDPAN.2, IDPAN.3 and
IDPAN.4 that in morphotyping were classified as an E-strain. The ascomycete W. mikolae was predominant, whereas the basidiomycetes were less
common. ECM community of Norway spruce seedlings from bare-root nurseries were not structured in association with soil pH and nutrient status of
the host plant. The consistent presence of inocula of ascomycete symbionts in the substrate from all nurseries is quite notable and may reflect their
adaptation and/or resistance to highly transformed nursery soil substrates compared to symbionts from the Basidiomycota.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Picea abies; Ectomycorrhiza; Morphotype; PCR–RFLP; Nursery
1. Introduction
Norway spruce (Picea abies (L.) Karst.) has an extensive
geographic range in Europe, growing from Scandinavia to the
Balkans, the Alps and the Carpathians. In Poland, Norway
spruce is a native species and after Scots pine, the most
commonly planted conifer, accounting for 5.8% of Polish
forests. As with most tree species in boreal and temperate
forests, ectomycorrhizal (ECM) fungi extensively colonize the
fine roots of Norway spruce. These ectomycorrhizal associa-
tions are ubiquitous and are believed to be critical to successful
seedling establishment and tree growth, facilitating both
nutrient and water uptake, increasing resistance to certain root
diseases, and enhancing the tolerance of the tree to stress
(Harley and Smith, 1983; Allen, 1991). Mycorrhizal formation
is usually adequate under natural conditions. ECM fungi live in
the forest soil and litter layer and readily colonize new tree
seedlings. Determination of the ECM community structure of
* Corresponding author. Tel.: +48 61 8170033; fax: +48 61 8170166.
E-mail address: [email protected] (M. Rudawska).
0378-1127/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.foreco.2006.09.066
young and mature Norway spruce stands has received much
attention recently (Egli et al., 1993; Mehmann et al., 1995;
Karen and Nylund, 1996; Kraigher et al., 1996; Dahlberg et al.,
1997; Erland et al., 1999; Kraigher, 1999; Mahmood et al.,
1999; Fransson et al., 2000; Jonsson et al., 2000; Peter et al.,
2001a,b; Haug, 2002; Kieliszewska-Rokicka et al., 2003).
As an alternative to natural regeneration, Norway spruce
seedlings are grown for 3 or 4 years in nurseries before
outplanting. Nursery managers have long recognized the
importance of well-developed mycorrhizas for healthy seedling
growth in the nursery and desired performance after out-
planting. There are currently around 1200 bare-root forest
nurseries in Poland, producing close to 200 million Norway
spruce seedlings each year for reforestation and afforestation
(Lenart, 2000). Very little is known about the ECM symbionts
that associate with spruce seedlings in nurseries and how this
relates to seedling and ecosystem function during the first years
following outplanting. Only a few reports describe the ECM
community structure of several commercially important Picea
species originating as bare-root or containerized seedling
nursery stock, such as Picea sitchensis or Picea glauca
(Thomas and Jackson, 1979; Danielson and Visser, 1989;
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384376
Grogan et al., 1994; Krasowski et al., 1999; Kernaghan et al.,
2003). The first record of nursery mycorrhizas of P. abies
appears to be that of Weiss and Agerer (1988) who described
three unidentified mycorrhizas of Norway spruce cuttings
grown in a Bavarian nursery. Recently, fungal community
structure in fine roots of P. abies seedlings under different
nursery cultivation system in Lithuania was demonstrated by
Menkis et al. (2005).
Identification of the indigenous mycorrhizal populations on
seedlings produced in bare-root nurseries is complicated because
nursery management practices such as fertilization, fumigation,
and use of fungicides are known to affect mycorrhizal
colonization (Molina and Trappe, 1982). The intensive cultiva-
tion of soil (i.e. ploughing, weeding, root pruning, etc.) breaks up
the delicate fungus–soil network, leading to a significant
reduction in ECM colonization and makes sporophores
production near unfeasible (Xavier and Germida, 1999). Thus,
the species composition and relative abundance of fungi that
colonize seedlings in bare-root nurseries have to be estimated
directly by observing roots. Molecular techniques have greatly
advanced the opportunities to identify fungal species and strains
from even single ectomycorrhizal root tips (Gardes and Bruns,
1993). Using this approach, we have examined the species
richness and abundance of ECM community associated to spruce
seedlings grown in bare-root nurseries in Poland. The aims of this
study were (1) to obtain comprehensive insight into the ECM
fungi that form mycorrhizal associations in the condition of
nursery and (2) to determine whether the detected ECM fungi
diversity would differ according to the nursery stock sample, age
of the seedlings, pH of the nursery bed soil and nutritional status
of seedlings, estimated as the foliar content of macroelements (N,
P, K, Ca, and Mg).
2. Materials and methods
2.1. Nurseries and seedlings sampled
We surveyed 16 nurseries that were each active in supplying
planting stock for the restocking of forests and the reforestation
of post-agricultural land. The sampled nurseries belonged to
Forest Districts located in northwestern Poland (Fig. 1). They
are all rather large provincial nurseries, ranging in size from
Fig. 1. Location of sampled nurseries. The names of nurseries are labeled as: Ch, C
Mm, Milomlyn; Mr, Miradz; Ok, Okonek; Ol, Olsztynek; Pr, Przymuszewo; Rn, R
five to 10 ha, and separated into several compartments with four
to six standard nursery seedbeds each. Norway spruce seedlings
were all precision seeded by machine and fertilized following a
schedule designed to satisfy their nutrient requirements and
based on soil analysis of each nursery.
Depending on the current nursery production, P. abies stock
(1-, 2-, 3- and 4-year-old) or transplants (1-, 1.5- and 2-year-old
trees transferred to transplant beds) were harvested, between
the beginning of September and middle of October during 3
consecutive years (2001, 2002, 2003). Each stock was classified
according to seedling age and growing location. For example, a
1 + 0 classification represents stock that has been grown for 1
year in a seedbed and 0 years in a transplant bed; a 1 + 1 is
grown for 1 year in a seedbed and 1 year in a transplant bed, etc.
2.2. Analysis
The pH of the nursery-bed soil was determined by mixing
20 ml of soil substrate with 40 ml of de-ionized water. After
1 h, the pH was determined with a calibrated pH meter
equipped with a glass electrode.
The foliar content of macronutrients (N, P, K, Ca, and Mg)
was determined using three composite samples. Each sample
consisted of five seedlings lifted from the Norway spruce
seedbeds of a given age. Milled samples of spruce needles, of
approximately 2.5 g dry mass each, were digested in a mixture
of spectrally pure concentrated acids: HNO3 and HClO4 in a
proportion of 4:1 (v:v) and diluted with bi-distilled water to
make 25 ml. Nitrogen was analyzed by the micro-Kjeldahl
method. The remaining macroelements were measured by
atomic absorption spectroscopy (Varian 220 FS) with
atomization in an air–acetylene flame. The accuracy of the
analyses was checked against standard reference material,
namely pine needles 1575 and tomato leaves SRM 1573a
(National Institute of Standards and Technology, USA).
2.3. Ectomycorrhizal assessment
Four subsamples, each comprised of five seedlings lifted
from the Norway spruce seedbeds of a given age were randomly
sampled. The base unit for data analysis was the average of
these four replicates (=nursery stock sample, NSS). A total of
hojna; Dr, Dobrocin; Dw, Dabrowa; Iw, Ilawa; Kt, Kartuzy; Mk, Mieszkowice;
unowo; SK, Solec Kujawski; Sw, Swidnica; Wr, Wronki; Zt, Zlotow.
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384 377
32 nursery stock samples and 640 seedlings were analyzed.
Seedlings were removed from the nursery together with an
adjacent soil sample and transported immediately to the
laboratory in plastic bags.
The root system was gently washed in tap water to remove
most of the soil and organic debris, minimizing any damage to
the ectomycorrhizas. Tightly adhering materials were removed
with forceps. Because of the large number of root tips present
on each seedling (usually 300–600, but up to 1000 per
seedling), assessments of morphotype frequencies by counting
all root tips was too time consuming. Therefore, the clean roots
were cut into approximately 2.5-cm long sections and placed in
a Petri dish filled with water. Sections were randomly selected
and the numbers of all active root tips colonized by each
morphotype were counted. Successive root sections were
examined until 300 root tips had been counted in each of the
replicates.
Different morphotypes were distinguished by their macro-
scopic and microscopic characteristics and named if matched to
published descriptions (Agerer, 1987–2002; Danielson and
Visser, 1989; Ingleby et al., 1990; Massicotte, 1994; Shihido
et al., 1996). Each root tip was examined under a microscope
(6–90� magnification) for features such as color, shape, size,
texture, presence or absence of mycelial strands or rhizo-
morphs; presence of cystidia, etc. Mycorrhizal colonization
was confirmed by microscopic (500�) examination of whole
mounts of root tips to determine the presence of a mantle and a
Hartig net. Each distinct mycorrhizal morphotype was
described and photographed for further reference.
For DNA analysis, fragments of the root system with the
same mycorrhizal types were placed in Eppendorf-tubes in
cetyltrimethyl ammonium bromide (CTAB) buffer and stored at
room temperature until processing.
2.4. Molecular identification of ectomycorrhizas
For molecular identification, three to six root tips per
morphotypes from each NSS were analyzed. In case of
morphotypes that in preliminary analysis represented mixtures
of taxa (different RFLP-types) additional analysis were
performed consisting five root tips of this morphotypes from
each subsample (n = 20) within NSS. Obtained results were a
basis to calculate relative abundance of mycorrhizal species.
Table 1
Macroelement concentration (%) in foliage of Norway spruce seedlings of varying a
samples for 1-, 2-, 3–4-year-old seedlings, respectively; SE in brackets)
1-year-old 2-year-old
Min–max Mean Min–max
N 1.62–2.85 2.24 (0.34) a 1.76–2.30
P 0.12–0.38 0.27 (0.06) a 0.10–0.40
K 0.43–1.26 0.81 (0.22) a 0.19–0.91
Ca 0.51–1.57 0.79 (0.35) a 0.32–1.16
Mg 0.10–0.24 0.14 (0.04) a 0.08–0.13
pHH2O 4.18–8.14 6.06 (1.18) a 4.22–7.07
pHKCl 3.57–7.84 5.87 (1.29) a 3.77–6.75
Mean values within a row followed by the same letter do not differ at P < 0.05 (T
Ectomycorrhizal fungi on mycorrhizal root tips were
identified using restriction fragment length polymorphism
(RFLP) of the PCR-amplified internal transcribed spacer of
DNA. Each sample consisted of a single mycorrhiza. DNA was
extracted using the miniprep method developed by Gardes and
Bruns (1996); the amplification followed the protocol of
Henrion et al. (1994) as modified by Karen et al. (1997). RFLP-
patterns were obtained using the restriction enzymes MboI,
HinfI, and TaqI. Restriction fragments were separated using 2%
agarose gel electrophoresis (4 h; 100 mV), stained with 0.5%
ethidium bromide, and recorded on black and white PolaroidTM
film. Morphotypes were identified by matching the sample and
reference specimens (obtained from a regional collection of
sporocarps or pure culture) using the Taxotron1 software
package (Pasteur Institute, Paris, France). Different restriction
fragment length polymorphism (RFLP) patterns were denoted
as separate species if the fragments varied above 4%.
2.5. Data analysis
The diversity of the ectomycorrhizas on the seedlings was
expressed as the number of identified ECM species (species
richness). The relative abundance of previously identified ECM
fungal species was calculated as number of tips of given ECM
species per total number of mycorrhizal tips extracted in NSS.
Because the mycorrhizal species richness and relative
abundance values were not normally distributed and typical
transformations did not correct the distribution, nonparametric
Kruskal–Wallis tests were used to test the effects of nursery
stock sample, age and seedling transplant type. The test could
be applied only for the nine fungal species that occurred in four
or more of the examined NSSs. The remaining taxa occurred
too infrequently to apply statistical tests. ECM species richness
in relation to macroelement contents in foliage of Norway
spruce seedlings and pH of the nursery-bed soil were correlated
using the Spearman rank correlation test. Macronutrient status
between Norway spruce seedlings of varying age and pH of the
nursery soil substrate were compared by ANOVA (Table 1).
When a significant difference was observed, Tukey’s post hoc
test was applied.
Canonical correspondence analysis (CCA) was used to
check if relative abundance is related with tested variables
(nutritional status of Norway spruce seedlings and nursery-bed
ge and pH of the nursery soil substrate (means from 33, 27 or 36 of composite
3–4 year-old
Mean Min–max Mean
2.04 (0.21) a,b 1.12–2.66 1.96 (0.39) b
0.25 (0.10) a 0.18–0.28 0.24 (0.03) a
0.59 (0.24) b 0.31–0.72 0.53 (0.14) b
0.82 (0.30) a 0.74–1.72 0.94 (0.26) a
0.10 (0.02) b 0.07–0.14 0.10 (0.02) b
5.86 (0.80) a 4.72–7.93 6.07 (1.05) a
5.49 (0.88) a 4.26–7.59 5.84 (1.26) a
ukey test).
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384378
soil pH). An unrestricted Monte Carlo permutation test (999
permutations) was performed to determine if tested relation-
ships were statistically significant for the first canonical axis
and for all four of the extracted axes combined (ter Braak and
Smilauer, 1998). Relative abundance values were transformed
(log10 + 1) before analysis, and the CCA was performed with
CANOCO 4 software.
3. Results
3.1. Mycorrhizal community composition
The mycorrhizal colonization of all tested samples was
nearly 100%. Very small proportions of the root tips appeared
dark and less turgid and were omitted in the analysis. Overall,
11 distinct ECM morphotypes were recorded. Among these 11
morphotypes, RFLP-analysis distinguished 17 unique RFLP-
patterns. Upon comparison with the available reference
material, twelve of these could be identified at least to genus.
These were Amphinema byssoides, Hebeloma crustuliniforme,
H. longicaudum, Paxillus involutus, Thelephora terrestris,
Cenococcum geophilum, Phialophora finlandia, Tuber sp.,
Wilcoxina mikolae, and Tricharina ochroleuca. Two RFLP-
patterns associated with an E-strain morphotypes that matched
closely, but not identically with W. mikolae (similarity level
with data base less than 96%), were designated as Wiloxina sp.
1 and Wiloxina sp.2. Five morphotypes remained unidentified.
Table 2
Description and identification of Norway spruce ECM morphotypes originating fr
ECM morphotype Description
1. Amphinema byssoides Comparable with published descriptions for
byssoides (Ingleby et al., 1990;
Agerer, 1987–2002)
2. Hebeloma-like Comparable with descriptions for Hebeloma
(Ingleby et al., 1990; Agerer, 1987–2002)
3. Paxillus-like Comparable with published descriptions
for Paxillus involutus (Ingleby et al., 1990;
Agerer, 1987–2002)
4. Thelephora-like Comparable with published descriptions
for Thelephora terrestris (Ingleby et al.,
1990; Agerer, 1987–2002)
5. Dark brown Conforms to the published descriptions
for ITE.5 (Ingleby et al., 1990)
6. Cenococum geophillum Comparable with published descriptions
for Cenococum geophilum (Ingleby et al.,
1990; Agerer, 1987–2002)
7. Mycelium radicis atrovirens Conforms to the published descriptions
for ITE.3 (Ingleby et al., 1990)
8. Tuber-like Monopodial–pyramidal, lemon-yellow or pal
brown, mantle surface smooth and spiny
9. E-strain-like 1 Conforms to the published descriptions
for Humaria hemispherica (Ingleby et al., 19
10. E-strain-like 2 Conforms to the published descriptions
for Tricharina gilva (Ingleby et al., 1990)
11. E-strain-like 3 Mycorrhizas single to pinnate, orange brown
pale yellowish-brown to dark reddish-brown
or blackish-brown with whitish-brown eman
hyphae, moderately thick-walled, verrucose,
septate, no clamps, rhizomorphs not observe
These were basidiomycete ITE.5 (Ingleby et al., 1990) and four
ascomycetes designated as IDPAN.1, IDPAN.2, IDPAN.3 and
IDPAN.4 that were classified as an E-strain in morphotyping
(Table 2, Fig. 2). The RFLP-patterns of these morphotypes did
not match the ECM fungal species in our database.
3.2. Species richness, frequency and relative abundance
ECM species richness among the tested nurseries was quite
variable and ranged from 1 to 8 per NSS with an average of 2.7
species per NSS (Table 3). Statistics based on the Kruskal–
Wallis tests indicated no effect of seedling age and transplant
type on ECM species richness of Norway spruce seedlings;
however, significant differences (P = 0.0127) were found
among NSS (Table 3).
The relative abundances and distributions of ECM taxa in
the screened nurseries are presented in Table 3. The Kruskal–
Wallis test for nine most frequent fungal species indicated that
relative abundance of this species differed significantly among
NSS (P < 0.001). W. mikolae was a dominant ECM species on
1- and 2-year-old seedlings (mean relative abundance of 56.5
and 37%, respectively), and less abundant on 3- and 4-year-old
seedlings (mean relative abundance of 21%); however, age
effect was statistically not significant (Table 3). W. mikolae was
very often the only species found on seedlings from the tested
nurseries (100% relative abundance). Several infrequently
observed taxa (Wilcoxina sp.2, IDPAN.1, IDPAN.2, IDPAN.3)
om bare-root forest tree nurseries in Poland
Identification ECM species code
A. A. byssoides Ab
Hebeloma crustuliniforme
Hebeloma longicaudum
Hc, Hl
P. involutus Pi
T. terrestris Tt
ITE.5 ITE.5
C. geophilum Cg
Phialophora finlandia Pf
e Tuber sp. Tsp.
90)
Wlicoxina mikolae Wilcoxina sp.2,
ID PAN.1, ID PAN.2
Wm, Wsp.2,
ID PAN.1 ID PAN.2
Wilcoxina sp. 1 Tricharina ochroleuca Wsp.1 To
,
ating
d
ID PAN.3, ID PAN.4 ID PAN.3 ID PAN.4
Fig. 2. Ectomycorrhizas observed on Norway spruce seedlings from bare-root forest tree nurseries. The coding follows that shown in Table 2. Bars, 1 mm.
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384 379
reached very high (>60%) relative abundances in certain
nurseries. Other rare species such as H. longicaudum, P.
involutus, and ITE.5 were less abundant (<20%). The
frequencies of ECM species observed on roots of screened
nursery stock are presented in Fig. 3. The most frequent species
on 1- and 2-year-old seedlings was W. mikolae, which was
noted in more than 70% of the NSS’s but was less frequent on 3-
and 4-year-old seedlings. Some ECM taxa such as T. terrestris,
P. finlandia, Wilcoxina sp. 1, A. byssoides, C. geophilum, and
Tuber sp. were consistently present in samples of all seedling
age classes (Fig. 3). During the study, these species collectively
occurred at a frequency of �10%. Basidiomycete taxa P.
involutus and ITE.5 were exclusively found on 1 + 0 seedlings.
Both Hebeloma species (H. crustuliniforme and H. long-
icaudum) were absent on 1 + 0 stock, but present on older
seedlings. The remaining five ascomycete taxa (IDPAN.1–
IDPAN.4 and T. ochroleuca) were unevenly distributed among
the tested nursery stock.
3.3. Foliage nutrient concentration and pH of the nursery-
bed soil
Table 1 summarizes the minimum, maximum and mean
contents of macronutrients in the foliage of Norway spruce
seedlings as well as the pH of the nursery-bed soil. The mean
concentration of nitrogen (N) decreased with age of seedlings;
however, significant difference was found only for the 3–4-
year-old plants. Regardless of seedling age, phosphorus (P) and
calcium (Ca) concentration in the needles of the tested
seedlings did not differ significantly. The mean concentration of
Table 3
Species richness and relative abundance (%) of ECM fungi species found on 1-, 2-, and 3–4-year-old Norway spruce seedlings from bare-root forest nurs ies (n = 20 per NSS, SE in brackets)
Nursery
symbol/stage
Richness A. byssoides H. crustuliniforme Hebeloma
longicadum
P. involutus T. terrestris ITE.5 Cenococcum
geophillum
P. finlandia Tuber sp. Wilcoxina
mikolae
Wilcoxina sp. 1 Wicox a sp. 2 T. ochroleuca IDPAN.1 IDPAN.2 IDPAN.3 IDPAN.4
1-year-old
Dr 1–0 1 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0 0 0 0 0
Iw 1–0 1 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0 0 0 0 0
Kt 1–0 2 0 0 0 0 0 15 (3.3) 0 0 0 85 (12.5) 0 0 0 0 0 0 0
Mm 1–0 1 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0 0 0 0 0
Ok 1–0 2 76 (11.2) 0 0 0 0 0 0 0 0 0 24 (12.2) 0 0 0 0 0 0
Rn 1–0 3 0 0 0 0 10 (7.1) 0 0 0 7 (2.2) 0 0 83 (11 ) 0 0 0 0 0
SK 1–0 3 0 0 0 0 8 (3.7) 0 0 3 (3.0) 0 89 (10.2) 0 0 0 0 0 0 0
Sw 1–0 4 0 0 0 18 (12.1) 34 (11.2) 0 0 0 0 45 (10.0) 0 0 0 0 0 0 3 (2.4)
Sw 1–0 4 0 0 0 0 0 0 10 (5.5) 4 0 38 (7.8) 0 0 0 48 (11.1) 0 0 0
Wr 1–0 3 0 0 0 0 0 0 0 0 0 65 (1.1) 20 (3.3) 0 0 0 0 0 15 (5.5)
Ch 1–0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0
2-year-old
Dw 2–0 2 0 0 0 0 0 0 0 0 0 20 (6.6) 0 0 80 (21.2) 0 0 0 0
Iw 1–1 2 0 0 0 0 0 0 6 (2.1) 0 0 94 (5.9) 0 0 0 0 0 0 0
Mk 2–0 4 0 0 0 0 0 0 0 0 5 (4.5) 13 (7.8) 42 (5.5) 0 0 0 0 35 (4.5) 0
Mm 1.5–0.5 2 0 0 0 0 0 0 0 0 5 (3.3) 0 0 0 0 0 95 (4.6) 0 0
Mr 2–0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 (0.0) 0
Pr 2–0 5 0 15 (2.4) 0 0 14 (1.1) 0 2 (5.3) 4 (2.1) 0 63 (5.9) 0 0 0 0 0 0 0
Rn 2–0 3 0 0 0 0 20 (2.3) 0 0 0 15 (8.3) 65 (9.9) 0 0 0 0 0 0 0
Ok 2–0 8 10 (3.5) 0 0 0 15 (5.6) 0 5 (2.2) 3 (1.1) 45 (7.3) 17 (2.3) 3 (2.2) 0 2 (3.3) 0 0 0 0
Wr 2–0 3 25 (9.2) 0 0 0 0 0 0 0 0 60 (16.2) 15 (5.7) 0 0 0 0 0 0
3 and 4-year-old
Dr 2–1 1 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0 0 0 0 0
Iw 1–2 1 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0 0 0 0 0 0
Mm 2–1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 28 (6.8) 72 (15.3) 0 0
Mr 2–1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 (0.0) 0 0
Mr 3–0 4 0 0 0 0 65 (11.3) 0 3 4 (2.2) 0 0 0 0 0 0 28 (9.1) 0 0
Ot 1.5–1.5 2 37 (3.3) 0 0 0 0 0 0 0 63 (11.1) 0 0 0 0 0 0 0 0
SK 1–2 4 0 3 (13.3) 0 0 77 (7.8) 0 19 (6.2) 4 (2.4) 0 0 0 0 0 0 0 0 0
Ch 3–0 2 0 84 (2.2) 0 0 0 0 0 0 16 (2.2) 0 0 0 0 0 0 0 0
Wr 3–0 3 0 0 0 0 0 0 0 0 0 56 (8.8) 25 (4.9) 0 0 0 0 0 19 (3.5)
Zt 3–0 4 0 0 0 0 0 0 7 (2.3) 30 (6.7) 60 (14.3) 3 (4.2) 0 0 0 0 0 0 0
Mm 4–0 3 0 0 14 (10.3) 0 0 0 0 0 0 0 63 (17.4) 23 (6. 0 0 0 0 0
Pr 2–2 4 0 62 (5.1) 0 0 14 (4.5) 0 0 0 5 0 0 19 (7. 0 0 0 0 0
Source
NSS * *** nt nt nt nt *** *** *** *** *** nt nt nt *** nt nt ***
Age ns ns nt nt nt nt ns ns ns ns ns nt nt nt ns nt nt ns
Transplant type ns ns nt nt nt nt ns ns ns ns ns nt nt nt ns nt nt ns
Kruskal–Wallis test: ***P < 0.001; *P < 0.05; ns, not significant, P > 0.05; nt, not tested.
M.
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eta
l./Fo
restE
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an
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an
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7)
Table 4
Spearman’s rank correlations between ECM species richness and the macro-
element concentration of foliage and pH of the nursery soil
Rs t (N � 2) P
N �0.13 �1.07 0.29
P 0.13 1.03 0.31
K �0.13 �1.05 0.29
Ca �0.12 �0.93 0.35
Mg 0.37 3.16 0.002
pHH2O �0.13 �1.06 0.29
pHKCl �0.15 �1.21 0.23
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384 381
potassium and magnesium was significantly higher in 1-year-
old seedlings compared to the older ones. The mean pH of the
nursery-bed soil ranged from slightly acidic (4.18 in H2O and
3.57 in KCl) to slightly alkaline (8.14 in H2O and 7.84 in KCl)
and did not differ in dependence on the age of the seedlings.
3.4. Relationships between the pH of the nursery-bed soil,
foliar macroelement concentration, and ECM richness and
abundance
The Spearman rank correlation coefficient was used to
examine the association of the plant and soil variables (foliar
macroelement concentration and pH of the nursery-bed soil)
with the species richness of the ECM fungi. The only positive
rank correlation was found for magnesium concentration in the
needles of the tested spruce seedlings (P = 0.002, Table 4).
Fig. 3. Frequency of occurrence of mycorrhizal species in nursery stock
samples (NSS) of 1-year-old (a); 2-year-old (b); and 3–4-year-old; (c) Norway
spruce seedlings (n = 11, 9 or 12 for 1-, 2-, and 3–4-year-old seedlings,
respectively). The coding follows that shown in Table 2.
The abundances of nine fungal species that occurred in four
or more of the examined NSSs and their corresponding CCA
species scores were plotted together with the examined plant
and soil variables of foliar nutrient concentration and pH of the
nursery-bed soil. The first two axes of the CCA, with
eigenvalues of 0.234 and 0.198, explained 14.4% of the
species variance and 64% of the species–environmental
relationship. Along with the low percentage of species variance
explained, a not significant relationship between species
distributions and the measured environmental variables was
revealed for the first axis (F = 1.86, P = 0.87) and all four axes
combined (F = 0.914, P = 0.58) (figure not shown).
4. Discussion
4.1. Ectomycorrhizal diversity
While natural mycorrhizal colonization is common in bare-
root nurseries, the fungal diversity of colonization can be quite
variable (Danielson and Visser, 1989; Letho, 1989; Ursic and
Peterson, 1997; Ursic et al., 1997; Rudawska et al., 2001;
Menkis et al., 2005). We found that 17 fungi species might
contribute to the ECM community structure of 1–4-year-old
Norway spruce seedlings. Due to the more precise method of
identification in the present study, based on molecular analysis,
the total number of mycorrhizal species in the screened
nurseries was higher than reported in previous studies based on
morphotyping or microscopic examination (e.g. Thomas and
Jackson, 1979; Danielson and Visser, 1990; Ursic and Peterson,
1997) but comparable with molecular studies of Menkis et al.
(2005) for Lithuanian nurseries. Given the fact that we found no
statistically significant effect of seedling age on ectomycor-
rhizal species richness of Norway spruce seedlings (Table 3), it
might be interpreted that one to 4-year-old seedlings have a
comparable physiological potential to host mycorrhizal
symbionts. The fact that 17 ECM species were found in the
tested nurseries likely reflects the high potential of Norway
spruce seedlings to form ectomycorrhiza. However, individual
nursery stock samples significantly differed in terms of species
richness and abundance and varies from 1 to 8 per nursery stock
sample with an average of 2.7. It raises the question about the
causes of such a limited species associations of ECM fungi with
spruce seedlings of particular NSS. Kranabetter (2004) studied
pioneer ECM community of hybrid spruce seedlings and
indicate a better growth response by the less diverse, more
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384382
unevenly distributed pioneer ECM communities in the earliest
stand conditions. Spearman rank correlation test (Table 4) show
that the ECM richness were not structured in association with
soil pH and nutrient status (N, P, K, Ca concentration) of the
host plant in the conditions of the bare-root nursery. The only
weak correlations with magnesium may be connected with
magnesium shortage in the foliage of nursery seedlings.
Positive correlations between species richness and increasing
magnesium concentration were recorded by Humphrey et al.
(2003) for Sitka spruce stands. Ordering of the data by CCA
also did not indicate linkage of abundance of some fungal
species to tested variables (figure not presented). Similarly,
Flynn et al. (1998) found no significant correlations between
ECM colonization and soil pH, loss-on-ignition, or water
content on Sitka spruce seedlings in a Scottish plantation forest.
Krasowski et al. (1999) concluded that the differences in the
composition and abundance of ectomycorrhizas on white
spruce seedlings appeared to be related more to root vigor than
to differences in root growth media and fertilizer treatments.
Although several authors have reported that high fertilizer
levels decrease mycorrhiza formation (for review see Smith and
Read, 1997), Molina and Chamard (1983) and Danielson et al.
(1984) reported that ectomycorrhiza formation was not affected
by fertilization treatments. As shown by Brunner and Brodbeck
(2001) Norway spruce can tolerate N at rather high application
rates. The highest application rates of N influenced mycor-
rhization, but at levels (800 kg N ha�1 year�1) never used in
Polish bare-root nurseries (Rudawska, unpublished data).
Regardless of seedling age, the mean concentration of nitrogen
(N) in the needles of the tested seedlings fell within the range
considered optimal for spruce seedlings (1.80–2.40%) (Inges-
tad, 1962). The inconsistency in the ECM community structure
of planting stock from different nurseries may be the variation
among the different trials in fertilizer formulas, stock types,
site-specific soil characteristics, host physiology, and the
availability of inoculum among others (Perry et al., 1987). The
processes that control ECM diversity in bare-root forest tree
nurseries are still poorly understood. Other factors, including
competitive interactions among different ECM species and the
availability of different sources of inoculum (Bruns, 1995), may
well have contributed to the observed differences in the
mycorrhizal fungi communities among the different nurseries.
4.2. Ecology and epidemiology of nursery fungi
W. mikolae was the most common ascomycete mycobiont
detected in our study, present in high abundances on more than
60% of the nursery stock samples. Wilcoxina spp. are known as
common colonizers of nursery seedlings (Danielson, 1991;
Egger, 1995). Presumably, W. mikolae, classified into the ‘E-
strain’ fungi, was also the most common mycorrhizal type
found by Grogan et al. (1994) in Irish nurseries on Sitka spruce.
Mycorrhizas present on Sitka spruce in forest tree nurseries,
morphologically resembling W. mikolae from our studies, were
classified by Ingleby et al. (1990) as Humaria hemisphaerica
and Tricharina gilva. According to Fay and Mitchell (1999),
mycorrhizas of H. hemisphaerica are commonly associated
with conifer seedlings in nurseries, but do not persist on the
roots after outplanting onto forest sites. In our studies a small
proportion of the mycorrhizas from the E-strain group matched
T. ochroleuca, but none of E-strain morphotypes matched the
fruitbodies of H. hemisphaerica. This fungus was also absent in
containerized P. glauca seedlings from four nurseries in
northern Alberta, Canada (Kernaghan et al., 2003). W. mikolae
was quite frequent there instead. It seems that W. mikolae and
several other closely related species comprise a group of
symbionts best suited to nursery conditions. Wilcoxina sp. is a
member of the ascomycete fungi, which contribute to the
resistant propagule community, and in terms of life strategy
correspond with the ruderal model in plants (Taylor and Bruns,
1999). These fungi do not compete well in the stable
environment of a mature forest, but they have persistent
propagules and respond rapidly to disturbance. Mycorrhizas of
Wilcoxina have been shown to persist for longer periods in
habitats where competitors are reduced (Danielson and Pruden,
1990). The observation that certain types of E-strain fungi
produce thick-walled chlamydospores (Danielson, 1982) that
remain viable in the soil for extended periods of time support
this designation. The high abundance of W. mikolae and several
closely related fungi (Wilcoxina sp. 1 and 2) indicate that most
nursery soils may be considered a disturbed habitat. W. mikolae
is also the most common mycorrhizal symbiont found on other
conifers such as Scots pine and European larch in Polish and
Lithuanian bare-root nurseries (Iwanski et al., 2006; Menkis
et al., 2005; Rudawska, unpublished data). Other ECM taxa
consistently present in the tested nurseries were P. finlandia, C.
geophilum, Tuber sp., A. byssoides, and T. terrestris.
P. finlandia conforms to the published descriptions for ITE.3
and the fungus associated with this mycorrhiza is a member of
the Mycelium radicis atrovirens group. According to Ingleby
et al. (1990) some fungi of the M. radicis atrovirens group may
be pathogenic. Their persistence on nursery-grown seedlings
may be associated with higher abundance of moribund roots
due to mechanical injury of the root system arising from
mechanized nursery-bed practices (Ingleby et al., 1990).
C. geophilum is distinguished morphologically by black,
club-shaped mycorrhizas and thick emanating hyphae. Cen-
ococcum is one of the most abundant EM fungal taxa in many
studies and apparently has a high fitness or competitive ability
relative to other EM fungal species (LoBuglio, 1999). Its lack of
known sexual reproductive structures suggests that this species
can spread effectively through mycelial growth or asexual soil-
borne sclerotia (Jonsson et al., 2000). The Tuber sp. was also
widespread, but considerably less abundant than other
ascomycetes from the genus Wilcoxina. This may reflect
limited distribution of the field inoculum of Tuber sp. or its poor
competitive ability relative to other species in nursery soil.
Different authors have consistently identified Tuber-type
mycorrhizas on young seedlings of various host species (e.g.
Ingleby et al., 1990; Ursic and Peterson, 1997). The Tuber-type
mycorrhizas we found in forest nurseries bear a strong
morphological resemblance to that of T. puberulum, as
described by Agerer (1987–2002) on mature Norway spruce
trees. However, our molecular identification did not match this
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384 383
species but other from our data base not identified to genera.
Trappe (1969) described T. maculatum in Pinus strobus nursery
beds and T. maculatum colonized P. strobus in northern Italian
nurseries (Fassi and De Vecchi, 1963); however, T. maculatum
has not been found in Poland (Ławrynowicz, 1988). Recently
Menkis et al. (2005) reported close matching of Tuber-type
mycorrhiza from nursery Norway spruce seedlings with Tuber
rapaeodorum. The resolution of taxonomic position of our
Tuber-type mycorrhiza needs further studies.
In total, 11 ascomycete RLFP types were detected among
the tested seedlings. The consistent presence of inocula of
ascomycete symbionts in the substrate from all nurseries is
quite notable and may reflect their adaptation and/or resistance
to highly transformed nursery soil substrates compared to
symbionts from the Basidiomycota. This result is in contrast
with the findings of Kernaghan et al. (2003) on containerized P.
glauca seedlings where the ectomycorrhizal basidiomycetes
Thelephora americana and A. byssoides were dominant and the
ascomycetes were less common.
ECM symbionts from the Basidiomycota (H. crustulini-
forme, H. longicaudum, A. byssoides P. involutus, T. terrestris
and ITE.5) occurred infrequently in the surveyed nurseries.
They are all classified as pioneer or multistage species,
occurring in nurseries and young forest plantations with a low
humus content and in disturbed habitats (Last et al., 1983;
Ingleby et al., 1990; Deacon and Fleming, 1992; Kranabetter,
2004; Marmeisse et al., 2004).
The PCR–RFLP procedure is a promising method for
identifying ectomycorrhizal fungal partners. However, the
number of mycorrhizas identified to the species level in the
Ascomycota is still limited owing to a lack of fungal reference
material. It is highly probable that additional species, especially
those of the Ascomycota group, remain to be identified at bare-
root forest tree nurseries, which in the present study were
surveyed over a limited 3-year period. Continued observation
of ectomycorrhizas and ectomycorrhizal fungi in forest tree
nurseries is necessary before the effects of forest nursery
practices on particular ectomycorrhizas can be predicted and
managed.
Acknowledgements
The authors wish to thank the Board of State Forest District
in Gdansk, Olsztyn, Pila, Szczecin, Torun and Wroclaw for
encouragement to these studies and providing the plant
material. We thank Dr. Mark Tjoelker for critical reading of
the manuscript and English correction and Dr. Marek
Kasprowicz for statistical advice. We also acknowledge the
helpful comments of two anonymous reviewers. This research
was supported by KBN Grant no. 6 PO6L 027 22.
References
Agerer, R., 1987–2002. Colour Atlas of Ectomycorrhizae, 1–12th ed. Einhorn–
Verlag, Schwabisch Gmund, Germany.
Allen, M.F., 1991. The Ecology of Mycorrhizae. Cambridge University Press,
Cambridge.
Brunner, I., Brodbeck, S., 2001. Response of mycorrhizal Norway spruce
seedlings to various nitrogen loads and sources. Environ. Pollut. 114,
223–233.
Bruns, T.D, 1995. Thoughts on the process that maintain local species diversity
of ectomycorrhizal fungi. Plant Soil 170, 63–73.
Dahlberg, A., Jonsson, L., Nylund, J.-E., 1997. Species diversity and distribu-
tion of biomass above and belowground among ectomycorrhizal fungi in
an old Norway spruce forest in south Sweden. Can. J. Bot. 75, 1323–1335.
Danielson, R.M., 1982. Taxonomic affinities and criteria for identification of the
common ectendomycorrhizal symbiont of pines. Can. J. Bot. 60, 7–18.
Danielson, R.M., 1991. Temporal changes and effects of amendments on the
occurrence of sheathing (ecto-) mycorrhizas of conifers growing in oil sands
tailings and coal spoil. Agr. Ecosyst. Environ. 35, 261–281.
Danielson, R.M., Pruden, M., 1990. Ectomycorrhizae of spruce seedlings
growing in disturbed soils and in undisturbed mature forests. In: Allen,
M.F., Williams, S.E. (Eds.), Abstracts in the Proceedings of the 8th North
American Conference on Mycorrhizae. Jackson, Wyoming, p. p. 68.
Danielson, R.M., Visser, S., 1989. Effects of forest soil acidification on
ectomycorrhizal and vesicular–arbuscular mycorrhizal development. New
Phytol. 112, 41–48.
Danielson, R.M., Visser, S., 1990. The mycorrhizal and nodulation status of
container-grown trees and shrubs reared in commercial nurseries. Can. J.
For. Res. 20, 609–614.
Danielson, R.M., Griffiths, C.L., Parkinson, D., 1984. Effects of fertilization on
the growth and mycorrhizal development of container-grown jack pine
seedlings. For. Sci. 30, 828–835.
Deacon, J.W., Fleming, L.V., 1992. Interactions of ectomycorrhizal fungi. In:
Allen, M.F. (Ed.), Mycorrhizal Functioning: An Integrative Plant–Fungal
Process. Chapman & Hall, New York, pp. 249–300.
Egger, K.N., 1995. Molecular analysis of ectomycorrhizal fungal communities.
Can. J. Bot. 73 (Suppl. 1), 1415–1422.
Egli, S., Amiet, R., Zollinger, M., Schneider, B., 1993. Characterization of
Picea abies ectomycorrhizas: discrepancy between classification according
to macroscopic versus microscopic features. Trees 7, 123–129.
Erland, S., Jonsson, L., Mahmood, T., Finlay, R., 1999. Below-ground ecto-
mycorrhizal community structure in two Picea abies forests in southern
Sweden. Scand. J. For. Res. 14, 209–217.
Fassi, B., De Vecchi, E., 1963. Researches in ectotrophic mycorrhizae of Pinus
strobus in nurseries. Part I. Description of some of the most common forms
in Piedmont (summary). Allionia 8, 133–152.
Fay, D.A., Mitchell, D.T., 1999. A preliminary study of the mycorrhizal
associations of tree seedlings growing on mine spoil atavoca, Co. Wicklow.
Biology and Environment. Proc. Roy. Irish Acad. 99b (1), 19–26.
Flynn, D., Newton, A.C., Ingleby, K., 1998. Ectomycorrhizal colonisation of
Sitka spruce (Picea sitchensis (Bong.) Carr) seedlings in a Scottish planta-
tion forest. Mycorrhiza 7, 313–317.
Fransson, P.M.A., Taylor, A.F.S., Finlay, R.D., 2000. Effects of continuous
optimal fertilization on belowground ectomycorrhizal community structure
in a Norway spruce forest. Tree Physiol. 20, 599–606.
Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for
basidiomycetes—application to the identification of mycorrhizae and rusts.
Mol. Ecol. 2, 113–118.
Gardes, M., Bruns, T.D., 1996. ITS-RFLP matching for identification of fungi.
Meth. Mol. Biol. 50, 177–186.
Grogan, A., O’Neill, J.J.M., Mitchell, D.T., 1994. Mycorrhizal associations of
Sitka spruce seedlings propagated in Irish tree nurseries. Eur. J. For. Pathol.
24, 335–344.
Harley, J.L., Smith, S.E., 1983. Mycorrhizal Symbiosis. Academic Press, London.
Haug, I., 2002. Identification of Picea-ectomycorrhizae by comparing DNA-
sequences. Mycol. Prog. 1 (2), 167–178.
Henrion, B., Di Battista, C., Bouchard, D., Vairelles, D., Thompson, B.D., Le
Tacon, F., Martin, F., 1994. Monitoring the persistence of Laccaria bicolor
as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the
rDNA intergenic spacer. Mol. Ecol. 3, 571–580.
Humphrey, J., Ferris, R., Newton, A., Peace, A., 2003. The value of conifer
plantations as a habitat for macrofungi. In: Humphrey, J., Ferris, R., Quine,
C.P (Eds.), Biodiversity in Britain’s Planted Forests. Forestry Commission,
Edinburgh, p. 118 pp.
M. Rudawska et al. / Forest Ecology and Management 236 (2006) 375–384384
Ingestad, T., 1962. Macroelement nutrition of pine, spruce and birch seedlings
in nutrient solutions. Medd. Skogsforskn. Inst. Stockholm 51 (7), 1–50.
Ingleby, K., Mason, P.A., Last, F.T., Fleming, L.V., 1990. Identification of
ectomycorrhizas. ITE Research, De la Bastide and Kendrick Publication no.
5. HMSO, London.
Iwanski, M., Rudawska, M., Leski, T., 2006. Mycorrhizal associations of
nursery grown Scots pine (Pinus sylvestris L.) seedlings in Poland. Ann.
For. Sci. 63, 715–723.
Jonsson, L., Dahlberg, A., Tor-Erik, B., 2000. Spatiotemporal distribution of an
ectomycorrhizal community in an oligotrophic Swedish Picea abies forest
subjected to experimental nitrogen addition: above- and below-ground
views. For. Ecol. Manage. 132, 143–156.
Karen, O., Nylund, J.-E., 1996. Effects of N-free fertilization on ectomycorrhiza
community structure in Norway spruce stands in southern Sweden. Plant
Soil 181, 295–305.
Karen, O., Hogberg, N., Dahlberg, A., Jonsson, L., Nylund, J.E., 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.
Kernaghan, G., Sigler, L., Khasa, D., 2003. Mycorrhizal and root endophytic
fungi of containerized Picea glauca seedlings assessed by rDNA sequence
analysis. Microbial. Ecol. 45, 128–136.
Kieliszewska-Rokicka, B., Rudawska, M., Staszewski, T., Kurczynska, E.,
Karlinski, L., Kubiesa, P., 2003. Ectomycorrhizal associations in Norway
spruce stands influenced by long lasting air pollution (Silesian Beskid
Mountains, Poland). Ekol. Bratislava 23, 142–149.
Kraigher, H., 1999. Diversity of types of ectomycorrhizae on Norway spruce in
Slovenia. Phyton 39, 199–202.
Kraigher, H., Batic, F., Agerer, R., 1996. Types of ectomycorrhizae and
mycobioindication of forest site pollution. Phyton 36, 115–120.
Kranabetter, J.M., 2004. Ectomycorrhizal community effects on hybrid spruce
seedling growth and nutrition in clearcuts. Can. J. Bot. 82, 983–991.
Krasowski, M.J., Owens, J.N., Tackaberry, L.E, Massicotte, H.B., 1999. Above-
and below-ground growth of white spruce seedlings with roots divided into
different substrates with or without controlled-release fertilizer. Plant Soil
217, 131–143.
Last, F.T., Mason, P.A., Wilson, J., Deacon, J.W., 1983. Fine roots and sheathing
mycorrhizas. Their formation, function and dynamics. Plant Soil 71, 9–21.
Ławrynowicz, M., 1988. Grzyby (Mycota), vol. XVIII. PWN, Warszawa,
Krakow.
Lenart, G.A., 2000. In: Milewski, W. (Ed.), Forests in Poland. The State Forests
Information Centre, 48 pp.
Letho, T., 1989. Mycorrhizal status of Scots pine nursery stock in Finland. Folia
Forest 726, 1–15.
LoBuglio, K.F., 1999. Cenococcum geophilum. In: Cairney, J.W.G., Chambers,
S.M. (Eds.), Ectomycorrhizal Fungi: Key Genera in Profile. Springer, New
York, pp. 287–309.
Mahmood, S., Finlay, R.D., Erland, S., 1999. Effects of repeated harvesting of
forest residues on the ectomycorrhizal community in a Swedish spruce
forest. New Phytol. 142, 577–585.
Marmeisse, R., Guidot, A., Gay, G., Lambilliotte, R., Sentenac, H., Combier, J.-
P., Melayah, D., Fraissinet-Tachet, L., Debaud, J.C., 2004. Hebeloma
cylindrosporum—a model species to study ectomycorrhizal symbiosis from
gene to ecosystem. New Phytol. 163, 481–498.
Massicotte, H.B., 1994. Characterization of ectomycorrhizal morphotypes. In:
Brundrett, M., Melville, L., Peterson, L. (Eds.), Practical Methods in
Mycorrhiza Research. Mycologue Publications, Chapter 11, pp. 88–94.
Mehmann, B., Egli, S., Braus, G.H., Brummer, I., 1995. Coincidence between
molecularly or morphologically classified ectomycorrhizal morphotypes
and fruitbodies in a spruce forest. In: Stocchi, V. (Ed.), Biotechnology of
Ectomycorrhizae. Plenum Press, New York, pp. 41–52.
Menkis, A., Vasiliauskas, R., Taylor, A.F.S., Stenlid, J., Finlay, R., 2005. Fungal
communities in mycorrhizal roots of conifer seedlings in forest nurseries
under different cultivation systems, assessed by morphotyping, direct
sequencing and mycelial isolation. Mycorrhiza 16, 33–41.
Molina, R., Chamard, J., 1983. Use of the ectomycorrhizal fungus Laccaria
laccata in forestry. Part II. Effects of fertilizer forms and levels on
ectomycorrhizal development and growth of container-grown Douglas-fir
and ponderosa pine seedlings. Can. J. For. Res. 13, 89–95.
Molina, R., Trappe, J.M., 1982. Patterns of ectomycorrhizal host specificity and
potential among Pacific Northwest conifers and fungi. For. Sci. 28, 423–
458.
Perry, D.A., Molina, R., Amaranthus, M.P., 1987. Mycorrhizae, mycorrhizo-
spheres and reforestation: current knowledge and research needs. Can. J.
Bot. 17, 929–940.
Peter, M., Ayer, F., Egli, S., 2001a. Nitrogen addition in a Norway spruce stand
altered macromycete sporocarp production and below-ground ectomycor-
rhizal species composition. New Phytol. 149, 311–325.
Peter, M., Ayer, F., Egli, S., Honneger, R., 2001b. Above- and below-ground
community structure of ectomycorrhizal fungi in three Norway spruce
(Picea abies) stands in Switzerland. Can. J. Bot. 79, 1134–1151.
Rudawska, M., Leski, T., Gornowicz, R., 2001. Mycorrhizal status of Pinus
sylvestris L. nursery stock in Poland as influenced by nitrogen fertilization.
Dendrobiology 46, 49–58.
Shihido, M., Massicotte, H.B., Chanway, C.B., 1996. Effect of plant growth
promoting Bacillus strains on pine and spruce seedling growth and mycor-
rhizal infection. Ann. Bot. 77, 433–441.
Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis, 2nd Ed. Academic
Press, London.
Taylor, D.L., Bruns, T.D., 1999. Community structure of ectomycorrhizal fungi
in a Pinus muricata forest: minimal overlap between the mature forest and
resistant propagules communities. Mol. Ecol. 8, 1837–1850.
ter Braak, C.J.F., Smilauer, P., 1998. CANOCO reference manual and user’s
guide to Canoco for Windows: software for canonical community ordina-
tion (Ver. 4) Microcomputer Power, Ithaca, NY.
Thomas, G.W., Jackson, R.M., 1979. Sheathing mycorrhizas of nursery grown
Picea sitchensis. Trans. Br. Mycol. Soc. 73 (1), 117–125.
Trappe, J.M., 1969. Mycorrhiza-forming ascomycetes. In: Proceedings of the
first North American Conference on Mycorrhizae, April, 1969, Misc.
Publication 1189. U.S. Department Agriculture, Forest Service, pp. 19–37.
Ursic, M., Peterson, R.L., 1997. Morphological and anatomical characterization
of ectomycorrhizas and ectendomycorrhizas on Pinus strobus seedlings in a
southern Ontario nursery. Can. J. Bot. 75, 2057–2072.
Ursic, M., Peterson, R.L., Husband, B., 1997. Relative abundance of mycor-
rhizal fungi and frequency of root rot on Pinus strobus seedlings in a
southern Ontario nursery. Can. J. For. Res. 27, 54–62.
Weiss, M., Agerer, R., 1988. Studien an Ektomykorrhizen XII Drei nichtiden-
tiefierte Mykorrhizen an Picea abies (L.) Karst. aus einer Baumschule. Eur.
J. For. Pathol. 18, 26–43.
Xavier, L.J.C., Germida, J.J., 1999. Impact of humans on soil microorganisms
microbial biosystems: new frontiers. In: Bell, C.R., Brylinsky, M., Johnson-
Green, P. (Eds.), Proceedings of the 8th International Symposium on
Microbial Ecology, Atlantic Canada Society for Microbial Ecology,
Halifax, Canada.