Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment

Abstract

This study aimed to elucidate the relationship between ericaceous understorey shrubs and the diversity and abundance of ectomycorrhizal fungi (EcMF) associated with the invasive Pinus strobus and native Pinus sylvestris. Seedlings of both pines were grown in mesocosms and subjected to three treatments simulating different forest microhabitats: (a) grown in isolation and grown with (b) Vaccinium myrtillus or (c) Vaccinium vitis-idaea. Ericaceous plants did not act as a species pool of pine mycobionts and inhibited the ability of the potentially shared species Meliniomyces bicolor to form ectomycorrhizae. Similarly, Ericaceae significantly reduced the formation of Thelephora terrestris ectomycorrhizae in P. sylvestris. EcMF species composition in the mesocosms was strongly affected by both the host species and the presence of an ericaceous neighbour. When grown in isolation, P. strobus root tips were predominantly colonised by Wilcoxina mikolae, whereas those of P. sylvestris were more commonly colonised by Suillus and Rhizopogon spp. Interestingly, these differences were less evident (Suillus + Rhizopogon spp.) or absent (W. mikolae) when the pines were grown with Ericaceae. P. strobus exclusively associated with Rhizopogon salebrosus s.l., suggesting the presence of host specificity at the intrageneric level. Ericaceous plants had a positive effect on colonisation of P. strobus root tips by R. salebrosus s.l. This study demonstrates that the interaction of selective factors such as host species and presence of ericaceous plants may affect the realised niche of the ectomycorrhizal fungi.

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ORIGINAL PAPER
Ericaceous dwarf shrubs affect ectomycorrhizal fungal
community of the invasive Pinus strobus and native Pinus
sylvestris in a pot experiment
Petr Kohout &Zuzana Sýkorová &
Mohammad Bahram &Věroslava Hadincová &
Jana Albrechtová &Leho Tedersoo &Martin Vohník
Received: 24 August 2010 /Accepted: 23 November 2010 /Published online: 14 December 2010
#Springer-Verlag 2010
Abstract This study aimed to elucidate the relationship
between ericaceous understorey shrubs and the diversity
and abundance of ectomycorrhizal fungi (EcMF) associated
with the invasive Pinus strobus and native Pinus sylvestris.
Seedlings of both pines were grown in mesocosms and
subjected to three treatments simulating different forest
microhabitats: (a) grown in isolation and grown with (b)
Vaccinium myrtillus or (c) Vaccinium vitis-idaea. Erica-
ceous plants did not act as a species pool of pine
mycobionts and inhibited the ability of the potentially
shared species Meliniomyces bicolor to form ectomycor-
rhizae. Similarly, Ericaceae significantly reduced the for-
mation of Thelephora terrestris ectomycorrhizae in P.
sylvestris. EcMF species composition in the mesocosms
was strongly affected by both the host species and the
presence of an ericaceous neighbour. When grown in
isolation, P. strobus root tips were predominantly colonised
by Wilcoxina mikolae, whereas those of P. sylvestris were
more commonly colonised by Suillus and Rhizopogon spp.
Interestingly, these differences were less evident (Suillus+
Rhizopogon spp.) or absent (W. mikolae) when the pines
were grown with Ericaceae. P. strobus exclusively associ-
ated with Rhizopogon salebrosus s.l., suggesting the
presence of host specificity at the intrageneric level.
Ericaceous plants had a positive effect on colonisation of
P. strobus root tips by R. salebrosus s.l. This study
demonstrates that the interaction of selective factors such
as host species and presence of ericaceous plants may affect
the realised niche of the ectomycorrhizal fungi.
Keywords Plant invasions .Seedlings establishment .
Ericoid mycorrhiza .Rhizoscyphus ericae aggregate .Suillus
spp. .Rhizopogon salebrosus complex .Meliniomyces
bicolor .Common mycorrhizal networks
Introduction
Invasive plants are among the greatest threats to species
biodiversity and ecosystem functioning (Mack et al. 2000)
and may have a marked economic effect (Pimentel et al.
2005). Invasiveness of plants depends on the environment
and species-specific ecological traits, such as growth rate,
Electronic supplementary material The online version of this article
(doi:10.1007/s00572-010-0350-2) contains supplementary material,
which is available to authorized users.
P. Kohout (*):Z. Sýkorová :J. Albrechtová :M. Vohník
Department of Mycorrhizal Symbioses,
Institute of Botany ASCR,
Zámek 1,
Průhonice 25243, Czech Republic
e-mail: kohout4@natur.cuni.cz
P. Kohout :J. Albrechtová :M. Vohník
Department of Experimental Plant Biology, Faculty of Science,
Charles University in Prague,
Viničná 5,
12843, Prague, Czech Republic
M. Bahram :L. Tedersoo
Institute of Ecology and Earth Sciences, Tartu University,
40 Lai,
51005, Tartu, Estonia
V. Hadincová
Department of Population Ecology, Institute of Botany ASCR,
25243, Průhonice, Czech Republic
M. Bahram :L. Tedersoo
Natural History Museum, Tartu University,
40 Lai,
51005, Tartu, Estonia
Mycorrhiza (2011) 21:403–412
DOI 10.1007/s00572-010-0350-2

escape from pathogens, production of allelochemicals, etc.
(Rejmánek 1989; Keane and Crawley 2002). However,
sometimes the mechanisms triggering invasiveness are
unpredictable and may depend on other factors or combi-
nations of factors including biotic interactions. In particular,
plant invasions may be facilitated by the presence of
compatible mutualists such as pollinators, seed dispersers
and mycorrhizal fungi (Richardson et al. 2000; Klironomos
2003; Pringle et al. 2009). In their new range, invaders
often lack suitable partners, but new host–mutualist
combinations may emerge (Richardson et al. 2000). Soil
biota may enhance plant invasions (Reinhart and Callaway
2006). Non-native plants, in turn, may alter soil microbial
communities via root exudates (van der Putten et al. 2007).
Ectomycorrhizal (EcM) symbioses benefit coniferous
trees by facilitating water and nutrient uptake, protecting
roots from pathogens and interconnecting single plants into
common mycorrhizal networks (CMNs; Selosse et al. 2006;
Smith and Read 2008). Fungi that are involved in EcM
symbiosis (EcMF) differ in their ability to exploit various
substrates, indicating certain-level functional complemen-
tarity (van der Heijden and Kuyper 2003; Courty et al.
2005). Accordingly, plant seedlings may benefit from
associating simultaneously with several EcMF particularly
when growing in complex substrates (Baxter and Dighton
2001; Jonsson et al. 2001). Naturally, the great diversity of
EcMF communities depends on both biotic and abiotic
factors, such as host plant, soil quality, pollution, etc.
(Lilleskov et al. 2002; Ishida et al. 2007; Morris et al. 2008;
Tedersoo et al. 2008a,b).
In the southern hemisphere, the establishment and
naturalisation of introduced Pinus spp. was retarded in
regions that lacked compatible EcMF (Mikola 1969; Read
1998; Richardson et al. 2000; Nuñez et al. 2009). Recent
studies suggest that introduced and/or invasive plants
associate with fewer EcMF than the native species
(Tedersoo et al. 2007; Nuñez et al. 2009; Dickie et al.
2010; Walbert et al. 2010). Native plants may share their
EcMF with Pinaceae (Horton et al. 1999; Richard et al.
2009), except in exotic habitats (Tedersoo et al. 2007).
Understorey vegetation in many boreal, temperate and
Mediterranean forests consists of ericaceous dwarf shrubs
that form ericoid mycorrhiza (ErM; Read 1991). ErM
mycobionts from the Rhizoscyphus ericae (Read) Zhuang
& Korf aggregate are taxonomically distinct from common
EcMF, but some of them are capable of forming EcM
symbiosis (Vrålstad et al. 2000,2002). This led to
hypotheses that ericaceous and ectomycorrhizal plants
might be functionally interconnected with CMNs (Vrålstad
2004) and that Ericaceae could serve as an inoculum
reservoir for Pinaceae. However, to date, only a single R.
ericae aggregate strain has been demonstrated to form EcM
and ErM simultaneously (Villarreal-Ruiz et al. 2004). ErM
fungi (ErMF) also occur as common root endophytes in non-
ericaceous hosts (Hambleton and Sigler 2005;Curlevskiet
al. 2009; Tedersoo et al. 2009).
Pinus strobus L. was introduced in the Czech Republic as
an ornamental tree as early as in 1784, and the first
plantations were established in the Elbe Sandstone Mountains
(NW Czech Republic) in 1789 (Nožička 1965). However,
reports of its invasion in different types of forests did not start
to accumulate until as late as the early 1990s (V. Hadincová
and co-workers, unpublished data). P. s t r o b u s is now
commonly found in native forests in many sandstone areas,
often regenerating within ericaceous understorey in the early
stages of its invasion. Due to a combination of effective seed
spread, high seedling recruitment, fast growth and enormous
litter production (Hadincová et al. 2007; Kubartová 2007), P.
strobus inhibits the growth of understorey vegetation and
regeneration of native trees, especially Pinus sylvestris L.
According to Carrillo-Gavilan and Vila (2010), P. strobus is
the only invasive conifer in Europe. The role of mutualistic
symbioses, including EcM, in the P. strobus,invasions
remain unknown.
This study aimed to address the potential role of EcMF
and ErMF in facilitating the invasion of P. strobus.In
particular, the EcMF resistant propagule communities
associated with the roots of both P. strobus and P. sylvestris
were studied in order to test the hypotheses that (a) P.
strobus associates with a less diverse and more generalist
EcMF spectrum than P. sylvestris, (b) EcMF communities
of both pine species are affected by the presence of
ericaceous plants and (c) there is an increase in the
abundance of ectomycorrhizal members of the R. ericae
aggregate in the presence of ericaceous plants, due to their
supposed co-association with Ericaceae.
Materials and methods
Experimental design and sampling
To test the above hypotheses, a mesocosm experiment was
set up because it has advantages over a field experiment: all
experimental units experience the same environmental
conditions, including exposure to soil fungi and the
confounding effect of spatial heterogeneity is avoided,
resulting in less variation among replicates (Pickles et al.
2010). A pot experiment may alter the realisable spectrum
of EcMF in favour of species dispersing predominantly by
spores. However, if the EcMF spore community introduced
into a pot experiment reflects, the EcMF spore community
occurring in the field (e.g. the inoculation is done with a
non-treated natural substrate), the differences in the realised
EcMF spectrum between a pot experiment and the situation
in the field should not be large. On the other hand, the
404 Mycorrhiza (2011) 21:403–412

mesocosm approach may select for generalists and against
fungi outside their ecophysiological optimum.
In May 2005, soil (mineral, organic and litter layer) was
collected at two sites in the Elbe Sandstone Mountains: (a)
Babylon (50°52.20′N; 14°22.90′E, 310 m above sea level)
and (b) Picket (50°52.84′N; 14°20.87′E, 380 m above sea
level) that are located ca. 5 km apart. Babylon is a forest
dominated by P. sylvestris; the closest P. strobus trees are
100 m away. Picket is a mixed forest dominated by P.
strobus,P. sylvestris and Picea abies L. Karst. Both sites
have a similar understorey of ericaceous shrubs (Vaccinium
myrtillus L. and Vaccinium vitis-idaea L.) and podzolic
soils. At both sites, 6 kg of soil was collected from five
cores located approximately 20 m apart. The soils from the
two localities were pooled (each layer separately), mixed
and used as a substrate in 1.5-l plastic pots (diameter
15 cm). This was done to maximise the diversity of EcMF
available in the substrate as an inoculum for the seedlings.
The soil in each pot consisted of three layers as in the field
(i.e. 1 cm Land 10 cm F+Hlayers, 5 cm sand drainage).
The experiment was two-factorial: (a) Pinus species (the
invasive P. strobus and native P. sylvestris) and (b) presence
of an ericaceous neighbour (V. myrtillus,V. vitis-idaea or
none). There were eight replicates of each treatment; thus,
the number of mesocosms totalled 48. Thirty seeds of either P.
sylvestris or P. strobus originating from the Elbe Sandstone
Mountains were sown in each of the mesocosms. P. sylvestris
seeds were sown in March 2006. As P. strobus seeds need
cold stratification, they were sown in September 2005. The
seeds germinated simultaneously in April 2006. Five healthy
seedlings were left per each pot. Single branches of
ericaceous shrubs along with their rhizomes, and roots were
transplanted from the sites where the soil was collected.
Thus, they were most likely pre-colonised by native ErMF.
Ericaceous plants were collected at the same time as the soil
and replanted in pots in May 2005.
The mesocosms were kept outdoors in the experimental
garden of the Institute of Botany ASCR at Průhonice (49°
59.86′N; 14°33.97′E; 300 m above sea level; mean annual
temperature 8.8°C, mean annual precipitation 565 mm).
The pots were randomly placed on the ground, shaded by
green plastic netting and watered when needed during hot
summer days. The mesocosms were randomly harvested in
September 2008. At the harvest, soil was carefully removed
from the roots using running tap water, and the roots of
Pinus spp. were carefully separated from the hairy roots of
ericaceous plants. The fine roots of Pinus species were then
cut into 5 cm lengths. For each mesocosm, 40 randomly
selected lengths of roots were examined under a dissecting
microscope which resulted in 2 m of root length analysed
per pot. EcM root tips were counted (bifurcated and
coralloid root tips were counted as a single root tip) and
separated into morphotypes based on surface texture, colour
and presence and type of hyphae and rhizomorphs. Morpho-
typing of all EcM root tips was done by the same evaluator
(P. K.). For each morphotype from each pot, three to 15 of the
most healthy-looking and cleanest ectomycorrhizae were
placed in 0.5 ml Eppendorf tubes containing 70% ethanol
and stored at 4°C until required for molecular analyses. In
total, 580 root tips were individually subjected to molecular
analyses. The molecular analysis indicated that each EcM
morphotype corresponded to a single fungal taxon. Only the
suilloid morphotype was separated into eight species of
Suillus and Rhizopogon based on 200 internal transcribed
spacer (ITS) sequences (Supplementary material S2).
Molecular analyses
Prior to DNA extraction, individual root tips were surface-
sterilised in a 100% commercial bleach solution (4.5%
available chlorine), containing 100 μl/l of Tween 20, for
30 s, followed by 30 s in 70% ethanol and 3×1-min rinses
in sterile water. Root tips were then ground in liquid
nitrogen. DNA was extracted from each root tip using the
DNeasy Plant Mini extraction kit (Qiagen GmbH, Hilden,
Germany) following the manufacturer’s instructions. DNA
was eluted in 75 μl of sterile ddH
2
O and kept at −20°C.
Polymerase chain reaction (PCR) amplification of the ITS
region was performed using the primers ITS1F (Gardes and
Bruns 1993) and ITS4 (White et al. 1990). Additionally,
samples from the basidiomycetous morphotypes were ampli-
fied using a combination of primer ITS1F and
Basidiomycetes-specific primer ITS4B (Gardes and Bruns
1993) or LB-W (Tedersoo et al. 2008a) in order to avoid co-
amplification of ascomycetes that commonly inhabit EcM
root tips (Tedersoo et al. 2009). The PCR mix included
2.5 μl of 10× PCR buffer without MgCl
2
,2μlofdNTPs
mixture (200 nM), 2.5 μlMgCl
2
(2 mM), 0.5 μlofeach
primer (10 mM), 1 U of Taq DNA polymerase (Fermentas
International Inc, Burlington, ON, Canada), 15.8 μlofsterile
ddH
2
Oand8μl of the template (DNA extract diluted 1:10 in
sterile water) in a final volume of 25 μl.
Thermal cycling parameters were as follows: initial
denaturation step of 4 min at 94°C, 35 cycles consisting
of a denaturation step at 94°C for 30 s, annealing at 55°C
for 30 s, extension at 72°C for 70 s and a final extension at
72°C for 10 min. The length and quality/quantity of the
PCR products were checked using gel electrophoresis (1%
agarose). Samples that yielded double-banded PCR prod-
ucts were excluded from further analyses. In the case of
barely visible PCR products, a semi-nested or nested PCR
was performed using primers ITS1 and ITS4 with 1 μlof
the ITS1F/ITS4 or ITS1F/LB-W PCR product as a template
(diluted 1:100 in sterile water). PCR products were purified
using the QIAquick PCR Purification Kit (Qiagen GmbH,
Hilden, Germany). Each sample was separately sequenced
Mycorrhiza (2011) 21:403–412 405

with the primer ITS1 or ITS1F in Macrogen Inc. (Seoul,
South Korea). The DNA sequences were checked for
possible machine errors and edited in Sequence Scanner
1.0 (Applied Biosystems, Forest City, CA, USA). Prelim-
inary identification of EcMF was achieved by conducting a
nucleotide Basic Local Alignment Search Tool (BLASTn)
search of the GenBank and UNITE (Abarenkov et al. 2010)
public sequence databases.
Sequence analyses
Alignment of sequences was performed using the CLUSTAL
W algorithm (Thompson et al. 1994) in BioEdit 7.0.9.0 (Hall
1999), followed by manual correction. Neighbour-joining
analyses were conducted using TOPALi version 2.5 (http://
www.topali.org/). To determine the phylogenetic affinities of
species belonging to the suilloid morphotype and R. ericae
aggregate, selected ITS sequences from identified fruit
bodies or pure cultures were downloaded from the sequence
databases. The sequences were aligned as above. Neighbour-
joining analyses were performed using MEGA 4 (Tamura et
al. 2007) with 1,000 bootstrap replicates. A value of 97.0%
similarity in the ITS region was used as a threshold for
species delimitation. Representative sequences of each
species or sequence type from each experimental treatment
were submitted to National Center for Biotechnology
Information (accession numbers FN678889–FN678898,
FN679001–FN679049, FN686777, FN686778, FN691763
and FN811647–FN811657).
Statistical analyses
The influence of EcM host plant (P. sylvestris or P. strobus)
and cultivation treatment (presence/absence of ericaceous
plants) on EcM colonisation and EcMF species richness was
analysed using the non-parametric Kruskal–Wallis test in
software package STATISTICA 8 (StatSoft, Inc., Tulsa, OK,
USA). The EcM colonisation was expressed as a number of
EcM root tips per 2 m of the root length. To determine the
effects on each EcMF species separately, Fisher’s exact test
based on their occurrence patterns in mesocosms was used.
Presence–absence data per pot were used instead of
abundance because it was not possible to quantify species
of Rhizopogon and Suillus within the suilloid morphotype;
thus, absence of a suilloid species may equally indicate that
its abundance was below the molecular detection limit.
The relative effect of EcM host plant, presence of
ericaceous shrubs and their interaction on the perceived EcMF
community structure were further tested using the ADONIS
routine and visualised using the non-metric multidimensional
scaling (NMDS) in the Vegan package of R (R Core
Development Team 2007). Species occurrence and Bray–
Curtis distance measure were used in both analyses.
Results
EcM fungi species richness and diversity
In all treatments, nearly all pine roots were ectomycor-
rhizal. In total, approximately 11,500 root tips from Pinus
spp. seedlings were analysed by morphotyping (Table 1and
Supplementary material S1), and 580 EcM root tips were
consequently used for DNA extraction and PCR. However,
only 330 root tips produced a sequence of sufficient quality
that could be matched to an EcM fungus. According to the
Kruskal–Wallis test, the total number of EcM root tips per
2 m root length in each mesocosm was similar in the two
Pinus spp. and three ericaceous plant treatments (Table 1).
In total, 13 fungal taxa were identified. Seven species of
EcMF (Cenococcum geophilum Fr., Inocybe sp. Fr.,
Rhizopogon luteolus Fr., Rhizopogon roseolus s.l. (Corda)
Th. Fr., Rhizopogon salebrosus s.l. A.H. Sm., Thelephora
terrestris Ehrh. ex Fr. and Wilcoxina mikolae Yang &
Wilcox) were identified based on their closest ITS BLAST
match (Table 2). The remaining six were members of the
genus Suillus and of the R. ericae aggregate and were
identified to species level based on phylogenetic analyses
(Fig. 1; Supplementary material S3).
There were 11 and 10 fungal species associated with the root
tips of P. sylvestris and P. s t r o b u s , respectively, of which eight
were shared. Of all root tips, 46% and 72% of those of P.
sylvestris and P. strobus, respectively, were colonised by
Ascomycota. There were no significant effects of pine species
or ericaceous neighbours on EcMF species richness (p>0.05).
Community composition of EcM fungi
The ascomycete W. mikolae was the most abundant EcMF
species associated with the roots of both pine species in all
treatments (on average 53.1%), except where P. sylvestris
was grown in isolation (Fig. 2). Meliniomyces bicolor
Hambl. & Sigler (a member of the R. ericae aggregate)
was almost exclusively detected in treatments where the pine
species were grown in isolation (Fisher’sexacttest:
p<0.001; Table 3), colonising on average 47.2% of the root
tips. Similarly, T. terrestris wasmainlyrecordedwhenthere
were no ericaceous neighbours (p=0.022). Conversely, R.
salebrosus s.l. was significantly more frequent in treatments
with an ericaceous neighbour (p=0.048). More suilloid fungi
(six subspecies, 37.4% of all root tips) were associated with
P. sylvestris when it was grown in isolation. In contrast, four
species of suilloid fungi colonised only 3.5% of root tips of
P. strobus grown without an ericaceous neighbour. The
ascomycetes W. mikola e and M. bicolor colonised more than
95% of all EcM root tips in this treatment.
Similarly, host plants had a strong effect on the occurrence
of individual EcMF species (Table 3). In particular, Suillus
406 Mycorrhiza (2011) 21:403–412

bovinus (Pers.) Roussel (p=0.023) and T. terrestris (p=
0.002) were significantly more frequently, but not exclusive-
ly, associated with P. sylvestris. Conversely, R. salebrosus s.l.
was only associated with P. strobus (p<0.001). According to
Fisher’s exact test, W. mikolae was significantly more
frequently associated with P. strobus (p=0.008). This result
was probably due to the complete absence of W. mikol a e
on the eight seedlings of P. sylvestris grown in isolation.
These results were supported at the community level,
where the host plant (ADONIS: F
1, 42
=14.38; p<0.001),
ericaceous neighbour (F
2, 42
=13.03; p<0.001) and their
interaction (F
2, 42
=4.59; p< 0.001), respectively, explained
15.7%, 28.44% and 10.02% of total variation in the EcMF
species distribution (Fig. 3).
Discussion
EcM communities associated with the native vs. invasive
pine
In this mesocosm study of a resistant propagule community,
there were no differences in species richness of EcMF
colonising the native and introduced species of Pinus. This
study along with those of Nuñez et al. (2009) and Tedersoo
et al. (2007) indicates that introduced or invasive EcM trees
may host a comparable number of symbionts when a
suitable species pool is present. However, the mesocosm
approach used in our study might select for generalists and
against fungi outside their ecophysiological amplitude.
The ascomycetes W. mikolae and M. bicolor and suilloid
fungi were the most abundant EcMF associated with both
pine species. This observation accords with previous
studies on resistant propagule and nursery communities of
conifer seedlings (Iwanski et al. 2006; Rusca et al. 2006).
Leski et al. (2010) showed that P. sylvestris seedling
survival is negatively correlated with the relative abundance
of W. mikolae but positively correlated with the relative
abundance of suilloid mycorrhizae. This indicates that
EcMF taxa may significantly differ in their effects on host
fitness and therefore have different roles in the pine
invasion processes. Interestingly, Ericaceae supported for-
mation of both W. mikolae (in P. sylvestris) and suilloid (in
P. strobus) ectomycorrhizae at the expense of M. bicolor,
underlining the differential effect of surrounding vegetation
on the native vs. introduced pine (see below).
In particular, host species had a strong effect on the
frequency of several EcMF species, which contrasts with
the results of most previous studies on intergeneric EcMF
selectivity (Molina and Trappe 1994; Walker et al. 2005;
Tedersoo et al. 2008a). However, Morris et al. (2008,2009)
demonstrated that host species is an important factor
Table 1 Total numbers of EcM root tips per treatment and numbers of EcM root tips colonised by different morphotypes in each treatment per
2 m root length (means±SD; n=8)
Suilloid M. bicolor W. mikolae Inocybe spp. T. terrestris C. geophilum Total no. of EcM root tips Nm
Sy 89± 42.8 111±53 0 0 35± 20.5 7±15.8 246± 42 0
SyVm 166± 109.4 2± 5 124± 124 2± 5.6 2±4.6 0 296± 42 0
SyVt 73± 32.4 0 75± 77.4 0.3± 0.7 1±3.9 0 165± 45 0
St 12± 23.8 163± 157.7 166 ± 124.1 0 0 0 341± 42 0.8± 1.2
StVm 35± 35.1 0.4± 1.1 178±61.1 8±15.9 1± 2.8 0 236±42 1 ± 1.8
StVt 95± 61 0.1± 0.4 104 ± 127.4 0 0 0 230± 45 2± 2.4
Each row represents a single treatment
Sy P. sylvestris grown in isolation, SyVm P. sylvestris gown with V. myrtillus,SyVv P. sylvestris grown with V. vitis-idaea,St P. strobus grown in
isolation, StVm P. strobus grown with V. myrtillus,StVv P. strobus grown with V. vitis-idaea,Nm non-mycorrhizal
EcM species EMBL accession number Best BLASTn species match: accession number,
similarity (%), species identity
Cenococcum geophilum FN686778 AY880936 (98%) Cenococcum geophilum
Inocybe sp. FN679046 AM882710 (99%) Inocybe jacobi
Rhizopogon luteolus FN679020 EU784397 (99%) Rhizopogon luteolus
Rhizopogon roseolus s.l. FN679014 EU784401 (98%) Rhizopogon roseolus
Rhizopogon salebrosus s.l. FN679024 FJ197209 (98%) Rhizopogon salebrosus
Thelephora terrestris FN679049 AF272921 (100%) Telephora terrestris
Wilcoxina mikolae FN679042 DQ093774 (99%) Wilcoxina mikolae
Table 2 Identification of EcM
fungi based on a BLASTn
search of the public sequence
databases GenBank and UNITE
Mycorrhiza (2011) 21:403–412 407

affecting the EcMF community composition in ecosystems
of two co-occurring Quercus species. Congruently,
Jacobson and Miller (1992) recorded the host specificity
among cryptic species of Suillus granulatus (L.) Roussel
for the subgenera Pinus and Strobus. It is probable that
incompatibility between invasive host plants and indige-
nous EcMF could shift communities of EcMF associating
with a mature forest dominated by invasive pines. These
changes could result in a large impact on functional traits
in soil as well as extinctions of crucial ecosystems
components, e.g. indigenous EcMF. However, more
detailed studies are needed to evaluate the impact of
invasive pines on indigenous EcMF communities.
We detected several EcMF that have not been found in
Europe based on sequenced fruit-body data. However, the
phylogenetic analyses indicate that the closest relatives of S.
granulatus 2 originated in Europe (Fig. 1). Similarly, a
sequence derived from Arctostaphylos uva-ursi (L.) Spreng.
from the Alps (Krpata et al. 2007) nested in our sequences of
R. salebrosus s.l. (Supplementary material S4). Thus, this
taxonomically overlooked species may be derived from the
communities of Pinus cembra L., which belongs to Pinus
subgenus Strobus and is a native of the Alps. It is speculated
here that R. salebrosus s.l. may have switched subsequently
to P. strobus, following its invasion. The only known host
plant of R. salebrosus s.l. in Europe is A. uva-ursi,whichis
also an understorey plant in The Elbe Sandstone Mountains.
Thus, A. uva-ursi may have facilitated the host switch from P.
cembra to P. s t r o b u s (Horton et al. 1999;Krpataetal.2007).
The effects of ericaceous plants
There were no statistically significant effects of ericaceous
plants on colonisation and richness of EcMF in the
mesocosm experiment. In field studies, ericaceous plants
inhibit EcM colonisation (Walker et al. 1999; Collier and
Fig. 1 Phylogenetic tree of a
part of the genus Suillus based
on a neighbour-joining analysis
of 494 characters of ITS1, 5,8S
rDNA and part of the ITS2
sequences. The numbers above
branches denote neighbour-
joining bootstrap values from
1,000 replications. The tree was
rooted using sequences of R.
luteolus and R. roseolus.
Sequences obtained in the pres-
ent study are shown in bold.
They are labelled with the
database accession number, the
host plant species from which
they were obtained and the
cultivation treatment (Sy P. syl-
vestris grown in isolation, SyVm
P. sylvestris gown with V.
myrtillus,SyVv P. sylvestris
grown with V. vitis-idaea,St P.
strobus grown in isolation, StVm
P. strobus grown with V.
myrtillus,StVv P. strobus grown
with V. vitis-idaea). The
parentheses show the
delimitation of the fungal taxa
408 Mycorrhiza (2011) 21:403–412

Bidartondo 2009) and/or reduce the number of EcMF
species (Collier and Bidartondo 2009). Nevertheless, in our
study, ericaceous plants had the strongest effect on EcMF
community composition.
The differential effect of ericaceous plants on the EcMF
communities of the two Pinus species can only partly be
attributable to host specificity. For example, W. mikolae did
not form an association with P. sylvestris but did with P.
strobus when it was grown in isolation. However, it heavily
colonised both pines when grown with ericaceous shrubs.
As W. mikolae is regarded as a poor competitor (Mikola
1969), the fungi preferring P. sylvestris may have out-
competed W. mikolae in favourable conditions in the
absence of ericaceous neighbours.
This indicates that factors such as host, surrounding
vegetation and soil conditions may interact to create
niches for EcMF. At the species level, ericaceous plants
promoted the proliferation of one EcMF species (R.
salebrosus s.l.) and inhibited two EcMF species (T.
terrestris and M. bicolor). The selective effect of erica-
ceous dwarf shrubs on EcMF is intriguing and deserves
further investigation because in some pine habitats,
Ericaceae form dense understorey and may therefore shift
EcMF communities available for pine seedlings. EcMF
species that are not inhibited by the presence of ericaceous
shrubs and ErMF are probably adapted to poor soil
conditions (Nilsson et al. 1993;Genneyetal.2000)and
form an important symbiont pool for seedling establish-
ment on heathlands (Collier and Bidartondo 2009)and
forest ground.
It is clear that ericaceous plants play an important role in
the formation of EcMF communities associated with the
(a)
0%
20%
40%
60%
80%
100%
Sy SyVm SyVv
Relative number of EcM root tips
suilloid morphotype Inocybe sp.
Thelephora terrestris Meliniomyces bicolor
Wilcoxina mikolae Cenococcum geophilum
(b)
0%
20%
40%
60%
80%
100%
St StVm StVv
Relative number of EcM root tips
suilloid morphotype Inocybe sp.
Thelephora terrestris Meliniomyces bicolor
Wilcoxina mikolae Cenococcum geophilum
Fig. 2 a Relative number of
EcM morphotypes associated
with the roots of P. sylvestris
grown in isolation (Sy) or grown
with V. myrtillus (SyVm)orV.
vitis-idaea (SyVv). bRelative
number of EcM morphotypes
associated with the roots of P.
strobus grown in isolation (St)
or grown with V. myrtillus
(StVm)orV. vitis-idaea (StVv)
Mycorrhiza (2011) 21:403–412 409

roots of P. sylvestris seedlings. However, the effect of these
changes on seedling survival and physiology needs further
investigation and evaluation. Besides host specificity
(Jacobson and Miller 1992), differential spore dormancy
(Bruns et al. 2009) and competitive hierarchy (Kennedy et
al. 2009), ericaceous plants may provide a niche for the
differentiation of suilloid fungi.
The hypothesis that there would be a greater abundance
of M. bicolor (a member of the R. ericae aggregate) and
other ErMF as ectomycorrhizal symbionts of the pines in
the Ericaceae treatments was not supported. Surprisingly,
M. bicolor was detected as forming ectomycorrhizae almost
exclusively when the pines were grown in isolation. Thus,
our results suggest that rather than being unable to form
ectomycorrhizal symbiosis, the competitive abilities of the
respective M. bicolor strains were inhibited by ericaceous
plants and/or ErMF. Alternatively, M. bicolor might display
a hitherto overlooked preference for ericaceous roots.
EcM species of the R. ericae aggregate are among the
dominant fungi in both young and mature boreal forests in
northern Europe (Genney et al. 2006; Toljander et al. 2006;
Korkama et al. 2007; Tedersoo et al. 2008b). Some of its
members are able to form ericoid mycorrhizae and
ectomycorrhizae (Vrålstad et al. 2002; Vohník et al.
2007a,b; Grelet et al. 2009) or simultaneously both types
in vitro (Villarreal-Ruiz et al. 2004). Our findings are
congruent with those of Collier and Bidartondo (2009) and
Richard et al. (2009) who recorded no ectomycorrhizae
formed by the R. ericae aggregate in roots of EcM
seedlings in areas dominated by Ericaceae. This suggests
that if any mycelial links between ectomycorrhizal and
ericoid mycorrhizal plants exist under natural conditions,
they are limited rather to fungi outside the R. ericae
aggregate.
Conclusions
Our findings reveal that both host plant species identity and
surrounding vegetation may influence EcMF assemblages
in roots of establishing pine seedlings. Contrary to our
hypotheses, the invasive and native pines associated with a
comparably species-rich EcMF community and the erica-
ceous plants did not act as a species pool of pine
mycobionts. On the contrary, the ericaceous plants inhibited
ectomycorrhizae formed by M. bicolor, a mycobiont that is
potentially shared between ectomycorrhizal plants and
Ericaceae. Such changes in EcMF communities might be
explained by selective stimulation/inhibition of some EcMF
by Ericaceae, or a preference of some mycobionts for
ericaceous to coniferous roots.
Acknowledgements We acknowledge Petra Wildová for excellent lab
assistance, Kateřina Štajerová for suggestions that improved the
manuscript and Tony Dixon for language correction. We would like to
thank two anonymous reviewers for their highly valuable comments on
our manuscript. Grant Agency of Charles University (9714/2009), COST
OC 10058 (Ministry of Education, Youth and Sports of the Czech
Republic) and Charles University SVV 261209/2010 provided financial
support. This study is a part of the Academy of Sciences of the Czech
Republic research programme AV0Z60050516.
Fig. 3 NMDS ordination of EcM community data from 48 meso-
cosms, illustrating the effect of host trees (filled triangles) and
cultivation treatments (open triangles). For better visualisation,
mesocosms are not shown
Table 3 Fisher’s exact test based on the pattern of each EcMF
occurrence in the microcosms
EcMF Host plant effect
(p)
Understorey effect
(p)
Suillus bovinus 0.023 >0.999
Suillus variegatus nd nd
Suillus granulatus 1nd nd
Suillus granulatus 2nd nd
Suillus luteus 0.666 0.143
Rhizopogon roseolus s.l. 0.740 0.173
Rhizopogon luteolus nd nd
Rhizopogon salebrosus s.l. <0.001 0.048
Meliniomyces bicolor >0.999 <0.001
Wilcoxina mikolae 0.008 0.075
Inocybe sp. nd nd
Thelephora terrestris 0.002 0.022
Cenococcum geophilum nd nd
Species which were not abundant (less than six pots) were excluded
from the analyses
nd not determined
410 Mycorrhiza (2011) 21:403–412

References
Abarenkov K, Nilsson RH, Larsson KH, Alexander IJ, Eberhardt U,
Erland S, Hoiland K, Kjøller R, Larsson E, Pennanen T, Sen R,
Taylor AFS, Tedersoo L, Ursing BM, Vrålstad T, Liimatainen K,
Peintner U, Koljalg U (2010) The UNITE database for molecular
identification of fungi—recent updates and future perspectives.
New Phytol 186:281–285
Baxter JW, Dighton J (2001) Ectomycorrhizal diversity alters growth and
nutrient acquisition of grey birch (Betula populifolia) seedlings in
host–symbiont culture conditions. New Phytol 152:139–149
Bruns TD, Peay KG, Boynton PJ, Grubisha LC, Hynson NA, Nguyen
NH, Rosenstock NP (2009) Inoculum potential of Rhizopogon
spores increases with time over the first 4 yr of a 99-yr spore
burial experiment. New Phytol 181:463–470
Carrillo-Gavilan MA, Vila M (2010) Little evidence of invasion by
alien conifers in Europe. Divers Distrib 16:203–213
Collier FA, Bidartondo MI (2009) Waiting for fungi: the ectomycor-
rhizal invasion of lowland heathlands. J Ecol 97:950–963
Courty PE, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005)
Activity profiling of ectomycorrhiza communities in two forest
soils using multiple enzymatic tests. New Phytol 167:309–319
Curlevski NJA, Chambers SM, Anderson IC, Cairney JWG (2009)
Identical genotypes of an ericoid mycorrhiza-forming fungus
occur in roots of Epacris pulchella (Ericaceae) and Leptosper-
mum polygalifolium (Myrtaceae) in an Australian sclerophyll
forest. FEMS Microbiol Ecol 67:411–420
Dickie IA, Bolstridge N, Cooper JA, Peltzer DA (2010) Co-invasion
by Pinus and its mycorrhizal fungi. New Phytol 187:475–484
Gardes M, Bruns TD (1993) Its primers with enhanced specificity for
basidiomycetes—application to the identification of mycorrhizae
and rusts. Mol Ecol 2:113–118
Genney DR, Alexander IJ, Hartley SE (2000) Exclusion of grass roots
from soil organic layers by Calluna: the role of ericoid
mycorrhizas. J Exp Bot 51:1117–1125
Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution
of pine ectomycorrhizas and their extramatrical mycelium. New
Phytol 170:381–390
Grelet GA, Johnson D, Paterson E, Anderson IC, Alexander IJ (2009)
Reciprocal carbon and nitrogen transfer between an ericaceous
dwarf shrub and fungi isolated from Piceirhiza bicolorata
ectomycorrhizas. New Phytol 182:359–366
Hadincová V, Köhnleinová I, Marešová J (2007) Invasive behaviour
of white pine (Pinus strobus L.) in sandstone areas in the Czech
Republic. In: Härtel H, Cílek V, Herben T, Jackson A, Williams
R (eds) Sandstones Landscapes. Academia, Prague, pp 219–224
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucl Acids
Symp Ser 41:95–98
Hambleton S, Sigler L (2005) Meliniomyces, anew anamorph genus
for root-associated fungi with phylogenetic affinities to Rhizo-
scyphus ericae (Hymenoscyphus ericae), Leotiomycetes. Stud
Mycol 53:1–27
Horton TR, Bruns TD, Parker VT (1999) Ectomycorrhizal fungi
associated with Arctostaphylos contribute to Pseudotsuga men-
ziesii establishment. Can J Bot 77:93–102
Ishida TA, Nara K, Hogetsu T (2007) Host effects on ectomycorrhizal
fungal communities: insight from eight host species in mixed
conifer–broadleaf forests. New Phytol 174:430–440
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
Jacobson KM, Miller OK (1992) Physiological variation between tree
associated populations of Suillus granulatus as determined by in
vitro mycorrhizal synthesis experiments. Can J Bot 70:26–31
Jonsson LM, Nilsson MC, Wardle DA, Zackrisson O (2001) Context
dependent effects of ectomycorrhizal species richness on tree
seedling productivity. Oikos 93:353–364
Keane RM, Crawley MJ (2002) Exotic plant invasions and the enemy
release hypothesis. Trends Ecol Evol 17:164–170
Kennedy PG, Peay KG, Bruns TD (2009) Root tip competition among
ectomycorrhizal fungi: are priority effects a rule or an exception?
Ecology 90:2098–2107
Klironomos JN (2003) Variation in plant response to native and exotic
arbuscular mycorrhizal fungi. Ecology 84:2292–2301
Korkama T, Fritze H, Pakkanen A, Pennanen T (2007) Interactions
between extraradical ectomycorrhizal mycelia, microbes associ-
ated with the mycelia and growth rate of Norway spruce (Picea
abies) clones. New Phytol 173:798–807
Krpata D, Muhlmann O, Kuhnert R, Ladurner H, Göbl F, Peintner U
(2007) High diversity of ectomycorrhizal fungi associated with
Arctostaphylos uva-ursi in subalpine and alpine zones: potential
inoculum for afforestation. For Ecol Manag 250:167–175
Kubartová A (2007) Decomposition of needle litter in Pinus sylvestris
and Pinus strobus forests in the Bohemian Switzerland National
Park (Czech Republic). In: Härtel H, Cílek V, Herben T, Jackson
A, Williams R (eds) Sandstones landscapes. Academia, Prague,
pp 234–237
Leski T, Aučina A, Skridaila A, Pietras M, Riepšas E, Rudawska M
(2010) Ectomycorrhizal community structure of different geno-
types of Scots pine under forest nursery conditions. Mycorrhiza
20:473–481
Lilleskov EA, Fahey TJ, Horton TR, Lovett GM (2002) Belowground
ectomycorrhizal fungal community change over a nitrogen
deposition gradient in Alaska. Ecology 83:104–115
Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M, Bazzaz
FA (2000) Biotic invasions: causes, epidemiology, global con-
sequences, and control. Ecol Appl 10:689–710
Mikola P (1969) Afforestation of treeless areas. Unasylva 23:S1–S20
Molina R, Trappe JM (1994) Biology of the ectomycorrhizal genus,
Rhizopogon. 1. Host associations, host-specificity and pure
culture syntheses. New Phytol 126:653–675
Morris MH, Smith ME, Rizzo DM, Rejmánek M, Bledsoe CS (2008)
Contrasting ectomycorrhizal fungal communities on the roots of
co-occurring oaks (Quercus spp.) in a California woodland. New
Phytol 178:167–176
Morris MH, Perez-Perez MA, Smith ME, Bledsoe CS (2009)
Influence of host species on ectomycorrhizal communities
associated with two co-occurring oaks (Quercus spp.) in a
tropical cloud forest. FEMS Microbiol Ecol 69:274–287
Nilsson MC, Hogberg P, Zackrisson O, Wang FY (1993) Allelopathic
effects by Empetrum hermaphroditum on development and
nitrogen uptake by roots and mycorrhizae of Pinus silvestris.
Can J Bot 71:620–628
Nožička J (1965) Zavádění vejmutovky v Českých zemích do r. 1938
(White pine introduction into the Czech countries up to year
1938). Práce výzkumného ústavu lesnického ČSSR 31:41–67
Nuñez MA, Horton TR, Simberloff D (2009) Lack of belowground
mutualisms hinders Pinaceae invasions. Ecology 90:2352–2359
Pickles BJ, Genney DR, Potts JM, Lennon JJ, Anderson IC,
Alexander IJ (2010) Spatial and temporal ecology of Scots pine
ectomycorrhizas. New Phytol 186:755–768
Pimentel D, Zuniga R, Morrison D (2005) Update on the environ-
mental and economic costs associated with alien-invasive species
in the United States. Ecol Econ 52:273–288
Pringle A, Bever JD, Gardes M, Parrent JL, Rillig MC, Klironomos
JN (2009) Mycorrhizal symbioses and plant invasions. Annu Rev
Ecol Evol S 40:699–715
R Core Development Team (2007) R: a language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna
Mycorrhiza (2011) 21:403–412 411

Read DJ (1991) Mycorrhizas in ecosystems. Experientia 47:376–391
Read DJ (1998) The mycorrhizal status of pines. In: Richardson DM
(ed) Ecology and biogeography of Pinus. Cambridge University
Press, Cambridge, pp 324–340
Reinhart KO, Callaway RM (2006) Soil biota and invasive plants.
New Phytol 170:445–457
Rejmánek M (1989) Invasibility of plant communities. In: Drake JA,
Mooney HA, di Castri F, Groves RH, Kruger FJ, Rejmánek M,
Williamson M (eds) Biological invasions. A global perspective.
SCOPE 37. Wiley, Chichester, pp 369–388
Richard F, Selosse MA, Gardes M (2009) Facilitated establishment of
Quercus ilex in shrub-dominated communities within a Mediter-
ranean ecosystem: do mycorrhizal partners matter? FEMS
Microbiol Ecol 68:14–24
Richardson DM, Allsopp N, D’Antonio CM, Milton SJ, Rejmánek M
(2000) Plant invasions—the role of mutualisms. Biol Rev 75:65–
93
Rusca TA, Kennedy PG, Bruns TD (2006) The effect of different pine
hosts on the sampling of Rhizopogon spore banks in five Eastern
Sierra Nevada forests. New Phytol 170:551–560
Selosse MA, Richard F, He XH, Simard SW (2006) Mycorrhizal
networks: des liaisons dangereuses? Trends Ecol Evol 21:621–
628
Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Elsevier,
Amsterdam
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0.
Mol Biol Evol 24:1596–1599
Tedersoo L, Suvi T, Beaver K, Koljalg U (2007) Ectomycorrhizal
fungi of the Seychelles: diversity patterns and host shifts from the
native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia
bijuga (Caesalpiniaceae) to the introduced Eucalyptus robusta
(Myrtaceae), but not Pinus caribea (Pinaceae). New Phytol
175:321–333
Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I,
Koljalg U (2008a) Strong host preference of ectomycorrhizal
fungi in a Tasmanian wet sclerophyll forest as revealed by DNA
barcoding and taxon-specific primers. New Phytol 180:479–490
Tedersoo L, Suvi T, Jairus T, Koljalg U (2008b) Forest microsite
effects on community composition of ectomycorrhizal fungi on
seedlings of Picea abies and Betula pendula. Environ Microbiol
10:1189–1201
Tedersoo L, Partel K, Jairus T, Gates G, Poldmaa K, Tamm H (2009)
Ascomycetes associated with ectomycorrhizas: molecular diver-
sity and ecology with particular reference to the Helotiales.
Environ Microbiol 11:3166–3178
Thompson JD, Higgins DG, Gibson TJ (1994) Clustal-W—improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22:4673–4680
Toljander JF, Eberhardt U, Toljander YK, Paul LR, Taylor AFS (2006)
Species composition of an ectomycorrhizal fungal community
along a local nutrient gradient in a boreal forest. New Phytol
170:873–883
van der Heijden EW, Kuyper TW (2003) Ecological strategies of
ectomycorrhizal fungi of Salix repens: root manipulation versus
root replacement. Oikos 103:668–680
van der Putten WH, Klironomos JN, Wardle DA (2007) Microbial
ecology of biological invasions. ISME J 1:28–37
Villarreal-Ruiz L, Anderson IC, Alexander IJ (2004) Interaction
between an isolate from the Hymenoscyphus ericae aggregate
and roots of Pinus and Vaccinium. New Phytol 164:183–192
Vohník M, Fendrych M, Albrechtová J, Vosátka M (2007a) Intracel-
lular colonisation of Rhododendron and Vaccinium roots by
Cenococcum geophilum,Geomyces pannorum and Meliniomyces
variabilis. Folia Microbiol 52:407–414
Vohník M, Fendrych M, Kolařík M, Gryndler M, Hršelová H,
Albrechtová J, Vosátka M (2007b) The ascomycete Meliniomyces
variabilis isolated from a sporocarp of Hydnotrya tulasnei
(Pezizales) intracellularly colonises roots of ecto- and ericoid
mycorrhizal host plants. Czech Mycol 59:215–226
Vrålstad T (2004) Are ericoid and ectomycorrhizal fungi part of a
common guild? New Phytol 164:7–10
Vrålstad T, Fossheim T, Schumacher T (2000) Piceirhiza bicolorata—
the ectomycorrhizal expression of the Hymenoscyphus ericae
aggregate? New Phytol 145:549–563
Vrålstad T, Schumacher T, Taylor AFS (2002) Mycorrhizal synthesis
between fungal strains of the Hymenoscyphus ericae aggregate
and potential ectomycorrhizal and ericoid hosts. New Phytol
153:143–152
Walbert K, Ramsfield TD, Ridgway HJ, Jones EE (2010) Ectomycor-
rhizal species associated with Pinus radiata in New Zealand
including novel associations determined by molecular analysis.
Mycorrhiza 20:209–215
Walker JF, Miller OK, Lei T, Semones S, Nilsen E, Clinton BD (1999)
Suppression of ectomycorrhizae on canopy tree seedlings in
Rhododendron maximum L.-(Ericaceae) thickets in the southern
Appalachians. Mycorrhiza 9:49–56
Walker JF, Miller OK, Horton JL (2005) Hyperdiversity of ectomy-
corrhizal fungus assemblages on oak seedlings in mixed forests
in the southern Appalachian Mountains. Mol Ecol 14:829–838
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and
direct sequencing of fungal ribosomal RNA genes for phyloge-
netics. In: Innis N, Gelfand D, Sninsky J, White T (eds) PCR—
protocols and applications—a laboratory manual. Academic,
New York, pp 315–322
412 Mycorrhiza (2011) 21:403–412