Mycorrhiza

DOI 10.1007/s00572-014-0615-2

ORIGINAL PAPER

Tricholoma matsutake can absorb and accumulate trace elements

directly from rock fragments in the shiro
Lu-Min Vaario & Taina Pennanen & Jinrong Lu & Jorma Palmn &
Jarkko Stenman & Jussi Leveinen & Petri Kilpelinen & Veikko Kitunen

Received: 4 August 2014 / Accepted: 21 October 2014

# Springer-Verlag Berlin Heidelberg 2014

Abstract Tricholoma matsutake, a highly valued delicacy in

Japan and East Asia, is an ectomycorrhizal fungus typically
found in a complex soil community of mycorrhizae, soil
microbes, and host-tree roots referred to as the shiro in
Japan. A curious characteristic of the shiro is an assortment
of small rock fragments that have been implicated as a direct
source of minerals and trace elements for the fungus. In this
study, we measured the mineral content of 14 samples of shiro
soil containing live matsutake mycelium and the extent to
which the fungus can absorb minerals directly from the rock
fragments. X-ray powder diffraction identified major phases
Electronic supplementary material The online version of this article
(doi:10.1007/s00572-014-0615-2) contains supplementary material,
which is available to authorized users.
L.<M. Vaario (*) : T. Pennanen : J. Lu : P. Kilpelinen : V. Kitunen
Finnish Forest Research Institute, PL 18, 01301 Vantaa, Finland
e-mail: lu-min.vaario@metla.fi
T. Pennanen
e-mail: taina.penannen@metla.fi
J. Lu
e-mail: Jinrong.lu@metla.fi
P. Kilpelinen
e-mail: petri.kilpelainen@metla.fi
V. Kitunen
e-mail: veikko.kitunen@metla.fi
J. Palmn : J. Leveinen
Aalto University, PL 16200, 00076 Aalto, Finland
J. Palmn
e-mail: jorma.palmen@aalto.fi
J. Leveinen
e-mail: jussi.leveinen@aalto.fi
J. Stenman
Finnish Natural History Museum, University of Helsinki, Helsinki,
Finland
e-mail: jarkko.stenman@helsinki.fi

of quartz, microcline, orthoclase, and albite in all shiro samples. PCR-denaturing gradient gel electrophoresis (DGGE)
fingerprinting and direct sequencing confirmed the presence
of T. matsutake on 32 of 33 rock fragments. Piloderma sp. cooccurred on 40 % of fragments and was positively correlated
with locations known to produce good mushroom crops. The
ability of T. matsutake to absorb trace elements directly from
rock fragments was examined in vitro on nutrient-agar plates
supplemented with rock fragments from the shiro. In comparison to the mineral content of tissues grown on control media,
the concentration of Al, Cu, Fe, Mn, P, and Zn increased from
1.1 to 106.4 times for both T. matsutake and Piloderma sp.
Mineral content of dried sporocarps sampled from the study
site partially reflected the results of the in vitro study. We
discuss the implications of our results with respect to the
natural development and artificial culture of this important
fungus.
Keywords Ectomycorrhizal fungi . Minerals . Shiro .
Tricholoma matsutake

Introduction
The ectomycorrhizal fungus Tricholoma matsutake (S. Ito et
Imai) produces commercially important mushrooms that have
iconic significance in the Far East and are highly valued as a
delicacy and medicine (Ogawa 1978; Wang et al. 1997). As
well as occurring in eastern Asia and northern North America,
this mushroom has a patchy distribution in northern Europe
(Kytvuori 1988). In response to dwindling harvests in Japan
and Korea, forest managers in northern Europe have begun
exploring the commercial feasibility of alternative and highvalue forest products such as matsutake mushrooms
(Nagasaka 2013).

Mycorrhiza

Research into the natural and artificial culture of matsutake

has gained increasing momentum over the past 100 years as a
result of a gradual decline in the natural productivity of the
fungus in Asia (Ogawa 1978; Gong et al. 1999; see review in
Wang et al. 2012). In northern Europe, this fungus was only
recognized as T. matsutake in 2000 (Bergius and Danell 2000)
and information concerning its natural distribution in
Fennoscandia is limited. It is important to realize that all
matsutake mushrooms are harvested from wild populations
living in natural settings. Currently, the commercial cultivation of matsutake is not possible partly due to a lack of
knowledge concerning the precise soil requirements and cues
of sporocarp formation. Within the host-tree rhizosphere, matsutake grows in concentric rings to form a massive mycorrhizal aggregate known as a shiro (Hosford et al. 1997). Unlike
many other ectomycorrhizal basidiomycetes, the shiro of
T. matsutake is a very dense aggregate with clear limits
(Hamada 1970). Development of an artificial shiro is considered to be the main problem in culturing the fungus under
controlled conditions (Kobayashi et al. 2009). As such, most
studies of matsutake are natural experiments relying on the
observation of correlated environmental variables and productivity. In spite of this research, many questions remain
concerning the requirements for mycelial growth and the
factors that regulate fruiting and yield.
Matsutake is typically found growing in a sandy, mineralrich soil layer in association with the roots of pine trees
(Hosford et al. 1997; Ogawa 1978; Gong et al. 1999; Vaario
et al. 2011). This fungus dominates the fungal community in
the shiro and is often observed growing in the company of
Piloderma sp. (Lian et al. 2006; Vaario et al. 2011). Ogawa
(1977) suggested a preference for sandy soil on the basis of
reduced competition for matsutake in a matrix that is typically
unsuitable for many other fungi (Ogawa 1977). A functional
connection between soil matrix and the growth and fruiting of
matsutake has yet to be demonstrated.
In Fennoscandia, the main host species are Norway spruce
(Picea abies), Scots pine (Pinus sylvestris), and birch (Betula
spp.). Soils tend to be young and nutrient-poor, and the
decomposition of litter can be slow in the cool and moist
climate (Hari et al. 2013). Soil development in this region
has been critically dependent on the weathering of raw mineral substrates to release essential nutrients for tree growth and
to improve soil moisture and nutrient storage. It is worth
noting that the interface between the tree and soil is mostly
formed by the symbiotic ectomycorrhizal fungi that cover
over 90 % of their root tips (Smith and Read 2008). Recent
studies of ectomycorrhizae have shed light on the possible
ways in which the fungal partner obtains minerals directly
from rock fragments in the soil and forwards them to the host
plant (Hoffland et al. 2004), and others have shown how ECM
fungi can act as mineral weathering agents (Landweert et al.
2001; Machuca et al. 2007; Smits et al. 2008). Species of

Piloderma associate with a wide variety of conifer and hardwood species to form ectomycorrhizae (Molina et al. 1992).
Soils dominated by Piloderma spp. typically had higher
amounts of Ca2+, Mg2+, and K+ compared to soils containing
microbial communities in which members of this genus were
scarce (Arocena et al. 1999).
In this study, we aim to explore any role that soil minerals
play in the shiro and how the fungus interacts with this
component of the environment. We focused on the fungal
community and mineral profile of shiro soils. We did not
compare our observations with the mineral profiles of
matsutake-free soils as the definition of a natural control
sample is very challenging; the absence of matsutake may
be due to other factors than soil chemistry. Nevertheless, the
following hypothesis can be proposed: a close association
between matsutake mycelium and rock fragments enables
the direct absorption of minerals and trace elements from the
soil matrix. We determined the identity of the fungal community attached to rock fragments in the shiro and compared the
growth of mycelium samples on nutrient agar media with and
without rock fragments. We also compared the composition of
trace elements in shiro soil and in dried matsutake sporocarps
in order to estimate the uptake and accumulation of these
micronutrients.

Materials and methods

Study site and sampling
The fieldwork was conducted in a mature and forest park in
southern Finland. This protected area occupies ca. 215 km2 of
forest and lakes and contains large areas of exposed crystalline
bedrock. Repeated glacial advance has exposed the underlying bedrock, and wave action in the Baltic basin
has redistributed glacial tills and created thin and sporadic sand-gravel deposits on the slopes of bedrock
outcrops. Most of the bedrock in this area was formed
2109 years ago and is composed of mainly microcline
granite, granodiorite, and the younger Bodom rapakivi
granite (Haavisto-Hyvrinen et al. 2001; GTK active
map explorer 2013). We surveyed a 100135-m area
in which matsutake sporocarps have been observed consistently since their discovery in 2000 (Miyazawa T.,
personal communication). Within the study site, there
were 37 P. sylvestris, 7 P. abies, and 8 Betula pendula
taller than 2 m (Vaario et al. 2013). Soil chemistry and
microbial (i.e., fungi+actinobacteria) communities have
been reported earlier (Vaario et al. 2011). Between 2008
and 2013, annual mean temperature in the study site
was 4.46.7 C; annual accumulated degree days were
13911738 d.d., and annual precipitation was 596
932 mm (Venlinen et al. 2005).

Mycorrhiza

From 2008 to 2013, we visited the site two to three times

per week during the fruiting season (i.e., mid-July to late
September) to count and map the emergent matsutake sporocarps. Based on the distribution of T. matsutake fruiting bodies, we estimated there to be five distinct fruiting patches in the
study site (see Fig. 1 in Vaario et al. 2012. Mean annual
fruiting percentage of each patch (the number of fruiting
bodies in each patch/total number of fruiting bodies in the
study site100 %) was calculated. In 2009, 69 sporocarps
were harvested at the site and shiro samples were taken from
33 randomly selected sporocarp locations to analyze the fungal community attached to rock fragments. Approximately

15 ml of material from the mineral soil layer was sampled

immediately after harvesting the sporocarp and transported to
the laboratory in an insulated container. A single rock
fragment of ca. 510-mm diameter was selected from
each of the 33 soil samples, shaken briefly to remove
excess soil particles, and then stored in a 1.5-ml
microcentrifuge tube at 20 C prior to subsequent
molecular analysis. The remainder of the mineral-soil
sample was pooled to create nine mixed samples and
subjected to an analysis of trace elements. Eleven sporocarps from the 20092011 harvests were stored at
20 C prior to the measurement of trace elements.

Fig. 1 X-ray diffraction (XRD) pattern of 14 shiro samples taken from the study site. The XRD peaks of each studied mineral are indicated according to
the ICDD PDF reference pear list

Mycorrhiza

Analysis of the fungal community associated with rock

fragments in the shiro
Genomic DNA was extracted from each rock fragment sample
with the PowerSoil DNA kit according to the manufacturers
instructions. The internal transcribed spacer (ITS) region of
the rDNA was amplified with a GC-clamped ITS1F (5-CGC
CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA
CGG GGG GCT TGG TCA TTT AGA GGA AGT AA-3)
and ITS2 primers (White et al. 1990). PCR amplification for
denaturing gradient gel electrophoresis (DGGE) analysis was
performed with Biotools polymerase (B & M Laboratories,
Madrid, Spain) following Korkama et al. (2006). The DCode
denaturing gradient gel system was used (Bio-Rad, Hercules,
CA, USA) for fungal community analyses. DGGE gels were
stained with SYBR Gold, visualized under low-power UV
light (Dark Reader Transilluminator; Clare Chemical
Research, Dolores, CO, USA), and digitally photographed.
DGGE gel photographs were screened for the presence (1) or
absence (0) of fungal bands using Alphaimager 2.1 (Alpha
Innotech Corp., CA, USA). Based on their mobility, two to
four bands of similar mobility were selected for sequencing
and subsequently excised, amplified, and run again in DGGE
three to five times until a single and high-quality band was
obtained. Finally, DGGE-PCR products were re-amplified
with 2325 cycles and purified with the High-Pure PCR
product purification kit (Roche, Germany). Direct sequencing
of the partial ITS (fungi) products was conducted by a commercial sequencing service (Macrogen Inc., The Netherlands)
with the same primers (ITS1F (Gardes and Bruns 1993) and
ITS2) used in amplification. Contigs representing consensus
DNA sequences were assembled and edited in Geneious Pro
(version 5.0.4) (Biomatters Ltd, New Zealand) and BLAST
searched against fungal sequences in GenBank. Operational
taxonomic units (OTUs) were defined on the basis of sequences that were at least 97 % identical and using UNITE
(Kljalg et al. 2005). Original sequences generated during the
course of the present study are deposited in GenBank under
accession numbers KF02744573, KJ6813723, and
KJ70258082.
Mineral properties of rock fragments in matsutake shiro
In October 2012, we randomly selected 14 of the mapped
locations where sporocarps were detected at least once during
20082012: one 2008 fruiting location, two from 2009, three
from 2010, five from 2011, and three from 2012. Shiro soil
samples were collected with a metal sample spoon from
natural subvertical funnels and transported to the laboratory
for mineralogical analysis and removal of rock fragments.
Small pieces of rock covered with matsutake mycelium were
removed with the aid of a stereo-microscope and ground in an
agate mortar to achieve a particle size of 2030 m prior to X-

ray powder diffraction (XRPD). Rock dust was then

suspended in acetone, placed on a glass slide, and measured
over the 2 range of 575 using graphite monochromatized
Cu K radiation (1.54184 ) at room temperature (1 divergence slit; 1 detector slit and 0.1 mm receiving slit, scanning
step 0.05, soller slit 0.04 rad, proportional counter, counting
time 1 s). The ICDD PDF-2 powder diffraction database
implemented in Panalytical HighScore was used for searchmatch phase identification analyses (International Centre for
Diffraction Data, ICDD-PDF2). XRPD measurements were
carried out using Philips XPert (PW3020) diffractometer
(45 kV, 35 mA) in Bragg-Brentano geometry.
Matsutake isolates maintained in vitro
In this study, we used four isolates of T. matsutake, a Japanese
isolate (JA: Tm0945 (Matsushita et al. 2005), and three
Finnish isolates from eastern (EF: GQ904716 (Vaario et al.
2010), southern (SF: JF346748 (Vaario et al. 2012)), and
northern Finland (NF: KF027474, this study). Cultures were
maintained on modified Modified Melin-Norkrans (MMN)
medium (Marx 1969; Vaario et al. 2010) with a cellophane
sheet placed on the surface. Piloderma fallax R-SP02 was
isolated from the ectomycorrhizal root tips of young Norway
spruce seedlings growing in a sand pit in southern Finland.
In vitro culture on artificial media supplemented with rock
fragments from the shiro
Rock fragments of ca. 510 mm diameter were selected from
shiro soil, rinsed under running tap water for 30 min, and then
dried at 50 C for 4 h. An ammonium tartrate-glucose (ATG)
medium containing 0.5 g/l ammonium tartrate, 1 g/l glucose,
and 12 g/l agar (Fluka 05040) was formulated and adjusted to
pH 5.3 with 1 M HCl. The media was then divided into 20-ml
aliquots, half of which each received 5 g of rock fragments,
and all were autoclaved prior to being loaded onto 9-cm petri
dishes. Sterilized cellophane sheets were placed onto the
cooled surface of the media, and six plugs (ca. 7 mm diameter)
of actively growing matsutake mycelium were then added to
each microcosm containing ATG or ATG+rock. Three to four
replicate microcosms were incubated for each isolate, and
plates of each media type were left without matsutake inoculum to serve as a control. All microcosms were incubated in
the dark at room temperature for 12 weeks.
Determination of concentration of trace elements
from microcosm fungal cultures, dried sporocarps,
and mineral soils
Dry weight of the mycelia grown in the microcosms
described above was determined after 12 weeks of incubation. Mycelial growth was observed as a thin layer

Mycorrhiza

on the cellophane surface. In order to minimize variation in the measurement of biomass, we removed both
the mycelial growth and the cellophane sheet and dried
both at 50 C for 24 h prior to weighing. Mineral (i.e.,
Al, B, Cr, Cd, Cu, Fe, K, Mg, Mn, Na, P, Pb, S, and
Zn) concentrations in culture+cellophane and cellophane
only (i.e., from control microcosms) were analyzed separately with an inductively coupled plasma atomic emission spectrometer (ICP-AES, Thermo Jarrell Ash IRIS
Advantage) according to protocol SFS-EN ISO 11885.
Dry weights and mineral concentrations were calculated
as the difference between cellophane+culture and cellophane in control microcosms. Mean values of
T. matsutake and P. fallax were reported in final.
Using the same method, we also measured trace elements from dried sporocarps and shiro soil samples after
drying at 50 C for 24 h. Bioaccumulation factor (BAF:
the ratio of the concentration of trace elements in sporocarp to the concentration in the ambient environment)
was calculated.

Statistical analyses
The main results were presented as mean values and their
standard errors. Normality and homogeneity of variance were
examined using Kolmogorov-Smirnov and Shapiro-Wilk tests
and Levenes test, respectively. The data were transformed
when necessary to obtain a normal distribution. The differences
in mean annual fruiting percentage among five fruiting patches
in the study site were examined with ANOVA (Tukeys test at
p < 0.05). DGGE gel images were analyzed with the
GelCompar II (ver. 5.1, Applied Maths BVBA, Belgium),
and a binary matrix (presence/absence data) was produced with
a band matching optimization of 0 % and band position tolerance of 1 %. The relative frequency of each OTU was calculated as the proportion of all samples examined in which it was
detected. We used the phi coefficient (Guilford and Perry 1951)
as a measure of association between the presence of OTUs and
sporocarp yield. Statistical significance of the association was
tested using a two-sided Fishers exact test. The difference in
mineral concentrations between dried sporocarps and shiro soil
samples was evaluated with Students t test (p<0.05). All
statistical analyses were performed with SPSS (version 15.0;
SPSS Inc., Chicago, Illinois).
The effect of rock fragments on the mineral content
of cultured mycelium was evaluated using a linear
mixed model fit with restricted maximum likelihood
(REML). Rock fragments and fungal species were treated as fixed effects and isolates as a random effect. The
analysis was performed in R (version R 2.15.1) (R
Development Core Team 2009).

Results
Mineral properties of the shiro
Based on the XRPD profiling, quartz, microcline, orthoclase,
and albite were identified as major phases in all 14 samples
(Fig. 1). Mica-, chlorite-, amphibole-, and pyroxene groups were
also present in all samples, but the exact mineral composition
could not be determined. The two amphibole group minerals
were similar to tremolite and hornblende, and the pyroxene
group was similar to augite. On the basis of the present data,
samples may also contain small amounts of titanite.
Matsutake fruiting patterns in different patches of the study
site
There was significant variation in fruiting among the five
matsutake patches during the 6-year survey. The annual sporocarp numbers varied from 7 to 108 in study site during
20082013, with a mean annual fruiting percentage/patch
from 3.4 % in patch 1 to 34.5 % in patch 4 (Fig. 2). Patches
3, 4, and 5 were designated as high-yield patches, and patches
1 and 2 were designated as low-yield patches.
Fungal communities associated with rock fragments
Nineteen of the 33 rock fragment samples were located in
high-yield patches and 14 in low-yield patches. In total,
DGGE band sequences were pooled as 33 fungal OTUs
comprising three phyla. Of these sequences, 39.4 % belonged
to Ascomycota, 51.5 % to Basidiomycota, 3.0 % (i.e., one
band) to Zygomycota, 3.0 % matched uncultured fungi, and
another 3.0 % was not successfully purified and remained
unknown (Fig. 3; Electronic supplementary material). The
OTU corresponding to T. matsutake was detected in 32 of
33 samples. The following four OTUs were also frequently
found in the DNA extractions from shiro rock fragments:

Fig. 2 Mean fruiting percentages for each patch 20082013. Common

letters indicate non-significant differences among the five fruiting patches
(n=6, Tukeys test, p<0.05)

Mycorrhiza
Fig. 3 Relative frequencies of
fungal operational taxonomic
units (OTUs) in rock fragment
samples. OTUs are in rank order

Pezizomycotina (42.4 % of samples), Tricholomaceae

(42.4 %), Piloderma sp. (39.4 %), and Russula sp. (39.4 %).
Of the remaining OTUs, 22 were detected in less than 10 % of
samples (Fig. 3). The presence of Piloderma sp. OTU correlated positively with high-yield patches (Fishers exact test,
p=0.047; symmetric measures phi=0.354).

Growth and mineral content of mycelium grown on ATG

medium supplemented with rock fragments
All isolates of T. matsutake and P. fallax grew on ATG
medium with or without rock fragments. While adding rock
fragments to the ATG medium did not significantly affect dry
weight of the culture biomass, some mineral concentrations in
cultured mycelia were significantly higher for both
T. matsutake and P. fallax (Table 1). Al concentration increased 27.7 times in T. matsutake and 106.4 times in
P. fallax growing on ATG media supplemented with shiro
rock fragments compared to controls. The second-highest
increase was for Fe: 3.7 times in T. matsutake and 6.9 times
in P. fallax. Cd and Pb were not detected in the mycelia of
either species. A small amount of Ni was detected in the NF
isolate (data not shown) and in P. fallax growing on ATG+
rock media. Concentrations of Al, Cu, Fe, Mn, P, and Zn were
significantly increased in mycelia of both species growing on
ATG+rock medium, and the effects of species on concentrations of Ca, Mg, Mn, and S were significant. In particular,
P. fallax seemed to accumulate much more Mn in the mycelium than T. matsutake did on ATG+rock medium (Table 1).
In line with the analyses above, the dried sporocarps and shiro
soil samples from the same site showed that matsutake accumulates Cu, P, and Zn but not Al. Of the 16 minerals measured, only B and Na concentrations were not significantly
different between dried sporocarps and shiro soil according to
the bioaccumulation factor. As such, BAF was also greater
than 1.0 for B, Cd, K, Na, and S (Table 2).

Discussion
Results presented here add to our earlier investigations of the
same site in which we have already profiled the soil microbial
community (Vaario et al. 2011), above-ground vegetation
(Vaario et al. 2013), and soil enzymatic characteristics
(Vaario et al. 2011). The present study has yielded the following key findings: (i) T. matsutake is the dominant species of
many soil fungi that have mycelial hyphae tightly attached to
rock fragments in the shiro; (ii) Piloderma sp. occurred on
about 40 % of rock fragments sampled from the shiro, and
significantly co-occurred with matsutake in high productivity
patches; (iii) T. matsutake and Piloderma sp. possess similar
abilities to absorb and accumulate minerals, and the mycelium
of both species contained more Al, Cu, Fe, Mn, P, and Zn
when grown on ATG media supplemented with shiro rock
fragments; and (iv) bioaccumulation factors indicate that matsutake sporocarps accumulate Cu, P, and Zn in nature, corresponding to the results of our microcosm experiment.

Fungal diversity associated with rock fragments in the shiro

Matsutake shiro has been described as a unique and massive
aggregate of mycorrhizal mycelium, host-plant roots, and soil
particles. While many reports state that this fungus is typically
found in sandy soils (Ogawa 1978; Gong et al. 1999), little
information exists concerning the precise geochemistry of
productive sites. Matsutake dominates either in the mineral
soil layer (Vaario et al. 2011) or at the root tips themselves
(Lian et al. 2006). Here, we discovered that the fungus is also
dominant among fungal species with mycelia growing in close
proximity or tightly attached to rock fragments. All loose soil
and organic matter was shaken off the rock fragment samples
prior to DNA extraction, a step taken to ensure that only those
microbes with a direct and strong attachment to the rock were
included in the subsequent DGGE sequence analysis.

1.7
29.077
4.638
12.144
0.058
2.4
1.0
1.382
0.050
1.725
0.035
0.8
2.81
1.503
0.752
0.000
0.000
0.9
1.467
0.015
1.582
0.049
0.9
1.9
8.647
0.532
1.675
0.072
5.2
0.9
351.000
13.000
361.000
2.309
1.0
7.800
0.462
10.300
1.097

27.7
1.813
0.182
0.017
0.001
106.4

0.6
20.700
1.620
20.500
1.328
1.0

1.0
2.532
0.188
2.720
0.147
0.9

0.9
1.806
0.143
0.000
0.000

2.4
6.431
0.944
2.939
0.101
2.2

3.7
454.383
60.971
66.000
1.992
6.9

1.0
2.867
0.361
2.172
0.003
1.3

Potential ability to absorb minerals directly from rock

fragments

mg/g dw

mg/kg dw
c

mg/fungal plug

ratio of R+/R

Rock fragment

Mean (n=3)
SE
Mean (n=3)
SE
ratio of R+/R
Rock fragment+
Piloderma fallax

Rock fragment

Interestingly, Piloderma sp. was one of the most frequently

co-occurring fungal OTUs on rock fragments and significantly co-occurred with matsutake in highly productive locations.
This is consistent with earlier results of a similar DGGEfingerprint study where we found Piloderma sp. to significantly co-occur with T. matsutake in two distant forest sites
(Vaario et al. 2011). In light of the present results, we are
intrigued by the seemingly positive interaction between these
ectomycorrhizal fungi and the possible role(s) played by rock
fragments in their physiology and nutrition.

1.1
1.893
0.288
1.745
0.199
1.1

26.637
2.579
15.941
1.562
1.185
0.047
1.193
0.087
0.523
0.236
0.186
0.135
3.076
0.217
1.599
0.195
Rock fragment+
Tricholoma matsutake

Mean (n=15)
SE
Mean (n=15)
SE

7.428
0.372
7.011
1.554

0.936
0.051
0.034
0.003

15.767
0.579
24.903
1.891

0.503
0.028
0.517
0.059

0.854
0.226
0.935
0.399

18.175
5.629
7.426
1.547

352.583
23.935
95.937
8.449

1.422
0.173
1.397
0.161

222.067
18.384
237.133
14.661

1.768
0.066
1.975
0.161

1.708
0.165
1.626
0.118

Znc
Sb
Pb
Nic
Nab
Mnc
Mgc
Kb
Fec
Cuc
Crc
Cab
Bc
Alb
Dry weighta
Treat
Species

Table 1 Mean values of concentrations of trace elements in Tricholoma matsutake and Piloderma fallax mycelia after 3-month incubation on ammonium tartrate-glucose agar medium with/without rock
fragments (R+/R)

Mycorrhiza

Here, we have shown that T. matsutake and P. fallax can

mobilize and absorb many important minerals and trace elements (e.g., Fe, Mn, Zn) but very little P and no K in artificial
culture and that fungal biomass is increased by the availability
of rock fragments. These results agree with earlier findings
concerning the extent to which Paxillus involutus is involved
in the weathering of hornblende (van Schll et al. 2006) and
suggest that mycelial dry weight is more heavily influenced by
the supply of an appropriate carbon source. Recently, the
clone of Scots pine forming well-developed T. matsutake
mycorrhizae was found to absorb more P, Al, Fe, Na, and
Zn than a clone supporting less mycorrhiza after 8 weeks of
microcosm incubation (Vaario et al. 2014). Thus, carbon
supply may significantly affect the ability to mobilize and
absorb minerals from rocks.
Secondly, it should be noted that the mycelial plugs used in
our microcosm experiment were taken from in vitro cultures
maintained on modified MMN medium. We analyzed the
concentrations of mineral elements in mycelium after
2 months of incubation on a modified MMN medium and
found them to be relatively high. We assume that the mycelium plugs had similar concentrations at the start of the microcosm experiment, and the failure of both test and control
mycelia to accumulate Ca, K, Mg, and S during incubation
of ATG media suggests that these trace elements are not
required for the in vitro growth of mycelium. This result is
consistent with previous reports concerning Piloderma sp.
(Hagerberg et al. 2005).
While the equivalence of in vitro micronutrient absorbance
and in vivo bioaccumulation seems intuitive, it remains to be
demonstrated empirically. Bioaccumulation factors (BAFs)
obtained from our field site indicate that, of the trace elements
absorbed by matsutake in artificial culture (i.e., Al, Cu, Fe,
Mn, Ni, P, and Zn), dry sporocarps harvested from the study
site only contained increased levels of Cu, P, and Zn and did
not accumulate Al, Fe, Mn, and Ni. Incidentally, dried sporocarps had also accumulated Cd, K, Na, and S. We compared
the trace element concentrations among a selection of Finnish
edible mushrooms (Pelkonen et al. 2008) and found that

53.582
6.636
15.922
0.347
3.4

mg/kg dw

mg/g dw

Bioaccumulation factor
c

Mineral soil

The significant difference between dried sporocarp and mineral soil was detected (Student t test, p<0.05)

5.952
0.704
0.068
0.018
88.2
0.095
0.015
0.897
0.061
<1
2.977
0.673
1.982
0.072
1.5
0.184
0.061
12.032
0.493
<1
Mean (n=11)
SE
Mean (n=9)
SE
BAFc
Dried sporocarps

23.192
2.572
0.592
0.028
39.2
202.882
63.533
11088.889
281.585
<1
28.527
2.794
6.500
0.456
4.4

566.000
67.058
1475.556
72.152
<1

8.280
1.227
59.556
3.832
<1

0.449
0.118
0.152
0.006
2.9

1.191
0.215
5.994
0.331
<1

3.532
0.469
0.167
0.020
21.2

0.579
0.417
21.467
1.417
<1

2.084
0.134
0.222
0.009
9.4

matsutake contained relatively high levels of Al (4.9 times)

compared to 12 common species. Although matsutake accumulated cadmium in the study site, the concentration of the
cadmium in sporocarp is very similar to the other edible
mushrooms in Finland, e.g., Boletus edulis which contains
ca. 6 mg/kg (Pelkonen et al. 2006). However, it should be
stressed that concentrations of trace elements in sporocarps
can be species- and substrate-dependent (Kala and Svoboda
2000).
Variation in soil acidity affects the solubility of Al ions and
thereby their concentration in the environment (Macdonald
and Martin 1988); a drop in pH from 5.8 to 4.8 is associated
with an increase in the concentration of free Al from 6.3 to
700 M (Bruce et al. 1988). In Asian countries, the pH of
shiro soil is typically between 4 and 5 (Ogawa 1978; Gong
et al. 1999) and ranged from 4.2 to 4.6 at our study site (Vaario
et al. 2012). The low pH of shiro soil may also indicate
relatively high concentrations of free Al. In boreal forests, a
cool climate leads to low rates of both evapotranspiration and
decomposition, with the result that acidic plant residues accumulate either as humus in the upper soil layers or as peat to a
considerable depth. This might explain why shiro soils and
matsutake sporocarps contain high levels of Al although BAF
of Al was <1.0 in the study site.

0.919
0.193
12.553
0.504
<1

Ka,d
Feb,d
Cub,d
Crb,d
Cdb,d
Caa,d
Bb
Ala,d
Sample

Table 2 Mean values of trace elements in Tricholoma matsutake sporocarps and shiro soil sampled from the study site

Mgb,d

Mnb,d

Naa

Nib,d

Pa,d

Pbb,d

Sa,d

Znb,d

Mycorrhiza

Potential applications of T. matsutake absorbing minerals

directly from rock fragments
Fungi, especially ectomycorrhizal fungi, have been implicated
as important weathering agents (Hoffland et al. 2004; Smits
et al. 2008; Rosling et al. 2009). The availability of mineral
nutrients, such as Ca, K, Mg, and P, are required for tree
growth. Cation levels measured in this study were low suggesting that the uptake of Mg and K may not have been greatly
affected by the fungi, but there was some evidence for bioaccumulation of K and P into the sporocarps. The host plant
might have a greater potential to absorb and accumulate trace
elements (Vaario et al. 2014). Recently, several studies focusing on the relationship between mineral weathering and
carbon-energy flux pointed out that biotic weathering rates
are driven by photosynthetic-energy fluxes directed from
below-ground mycorrhizal networks (Quirk et al. 2014;
Simts et al. 2014). The presence of intact connections to a
plant host that acts as a sink for mobilized elements could
remove weathering products that may otherwise create a negative feedback.
In many countries, T. matsutake and its close relatives have
been found in well-drained, sandy loams developed on a
variety of parent materials, the most common of which are
granite, slate, and sandstone (Ogawa 1978; Wang et al. 1997).
However, very little information is available concerning the
mineral characteristics of the matsutake shiro. Here, all 14
shiro samples had similar mineral profiles. Data from two sites

Mycorrhiza

studied by the Finnish Geology Research Center situated a

few hundred meters from our study site (GTK active map
explorer 2013) had peak trace element concentrations of
33,10033,940 ppm for Al, 26,00042,400 for Fe, and
860012,500 ppm for Mg. Thus, results from our microcosm
experiment are somewhat consistent with the general geochemistry of the area, but we are reluctant to draw conclusions
concerning the relative merits of different soils for the production of matsutake sporocarps. We did not compare the
mineral profile of a non-shiro soil, as the definition of a natural
control sample is very challenging. For example, the absence
of matsutake might be due to many factors other than soil
chemistry, not the least of which is the fact that matsutake is a
very rare species and chance surely plays an important role in
their distribution on a local scale.
Matsutake is a rare species with a patchy distribution in
Finland and other Nordic countries (Kytvuori 1988; Bergius
and Danell 2000). Our earlier work has shown that certain
above- and below-ground aspects such as litter cover, leaching
of organic carbon, and the co-occurrence of soil microbes are
essential for the growth and fruiting of T. matsutake (Vaario
et al. 2011, 2013). Here, we have demonstrated the capacity of
matsutake to acquire trace elements from rock fragments and
surmise that this trait plays a role in growth of the mycelium
and formation of the shiro.

Acknowledgments We thank Kristi Palmn and Matti Palmn for the

matsutake sporocarps from northern Finland, the Laboratory of Forest
Botany (U. Tokyo) for providing the Japanese isolate, Toyohiro
Miyazawa for the help in monitoring the fruiting period of matsutake at
the study site, Michael Hardman (Lucidia) for revising the English, Anne
Siika for preparing the illustrative material, and the two anonymous
reviewers for the useful comments.

References
Arocena JM, Glowa KR, Massicotte HB, Lavkulich L (1999) Chemical
and mineral composition of ectomycorrhizosphere soils of subalpine
fir (Abies lasiocarpa (Hook.) Nutt.) in the Ae horizon of Luvisol.
Can J Soil Sci 79:2535
Bergius N, Danell E (2000) The Swedish matsutake (Tricholoma
nauseosum syn. T. matsutake): distribution, abundance and ecology.
Scand J For Res 15:318325
Bruce RC, Warrell LA, Edwards DG, Bell LC (1988) Effects of aluminum and calcium in the soil solution of acid soils on root elongation
of Glycine max cv. Forrest. Aust J Agric Res 39:319338
Development Core Team R (2009) R: a language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna
Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for
basidiomycetesapplication to the identification of mycorrhizae
and rusts. Mol Ecol 2:113118
Gong MQ, Chen Y, Wang FZ, Chen YL (1999) Tricholoma matsutake.
In: Gong MQ et al. (eds), Tricholoma matsutake. Yunnan Science

and Technology Publishing House, Kunming, pp 1619 and pp 44

54 (In Chinese)
GTK active map explorer (2013) GTK active map explorer. http://
geomaps2.gtk.fi/activemap/. Accessed 20 May 2013
Guilford JP, Perry NC (1951) Estimation of other coefficients of correlation from the phi coefficient. Psychometrika 16:335346
Haavisto-Hyvrinen M, Grnholm S, Kielosto S, Stn C-G (2001)
Nuuksion jrviylnk: Geology of the Nuuksio lake upland. The
Geological Survey of Finland (GTK), Espoo, Finland, pp 3145
Hagerberg D, Pallon J, Wallander H (2005) The elemental content in the
mycelium of the ectomycorrhizal fungus Piloderma sp. during the
colonization of hardened wood ash. Mycorrhiza 15:387392
Hamada M (1970) Diaries on armillaria matsutake (5). Trans Mycol Soc
Jpn 11:8186
Hari P, Helivaara K, Kulmala L (eds) (2013) Physical and physiological
forest ecology. Springer, Dordrecht, p 531
Hoffland E, Kuyper TW, Wallander H, Plassard C, Gorbushina AA,
Haselwandter K, Holmstrom S, Landeweert R, Lundstrom US,
Rosling A, Sen R, Smits MM, van Hees PAW, van Breemen N (2004)
The role of fungi in weathering. Front Ecol Environ 2(5):258264
Hosford D, Plz D, Molina R, Amaranthus M (1997) Ecology and management of the commercially harvested American matsutake.
USDA general technical report PNW-GTR-412
Kala P, Svoboda L (2000) A review of trace element concentrations in
edible mushrooms. Food Chem 69:273281
Kobayashi H, Terasaki M, Yamada A (2009) The persistence of matsutake mycorrhizae two years after the transplantation of pine seedlings (CD-ROM). In: Proceedings of the 120th annual Japanese
forestry society meeting (in Japanese)
Kljalg U et al (2005) UNITE: a database providing web-based methods
for the molecular identification of ectomycorrhizal fungi. New
Phytol 166:10631068
Korkama T, Pakkanen A, Pennanen T (2006) Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones.
New Phytol 171:815824
Kytvuori I (1988) The Tricholoma caligatum group in Europe and North
Africa. Karstenia 28:6577
Landweert R, Hooffland E, Finlay RD, Kuyper TW, van Breemen N
(2001) Linking plants to rocks: ectomycorrhizal fungi mobilize
nutrients from minerals. Trends Ecol Evol 16:248254
Lian C, Narimatsu M, Nara K, Hogetsu T (2006) Tricholoma matsutake in a
natural Pinus densiflora forest: correspondence between above- and
below-ground genets, association with multiple host trees and alteration
of existing ectomycorrhizal communities. New Phytol 171:825836
Macdonald TL, Martin RB (1988) Aluminum ion in biological systems.
Trends Biochem Sci 13:1519
Machuca A, Pereira G, Aguiar A, Milagres AMF (2007) Metal-chelating
compounds produced by ectomycorrhizal fungi collected from pine
plantations. Lett Appl Microbiol 44:712
Marx DH (1969) The influence of ectotrophic mycorrhizal fungi on the
resistance of pine roots to pathogenic infections. I. Antagonism of
mycorrhizal fungi to root pathogenic fungi and soil bacteria.
Phytopathology 59:153163
Matsushita N, Kikuchi K, Sasaki Y, Guerin-Laguette A, Lapeyrie F,
Vaario L-M, Intini M, Suzuki K (2005) Genetic relationship of
Tricholoma matsutake and T. nauseosum from the Northern
Hemisphere based on analyses of ribosomal DNA spacer regions.
Mycoscience 46:9096
Molina R, Massicotte HB, Trappe JM (1992) Specificity phenomena in
mycorrhiza symbioses: community-ecological consequences and
practical implications. In: Allen MF (ed) Mycorrhizal functioning,
an integrative plant-fungal process. Routledge, Chapman and Hall,
New York, pp 357423
Nagasaka K (2013) Comparative economic value estimation of matsutake
mushroom and timber production in Swedish Scots pine forest.
Master Thesis n. 218, Swedish University of Agricultural Sciences

Mycorrhiza
Ogawa M (1977) Microbial ecology of Shiro In Tricholoma matsutake
(Ito et Imai) Sing and ItS allied speoes. V Trwholoma matsutake In
Tsuga steboldu forests Transactions of the Mycological Society of
Japan 18 3446 (In Japanese)
Ogawa M (1978) The biology of matsutake mushroom. Tsukiji Shokan,
Tokyo, p 326 (in Japanese)
Pelkonen R, Alfthan G, Jrvinen O (2006) Cadmium, lead, arsenic and
nickel in wild edible mushrooms. The Finnish environment 17,
Suomen ympristkeskus. Edita Prima Ltd, Helsinki, pp 2531
Pelkonen R, Alfthan G, Jrvinen O (2008) Element concentrations in wild
edible mushrooms in Finland. The finnish environment 25, Suomen
ympristkeskus. Edita Prima Ltd, Helsinki, pp 1826
Quirk J, Andrews MY, Leake JR, Banwart SA, Beerling DJ (2014)
Ectomycorrhizal fungi and past CO2 atmospheres enhance mineral
weathering through increased below-ground carbon-energy fluxes.
Biol Lett 10:20140375. doi:10.1098/rsbl.2014.0375
Rosling A, Roose T, Herrmann AM, Davidson FA, Finlay RD, Gadd GM
(2009) Approaches to modeling mineral weathering by fungi.
Fungal Biol Rev 23:138144
Simts MM, Johansson L, Wallander H (2014) Soil fungi appear to have a
retarding rather than a stimulating role on soil apatite weathering.
Plant Soil. doi:10.1007/s11104-014-2222-6
Smith SE, Read DJ (eds) (2008) Mycorrhizal symbiosis, 3rd edn.
Academic, London, pp 192197
Smits MM, Bonneville S, Haward S, Leake JR (2008) Ectomycorrhizal
weathering, a matter of scale? Mineral Mag 72:135138
Vaario L-M, Pennanen T, Sarjala T, Savonen E-M, Heinonsalo J (2010)
Ectomycorrhization of Tricholoma matsutake and two major conifers in Finlandan assessment of in vitro mycorrhiza formation.
Mycorrhiza 20:511518

Vaario L-M, Fritze H, Spetz P, Heinonsalo J, Pennanen T (2011)

Tricholoma matsutake dominates diverse microbial communities
in different forest soils. Appl Environ Microbiol 77(24):85238531
Vaario L-M, Heinonsalo J, Spetz P, Pennanen T, Heinonen J, Tervahauta
A, Fritze H (2012) The ectomycorrhizal fungus Tricholoma
matsutake is a facultative saprotroph in vitro. Mycorrhiza 22:
409418
Vaario L-M, Kiikkil O, Hamberg L (2013) The influences of litter cover
and understorey vegetation on fruitbody formation of Tricholoma
matsutake in southern Finland. Appl Soil Ecol 66:5660
Vaario L-M, Lu J, Koistinen A, Tervahauta A, Aronen T (2014) Variation
among matsutake ectomycorrhizae in four clones of Pinus sylvestris.
Mycorrhiza. doi:10.1007/s00572-014-0601-8
van Schll L, Smits MM, Hoffland E (2006) Ectomycorrhizal weathering
of the soil minerals muscovite and hornblende. New Phytol 171:
805813
Venlinen A, Tuomenvirta H, Pirinen P, Drebs A (2005) A basic Finnish
climate data set 19612000 description and illustrations. Finnish
Meteorological Institute, Reports 2005:5
Wang Y, Hall IR, Evans LA (1997) Ectomycorrhizal fungi with edible
fruit bodies. 1. Tricholoma matsutake and related fungi. Econ Bot
51:311327
Wang Y, Cummings N, Guerin-Laguette A (2012) Cultivation of basidiomycete edible ectomycorrhizal mushrooms: Tricholoma,
Lactarius, and Rhizopogon. In: Zambonelli, Bonito (eds) Edible
ectomycorrhizal mushrooms. Springer, Verlag, pp 281304
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis et al (eds) PCR protocols: a guide to methods and applications.
Academic, San Diego, pp 315322