Environmental Pollution 218 (2016) 176e185

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Invited paper

Bioaccumulation of heavy metals, metalloids, and chlorine in

ectomycorrhizae from smelter-polluted area*
Jaroslava Cejpkova
a, b, Milan Gryndler c, d, Hana Hrselova

c, Pavel Kotrba e,
Zdene 
k Randa a
, Iva Synkova cka a, f, *

a, b, Jan Borovi
a
Nuclear Physic Institute, v.v.i., Czech Academy of Sciences, Re z 130, CZ-25068 Husinec-Re
 z, Czech Republic
b
Institute of Geochemistry, Mineralogy and Mineral Resources, Faculty of Science, Charles University, Albertov 6, CZ-12843 Prague 2, Czech Republic
c
Institute of Microbiology, v.v.i., Czech Academy of Sciences, Vden
1083, CZ-14220 Prague 4, Czech Republic
 ska
d
Faculty of Sciences, J. E. Purkyn 
e University, Cesk

deze 8, CZ-40096 st nad Labem, Czech Republic
e mla
e
Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technicka
3, CZ-16628 Prague 6, Czech Republic
f
Institute of Geology, v.v.i., Academy of Sciences of the Czech Republic, CZ-16500 Prague 6, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: Ectomycorrhizal (ECM) fungi contribute to the survival of host trees on metal-rich soils by reducing the
Received 6 June 2016 transfer of toxic metals into roots. However, little is known about the ability of ECM fungi to accumulate
Received in revised form elements in ectomycorrhizae (ECMs). Here we report Ag, As, Cd, Cl, Cu, Sb, V, and Zn contents in wild-
26 July 2016
grown Norway spruce ECMs collected in a smelter-polluted area at Lhota near Prbram, Czech Repub-
Accepted 3 August 2016
Available online 26 August 2016
lic. The ECMs data were compared with the element concentrations determined in the corresponding
non-mycorrhizal ne roots, soils, and soil extracts. Bioaccumulation factors were calculated to differ-
entiate the element accumulation ability of ECMs inhabited by different mycobionts, which were
Keywords:
Ectomycorrhizal fungi
identied by ITS rDNA sequencing. Among the target elements, the highest contents were observed for
Ectomycorrhiza Ag, Cl, Cd, and Zn; Imleria badia ECMs showed the highest capability to accumulate these elements. ECMs
Quantitative real-time PCR of Amanita muscaria, but not of other species, accumulated V. The analysis of the proportions of I. badia
Roots and A. muscaria mycelia in ECMs by using species-specic quantitative real-time PCR revealed variable
Soil extent of the colonization of roots, with median values close to 5% (w/w). Calculated Ag, Cd, Zn and Cl
Cadmium concentrations in the mycelium of I. badia ECMs were 1 680, 1 510, 2 670, and 37,100 mg kg
1 dry weight,
Silver respectively, indicating substantial element accumulation capacity of hyphae of this species in ECMs. Our
data strengthen the idea of an active role of ECM fungi in soil-fungal-plant interactions in polluted
environments.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Ectomycorrhizal (ECM) fungi signicantly interfere in biogeo-
chemical cycles of elements in forest soils (Gadd, 2007). The major
active roles of ECM fungi in ecosystems involve, besides the
degradation and cycling of soil organic matter (Clemmensen et al.,
2013; Phillips et al., 2014), contribution to weathering processes
through excretion of organic acids and subsequent transfer of
* the host by forming a protective barrier against heavy metal
This paper has been recommended for acceptance by W. Wen-Xiong.
* Corresponding author. Nuclear Physic Institute, v.v.i., Czech Academy of
 
Sciences, Re z 130, CZ-25068 Husinec-Re z, Czech Republic; Institute of Geology,
v.v.i., Academy of Sciences of the Czech Republic, CZ-16500 Prague 6, Czech
Republic.
E-mail address: bore.bor@gmail.com (J. Borovi
cka).
mobilized nutrients towards the colonized plant roots (Gadd, 2007;
Schmalenberger et al., 2015), and trace element translocation and
accumulation (Clarholm and Skyllberg, 2013; Falandysz and
Borovi cka, 2013).
High concentrations of elements accumulated in fruit-bodies
suggest that ECM macrofungi substantially contribute to their
sequestration and cycling, Ag, As, Cd, Cl, Cu, Se, V, and Zn in
particular (Vetter, 2005; Falandysz and Borovicka, 2013). Ectomy-
corrhizae (ECMs) represent organs where the fungal-plant nutrient
exchange takes place; studies have also suggested that they benet
toxicity (Krupa and Kozdro
j, 2004, 2007; Gadd, 2007; Colpaert
et al., 2011). Extraradical mycelium is hardly separable from soil
under the natural conditions. However, analysis of ECMs might
represent an interesting tool for inspecting the soil-fungal-plant

http://dx.doi.org/10.1016/j.envpol.2016.08.009
0269-7491/ 2016 Elsevier Ltd. All rights reserved.

et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova
interactions in situ.
Vinichuk (2013) recently reported a stepwise increase in the
concentration of Cd, Zn, and Cu from unpolluted soil to extraradical
mycelia (5, 2.4 and 2-fold increase relative to bulk soil, respectively)
and to the fruit-bodies of ECM fungi (1.8, 1.4 and 1.8-fold compared
to mycelia, respectively). However, the information about the
element content in ECMs and the mycelia directly colonizing plant
roots is scarce. To our knowledge, the largest dataset published so
far was a study by Kottke et al. (1998) who focused on nutrients and
essential metals P, K, Mg, Ca, Fe, Zn, Al, and Mn. Berthelsen et al.
(1995) and Krupa and Kozdro
j (2004, 2007) have reported sub-
stantially elevated concentrations of Cu, Cd, Pb and Zn in ECMs. In
contrast, the concentrations of Au (Borovi cka et al., 2010a) and U
(Kubrova
et al., 2014) were found to be low even in ECMs from
auriferous and U-polluted areas.
The fact that the specic ability of fungal species to accumulate
elements in ECMs has never been investigated prompted us to
perform a systematic investigation of element concentrations in
ECMs, ne roots and soils at Norway spruce forest plantation in a
smelter-polluted area in the Czech Republic. Element concentra-
tions in the ECMs were compared to those in the ne roots and
accumulation of elements in the ECMs was accessed by calculation
of the bioaccumulation factor (BAF). Additionally, fungal biomass
concentration was quantied by real-time PCR (qRT-PCR) approach
in ECMs of two species, Imleria badia and Amanita muscaria; it
enabled us to semi-quantify the element concentrations in fungal
hyphae. This is the rst example of the use of the qRT-PCR-based
molecular approach to determine the mycobiont proportions in
ECMs.
2. Materials and methods
2.1. Investigated area and sample collection
Norway spruce (Picea abies) forest plantation above
Fig. 1. Smelter-polluted area at Lhota near Prbram (Czech Republic) and the sampling sites.
177
sedimentary bedrock (greywacke) at Lhota near Prbram was
selected for this study; most of the trees were circa 70 years old.
Long-term Ag-Cu-Pb-Zn ore mining activities in the region and
decades of lead smelting (Sucharov
a and Suchara, 2003) resulted in
elevated levels of Ag, As, Cd, Cu, Pb, Sb, and Zn levels in soils (Ettler
et al., 2004, 2007; Koma
rek et al., 2007). Samples of ECMs, roots
and soils were collected from Oe horizon at 32 randomly selected
places distributed throughout the area (Fig. 1); GPS coordinates of
sampling points are listed in the Supplementary Table S1.
2.2. Sample preparation
ECMs and non-mycorrhizal ne roots were processed as
described by Kubrova
et al. (2014). Dried samples were heat-sealed
into polyethylene (PE) capsules; the sample weight was in the
range of 0.5e16 mg for ECMs and 35e62 mg for roots. Soil samples
(Oe horizon) were collected directly from the places where ECMs/
roots were sampled, dried, and sieved through a 2 mm stainless
steel mesh. A representative part of each sample was milled in an
agate mill (Fritsch, Germany) and aliquots of approximately 250 mg
were used for determination of total element contents.
2.3. Chemical analyses
To analyze the concentrations of Cl, Cu, and V in ECMs, roots,
soils, and macrofungal fruit-bodies, short-time neutron activation

analysis (INAA) was used according to Randa et al. (2005). Times of
irradiation-decay-counting of 2e10-10 min for ECMs, 1-10-10 min
for roots and fruit-bodies, and 1-13-13 for soils were used as op-
timum conditions. The measurement of gamma spectra was per-
formed using the high-purity germanium (HPGe) coaxial detector
PGT IGC 20 (20% relative efciency, resolution FWHM 1.75 for the
1332.5 keV photons of 60Co). After 4 weeks of decaying, ECMs and
roots were re-irradiated and further analyzed as described below.
Concentrations of other elements in ECMs and roots were
178
et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova
assessed by using long-time instrumental neutron activation
analysis with epithermal neutrons (ENAA). This was performed by
2.5e3 h irradiation in a specially designed Al/Cd box as described

by Randa (1976). The measurements of gamma spectra were per-
formed after 3e4 and 18e25 days of decaying using the HPGe co-
axial detector Canberra GC7020 (78% relative efciency, resolution
FWHM 1.87 for the 1332.5 keV photons of 60Co). Soil samples were

analyzed by INAA according to Randa and Ku cera (2004).
Variable counting geometries optimized for individual analyt-
ical modes were applied. Neutron irradiation was carried out in
the LVR-15 reactor of the Nuclear Research Institute Re  
z plc.
(Czech Republic) at uence rates of 8  1013 n cm
2 s
1 and
3  1013 n cm
2 s
1 for thermal and fast neutrons, respectively. In
order to verify the quality of chemical analysis, NIST standard
reference materials (SRMs) 1566b (Oyster Tissue), 2711 (Montana
Soil), and 2711a (Montana II Soil) were used throughout the study.
Procedural blanks were included in all procedures.
As the INAA determination of total Cd in soils does not provide
reliable data, soil samples were acid-digested in a microwave oven
similarly as described in Gryndler et al. (2012) and Cd was deter-
mined by inductively coupled plasma optical emission spectrom-
etry (ICP-OES, Agilent 5100); the detection limit of Cd soil content
analyzed by this method was 0.5 mg/kg. In order to inspect the acid
soluble fraction of selected elements in soils, 1 M HNO3 extraction
was conducted as described in Borovi cka et al. (2014). Element
concentrations in the extracts were determined by inductively
coupled plasma quadrupole-based mass spectrometry (ICP-QMS, X
Series 2, Thermo Scientic).
2.4. Molecular methods
The complete procedure of molecular identication of fungal
species in ECM samples was described in Kubrova
et al. (2014). All
obtained ITS rDNA sequences were identied to species/genus level
by querying the GenBank database, using the nucleotideenucleo-
tide (blastn) BLAST search option, available through the National
Center for Biotechnology Information (http://blast.ncbi.nlm.nih.
gov). Quality sequences were accessioned into the EMBL-Bank
database (Supplementary Table S2). Several collections of macro-
fungi used in this study were deposited in herbarium of the
Mycological department, National Museum, Prague (PRM).
In order to quantify fungal biomass in ECM roots, two cultivable
ECM species forming morphologically recognizable ECMs were
selected: Imleria badia (formerly classied as Boletus badius or
Xerocomus badius) and Amanita muscaria. ECMs of both species
were collected from roots of Picea abies growing in Oe soil horizon
at the investigated site in September 2014 in close vicinity of fruit-
bodies. They were cleaned in water under stereomicroscope, and
kept frozen until lyophilization followed by DNA extraction. Only
the fresh, turgescent, rigid ECMs with light apices and compact
hyphal mantles were used in this study (Fig. 2).
Extraradical mycelial cultures of I. badia (explanted from a
basidiocarp collected directly at the studied locality, PRM 923859,
EMBL-Bank LN877746), and A. muscaria (isolate AM17, maintained
by the Laboratory of Fungal Biology, Institute of Microbiology ASCR,
Prague; EMBL-Bank LN877747) were used to calibrate qRT-PCR
data. The samples of both ECM roots and mycelia were weighed
and DNA was extracted in the same way as described above. In
order to determine the extraction efciency, 2  1010 gene copies of
an internal standard (linearized plasmid carrying fragment of cas-
sava mosaic virus, GenBank accession AJ427910) were added into
each sample before extraction and quantied in the DNA extract as
described in Thonar et al. (2012).
We used qRT-PCR with two specic primer pairs for each spe-
cies. Primers ITS1F, ITS2, and ITS3 were used according to White
Fig. 2. Detached ectomycorrhizae of Imleria badia prepared for qRT-PCR (in water
bath).
et al. (1990) and Gardes and Bruns (1993). Specic primers for
I. badia designed in this study were BadiusF (forward, 50 -GGA AGG
ATC ATT ATC GAA CAA GAA-30 ) and BadiusR (reverse, 50 -AAG GCC
TTG CTT GTC CAC-30 ). For A. muscaria, specic primers AmusF1
(forward, 50 -TCT CTT GCT TGT TTC TTC A-30 ) and AmusR1 (reverse,
50 -CAA CAA TTG TTC ATG TAT GTA AAT-30 ) were designed in this
study.
Primers recognizing ITS rDNA of I. badia have been designed
based on the motifs present only in the sequences belonging to
I. badia (GenBank accession numbers LK932098 and HQ207696).
These sequences were not found in the ITS of related relevant
species of the genera Boletus, Buchwaldoboletus and Xerocomus s.l.:
B. reticulatus (KC422620), B. edulis (HM579927), B. pinophilus
(GU198987), B. lignicola (HM003619), X. chrysenteron (HQ207694),
X. communis (EF493247), X. ferrugineus (HQ207698), X. porosporus
(HM190086), X. pruinatus (HQ207695), X. rubellus (EF644119), and
X. subtomentosus (DQ131632). qRT-PCR with primer pairs ITS3/
BadiusR and BadiusF/ITS2 produced the amplicons of expected size
of 230 bp and 240 bp, respectively.
Primers specic for ITS of A. muscaria have been designed on the
basis of unique motifs found in GenBank-deposited sequences of
A. muscaria (EU071912, EU071897, EU071920, AB080983,
AB080984, AB080777, and AB080778) and absent from the se-
quences of closely related Amanita regalis (LT594941, collection
PRM 860899), A. gemmata (LT594942, collection PRM 922166), A.
pantherina (AB096046), and A. eliae (JF907763); however, the
primer pair AmusF1/AmusR1 was able to amplify DNA extracted
from A. regalis. As expected, the length of qRT-PCR products ob-
tained with primer pairs AmusF1/ITS2 and ITS1F/AmusR1 were
180 bp and 202 bp, respectively.
DNA samples extracted from ECM tips of I. badia or A. muscaria
were used as templates in standard PCR with specic primer pairs
BadiusF/BadiusR or AmusF1/AmusR1 with PCR conditions as
described in Borovicka et al. (2011). A few samples with none or
weak signal (indicating possible PCR inhibition) were excluded
from further processing. In total, 19 samples of I. badia ECM and 11
samples of A. muscaria ECM were then subjected to qRT-PCR. Ac-
cording to Janouskova
et al. (2015), 10x diluted DNA sample (2 ml)
was added to an 18 ml of Hot FirePolEvaGreen qPCR Supermix
(Solis BioDyne, Tartu, Estonia) prepared according to the manu-
facturer's instructions (0.4 ml primer 1, 0.4 ml primer 2, 13.2 ml H2O,
4 ml EvaGreen). The cycling conditions in the StepOnePlus instru-
ment (Applied Biosystems) were: initial denaturation for 12 min at
95  C, followed by 40 cycles of 15 s denaturation at 95  C, annealing

et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova 179

for 20 s at 55  C, and synthesis for 20 s at 72  C. The results for each
sample are based on 3 replicates and have been corrected for DNA
extraction efciency using the quantitation of the internal standard
added to the ECM samples before the DNA extraction as described
above.
2.5. Calculation of element concentrations in fungal mycelia
Expected concentrations of metals (mg kg
1) in the mycobionts
shown in Table 2 were calculated using the formula:
ECMte
ROOTte
 1000
ECMw  FMF
where ECMte is total element content (mg) in ECM, ROOTte is total
element content (mg) in plant biomass forming the ECM, ECMw is
weight (mg) of ECM, and FMF is fungal mass fraction in ectomy-
corrhizae (0.1 and 0.07 for I. badia and A. muscaria, respectively). For
calculation of ROOTte, element concentration in the corresponding
ne roots was used.
2.6. Data analysis
Element accumulation ability of ECMs was depicted by BAF
which was calculated as the quotient of element concentrations in
ECMs and corresponding soil nitric acid extractable fractions; BAF
related to the total element soil content was calculated only for Cl
(indicated as BAFt). Considering that the concentrations of ele-
ments found in both ECMs and roots were often not normally
distributed (as resulted from the Shapiro-Wilk normality test), non-
parametric and distribution free Kolmogorov-Smirnov test was
used to compare the datasets. When below detection, the value of
75% of the detection limit was used for calculation. Results at
p < 0.05 level were considered statistically signicant.
3. Results
3.1. Ectomycorrhizae
Identication of the analyzed ECMs based on the obtained ITS
rDNA sequences, including the identities they share with GenBank
entries, is presented in the Supplementary Table S2; 6 samples
remained unidentied. Among the identied ones, 11 distinct
operational taxonomic units (OTUs) were observed from which
those corresponding to Thelephora terrestris, I. badia, and Paxillus
involutus occurred with the highest frequency. The quantities of
fungal biomass in ECMs of I. badia and A. muscaria determined by
qRT-PCR are presented in Table 1A and 1B, respectively; Table 1C
depicts statistical analysis of this data. Although we detected
considerably varying concentration of fungal biomass in ECMs, the
median values were close to 5% (w/w) in both species.
3.2. Element accumulation in ECMs
The comparison between element concentrations in ECMs,
roots, soil extracts and soils is presented in box plots (Fig. 3). Data
for ECMs of I. badia and A. muscaria ECMs, in which the concen-
trations of metals (Ag and Cd in particular) were markedly
increased, are summarized in Table 2, along with the expected
concentrations of elements associated with the mycobiont hyphae.
Complete data sets for ECMs, roots, and soils can be found in the
Supplementary Tables S3eS6. Values of BAF and BAFt for particular
elements are summarized in Table 3. Results from the Kolmogorov-
Smirnov test are presented in the Supplementary Table S7.
According to the bioaccumulation factor, the most accumulated
elements in ECMs were Ag, Cd, Cl, and Zn. Concentrations of these
elements in ECMs were also signicantly elevated when compared
with the ne roots. Substantial accumulation was generally
observed for Ag. In ECMs of I. badia (47.7e385 mg kg
1) the BAF
reached 4700 and Ag concentrations calculated for fungal hyphae
ranged from 402 to 3767 mg kg
1. The highest (but obviously
outlying) value of Ag was found in a single ECM sample of
T. terrestris (544 mg kg
1).
Despite the wide variation range of 10e333 mg/kg of Cd con-
centrations in ECMs, the median value was found to be 47 mg kg
1
making Cd a readily accumulated metal. Concentrations exceeding
100 mg kg
1 were found in ECMs of 6 fungal species, with the
highest in I. badia (maximum of 333 mg kg
1; BAF 2.6e21; the
highest hyphal Cd concentration was calculated as 3157 mg kg
1).

Table 1
Concentrations of fungal biomass (% w/w of dry weight) in ectomycorrhizae of Imleria badia (A) and Amanita muscaria (B) calculated from qRT-PCR results (corrected for DNA
extraction efciency) with a statistical summary (C).

A: Imleria badia B: Amanita muscaria

Weight (mg) % (w/w) biomass in ECM root Weight (mg) % (w/w) biomass in ECM root

ITS3-BadiusR BadiusF-ITS2 Average AmusF1-ITS2 ITS1F-AmusR1 Average

1.39 20.5 16.9 18.7 3.38 9.25 7.54 8.40

3.18 1.46 1.37 1.42 2.36 23.5 18.4 21.0
2.73 22.6 22.0 22.3 0.79 7.97 7.04 7.51
4.02 0.56 0.44 0.50 1.79 4.63 2.70 3.67
14.1 2.20 2.10 2.15 1.29 5.24 4.39 4.81
11.4 5.87 5.60 5.73 0.68 9.23 6.57 7.90
3.44 12.7 11.5 12.1 5.50 2.68 2.04 2.36
1.74 3.81 1.81 2.81 3.14 13.3 9.62 11.4
3.89 11.5 10.1 10.8 1.93 0.91 0.71 0.81
3.76 38.4 35.2 36.8 3.47 1.58 1.46 1.52
9.38 4.98 4.26 4.62 10.7 3.92 3.68 3.80
13.9 1.53 1.26 1.40
3.91 1.35 1.08 1.22

2.54 15.4 12.8 14.1 C: Statistics, % (w/w) biomass in ECM roots

4.76 3.27 1.35 2.31 I. badia A. muscaria

6.56 15.6 12.3 13.9 minimum 0.50 0.81

0.95 20.3 23.3 21.8 median 5.73 4.81
1.37 6.03 3.99 5.01 mean 9.71 6.65
2.98 7.74 5.87 6.81 maximum 36.8 21.0
180
et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova

Fig. 3. Element concentrations (mg kg
1 in dry weight) in ectomycorrhizae, non-mycorrhizal ne roots, nitric acid soil extracts (not determined for Cl) and total soils from Lhota
near Prbram presented in box plots.

Table 2
Concentrations of elements (mg kg
1 in dry mass) in roots and ectomycorrhizae and calculated element concentrations in mycorrhizae-forming mycelia of Imleria badia and
Amanita muscaria at Lhota near Prbram.

Roots Ectomycorrhizae ECM mycelium (calculated)

1
1
Concentration (mg kg ) Concentration (mg kg ) Concentration (mg kg
1)

Minimum Mean Maximum Minimum Mean Maximum Minimum Mean Maximum

I. badia (n 6) (n 9)
Ag 0.69 7.25 9.95 47.7 175 358 402 1680 3767
Cd 6.10 19.3 34.5 22.1 170 333 166 1511 3157
Cu <19.4 76.7 170 62.8 120 162 21 401 1105
Zn 129 284 415 224 537 713 695 2668 4683
Cl 586 819 1259 3651 4413 5614 30,034 37,127 44,804
A. muscaria (n 2) (n 2)
Ag 1.54 e 14.7 31.0 e 56.4 422 e 610
Cd 9.85 e 21.4 43.6 e 79.2 492 e 837
Cu <24.3 e 27.0 37.6 e 38.7 178 e 354
Zn 191 e 324 236 e 321 281 e 2120
Cl 507 e 725 1695 e 2217 17,478 e 22,039
V 0.73 e 0.80 26.4 e 61.2 367 e 864

et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova 181

Table 3
Values of bioaccumulation factor related to extractable fractions (BAF) or total element contents (BAFt) in soils calculated for ECMs of particular fungal species.

Ag As
BAF or BAF range/values BAF or BAF range/values

Amanita muscaria 860e1085 Amanita muscaria 0.1e0.2

Amphinema byssoides 1358e2148 Amphinema byssoides 0.5e0.6
C. aff. olivaceofuscus 267 C. aff. olivaceofuscus 0.17
Russula ochroleuca 279 Russula ochroleuca 0.25
Tomentella ellisii 254 Tomentella ellisii 0.35
Tomentella sublilacina 1226e4578 Tomentella sublilacina 6.1e6.9

min median max min median max

Imleria badia 1327 1871 4662 Imleria badia 0.04 0.6 1.3
Paxillus involutus 148 265 4559 Paxillus involutus 0.03 0.1 1.7
Thelephora terrestris 99 627 9725 Thelephora terrestris 0.1 0.7 5.3

Cd Cu
BAF or BAF range/values BAF or BAF range/values

Amanita muscaria 2.9e5.5 Amanita muscaria 0.3e0.4

Amphinema byssoides 6.6e7 Amphinema byssoides 0.1e0.3
C. aff. olivaceofuscus 4.2 C. aff. olivaceofuscus 0.14
Russula ochroleuca 10.4 Russula ochroleuca 0.57
Tomentella ellisii 7.62 Tomentella ellisii 0.43
Tomentella sublilacina 1.2e9.1 Tomentella sublilacina 0.4e1

min median max min median max

Imleria badia 2.6 8.4 21 Imleria badia 0.2 0.3 0.7

Paxillus involutus 2.4 3.6 9.0 Paxillus involutus 0.2 0.5 1.1
Thelephora terrestris 3.4 6.0 20 Thelephora terrestris 0.1 0.3 0.8

Sb V
BAF or BAF range/values BAF or BAF range/values

Amanita muscaria 0.6e4 Amanita muscaria 3.2e5.8

Amphinema byssoides 2.6e3 Amphinema byssoides 0.1e0.2
C. aff. olivaceofuscus 1.48 C. aff. olivaceofuscus 0.5
Russula ochroleuca 1.67 Russula ochroleuca 0.2
Tomentella ellisii 1.72 Tomentella ellisii 0.1
Tomentella sublilacina 7.7e11.3 Tomentella sublilacina 0.5e0.54

min median max min median max

Imleria badia 0.2 2.6 5.7 Imleria badia 0.03 0.2 0.6
Paxillus involutus 0.1 0.6 3.7 Paxillus involutus 0.03 0.1 0.3
Thelephora terrestris 0.1 1.1 23 Thelephora terrestris 0.02 0.1 0.8

Zn Cl
BAF or BAF range/values BAFt or BAFt range/values

Amanita muscaria 1.5e2 Amanita muscaria 4.4e12

Amphinema byssoides 2e2.2 Amphinema byssoides 4.1e4.2
C. aff. olivaceofuscus 1.5 C. aff. olivaceofuscus 14.9
Russula ochroleuca 3.5 Russula ochroleuca 14.6
Tomentella ellisii 2.3 Tomentella ellisii 7.8
Tomentella sublilacina 1e3.5 Tomentella sublilacina 3.4e15

min median max min median max

Imleria badia 1.7 2.2 6.4 Imleria badia 13 15.5 20

Paxillus involutus 1.6 3.3 4.6 Paxillus involutus 8.2 10 11.6
Thelephora terrestris 1.6 2.9 7.6 Thelephora terrestris 5.5 10 20

The results for Cu reported for many samples of ECMs and roots
(Supplementary Tables S3eS4) are associated with a relatively large
uncertainty (10e30%) because the concentrations were close to the
detection limit of INAA. Nevertheless, the data were still considered
worth of evaluation. In ECMs, Cu concentrations did not exceed
162 mg kg
1 and no signicant difference was found between ECMs
and roots; the BAF values below 1 indicate bioexclusion of Cu. The
highest Cu contents were found in ECMs of I. badia (mean
120 mg kg
1, maximum expected Cu concentrations in hyphae of
401 mg kg
1) and rather low values were observed in P. involutus
ECMs (mean 67.3 mg kg
1).
Zinc was accumulated in ECMs and its concentrations were the
highest we detected among the investigated metals (maximum
1280 mg kg
1 in T. terrestris). However, the Zn accumulation did not
appear striking (BAF of up to 7.6) as the extractable soil Zn contents
were relatively high (90e400 mg kg
1). With a single exception, V
concentrations in ECMs and ne roots were similar and generally
lower than both total and extractable V in soils (BAF below 1).
However, V is distinctly elevated in ECMs of A. muscaria
(26.4e61.2 mg kg
1) and was apparently accumulated (BAF 3e6);
concentrations calculated in the fungal hyphae were 367 and
864 mg kg
1.
Concentrations of As in ECMs were signicantly higher than
those in the ne roots. On the other hand, the BAF was only
182
et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova
exceptionally above 1 (e.g., in Tomentella sublilacina), so As was not
accumulated. Notably, its concentrations in ECMs among the
identied fungal species were very variable, e.g., with a large range
of 3.98e376 mg kg
1 in I. badia. Antimony concentrations in ECMs
and ne roots are not signicantly different but when compared to
soils, accumulation of Sb was noted particularly in Thelephoraceae
(BAF up to 23).
In contrast to the ne roots with median Cl concentration of
962 mg kg
1, Cl is signicantly accumulated in ECMs (median
2700 mg kg
1; BAFt 3.4e20). However, the levels found in partic-
ular ECMs do not follow the pattern known from the fruit-bodies;
Table 4 shows results for Cl concentrations in fruit-bodies (un-
published data from our archive, fruit-bodies of various origins)
and ECMs of selected species from Lhota near Prbram. As seen
particularly in P. involutus, low concentrations in fruit-bodies are
not followed in ECMs. The highest Cl concentrations were found in
ECMs of I. badia whereas the Cl levels in ECMs of A. muscaria (which
accumulates Cl in fruit-bodies) were comparable with those of
P. involutus.
Concerning the quality of INAA and ICP-OES data, the results
obtained for the analyzed SRMs (Supplementary Table S8) were
mostly in good agreement with the certied/reference values, with
recoveries in the range of 90e110%.
4. Discussion
4.1. Fungal biomass in ectomycorrhizae
Quantitative real-time PCR is a useful tool to determine the
biomass of individual fungal species in soil (Landeweert et al.,
2003). This approach has been successfully used for quantica-
tion of soil mycelia of ECM and saprotrophic fungi both in eld
studies (Hortal et al., 2008; de la Varga et al., 2012; Borovicka et al.,
2014) and under laboratory conditions (Parlade
et al., 2007; Kurth
et al., 2013). Recently, Gryndler et al. (2013) detected and quanti-
ed mycelium of Tuber aestivum in soil and from non-mycorrhizal
roots of Carpinus betulus. In our hands, the median values of
mycobiont proportions in ECMs of I. badia and A. muscaria deter-
mined by qRT-PCR approach were close to 5% (w/w). This value is
smaller relative to the data presented by Antibus and Sinsabaugh
(1993) who reported proportion of fungal biomass in ECMs of
Betula populifolia and Pinus sylvestris within the range of 18e34%.
Slightly higher values were reported for ECMs of Tuber borchii on
Tilia platyphyllos (Zeppa et al., 2000).
Apparently, our median values are lower than those reported by
others but it appears reasonable to expect that the amount of the
mycobiont in ECMs will be affected by many environmental factors
as well as by the physiological state of the partners in the symbiosis.
However, with a single exception of the age of ECMs, there are no
relevant reports that would support this notion. Zeppa et al. (2000)
noted remarkably higher proportion of the symbiotic mycelium in
young ECMs; however, the difference they reported (51% in young
Table 4
Concentrations of Cl in fruit-bodies (mg kg
1 in dry mass) from various sites and in
ectomycorrhizae from Lhota near Prbram presented as concentration range (if
n 2) or arithmetical mean standard deviation.
Species Fruit-bodies Ectomycorrhizae
n Cl (mg kg
1) n Cl (mg kg
1)
Amanita muscaria 10 7021 3229 2 1695e2217
Imleria badia 7 1339 422 9 3632 1267
Paxillus involutus 17 171 70 7 2541 292
Russula ochroleuca 4 2527 626 1 3711
Thelephora terrestris 2 1038e1642 17 2799 829
and 35% in old ECMs) cannot fully explain prevailing low values in
our ECM samples.
It must be stressed that the investigated site is characterized by
extremely high level of metal(loid) pollution, with particularly high
Pb concentrations in the Oe horizon reaching the levels of units of %
(w/w) (Ettler et al., 2004). It can be speculated that such a toxic
environment may substantially affect fungal tness and the ability
to produce ECMs. Furthermore, non-homogeneity of soil (the
patchiness of pollutant distribution) may, at least partly, explain the
high variation in the proportion of fungal biomass in ECMs. It can be
speculated that the high concentrations noted both in I. badia and
A. muscaria might represent a normal level of colonization
whereas the prevailing low concentrations might be attributed to
the stress exerted by onsite pollution. Regrettably, no comparable
literature data on fungal biomass concentration in ECMs under
conditions of varying stress conditions are available.
Furthermore, the discrepancy between our data and the data of
Antibus and Sinsabaugh (1993) and Zeppa et al. (2000) might also
stem from different methodology that in these studies relied on the
use of ergosterol as the biochemical marker for fungi (Wallander
et al., 2013). Our notion that the use of qRT-PCR provided reliable
data is reinforced by the fact that the primers used in qRT-PCR did
not affect the results (two different primer pairs per each of the
fungal species under study were used) as the differences in fungal
proportions obtained by using the two independent primer pairs
are negligible. Furthermore, the concentration of the fungus in
ECMs seems to be independent on sample weight, as no signicant
correlation between these two parameters was observed: correla-
tion coefcients (R2) were 0.1343 and 0.0442 for I. badia and
A. muscaria ECMs, respectively (Table 1).
Regardless the methods used for quantication, the results on
fungal biomass concentration in ECMs depend on sample selection
and preparation, including the subjective denition of ECM by
researcher who detaches the colonized parts from the root. Though
we cannot absolutely exclude that a minuscule piece of the root
tissue (other than ectomycorrhiza) was included into analyses in
some cases, we were careful to eliminate this problem. Then, the
above mentioned source of variance is not likely. We therefore
assume that the observed proportion of mycobionts reects
different extent of the root colonization, i.e. the density of fungal
structures inside the analyzed ECMs.
4.2. Metals in ectomycorrhizae
Accumulation of metals in fruit-bodies depends on the ability of
fungi to take them up by hyphae from soil and transport them
through mycelia. Accumulation of metals is often strictly species-
dependent and may occur only in a few fungal species. This was
documented with V, which was effectively excluded in all ECMs
except of those of A. muscaria, the V hyperaccumulator (Vetter,
2005). In the fruit-bodies of A. muscaria, V is deposited into ama-
vadin, an organometallic compound of unknown biological func-
tion (Garner et al., 2000). High V contents in ECMs of A. muscaria
should thus be attributed to fungal activity but whether or not
amavadin is synthesized in mycelium and present in ECMs remains
to be investigated.
Unlike vanadium, Ag, Cd, Zn, and Cu are known to be effectively
accumulated in fruit-bodies of many fungal species. The greatest
accumulation ability was repeatedly demonstrated for Ag
(Borovicka et al., 2007, 2010b) and this was conrmed also in ECMs
(BAF 100e9700). High Ag levels were found e.g., in ECMs of I. badia
which, however, accumulates quite low concentrations of the metal
in fruit-bodies (mean concentration of 14.2 mg kg
1 at the inves-
tigated site; Borovicka et al., 2010b). Curiously, Ag is accumulated
regardless its low mobility in soils which was also noted by Kubrova


et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova
et al. (2014) and Benes et al. (2016). We therefore speculate that the
low Ag levels in the easily extractable soil fractions might be at least
in part due to the effective uptake by fungal hyphae.
Cadmium levels in macrofungi have been documented from a
variety of habitats. Relatively low concentrations are reported e.g.
in fruit-bodies of I. badia and P. involutus (Malinowska et al., 2004;
Brzostowski et al., 2011a-b), whereas A. muscaria efciently accu-
mulates Cd in both clean and polluted environments (Kala c et al.,
1991; Falandysz et al., 2007). Reported differences on the fruit-
body level were not reected in ECMs from Lhota near Prbram in
which were Cd concentrations generally high, irrespective of the
fungal species. Noteworthy, the observed Cd concentrations
correspond to those reported by Krupa and Kozdro
j (2004) in ECMs
of Betula.
Dubiously low Cd concentrations (mean 4.6 mg kg
1) were re-
ported in various ECM morphotypes from a Norwegian region
described as with high surface soil Cd levels (Berthelsen et al.,
1995). On the other hand, their data for Zn in ECMs
(105e1090 mg kg
1) are in good agreement with our observations
(197e1280 mg kg
1). Regarding the Cu concentrations in ECMs, our
results appear substantially lower than those published by
Berthelsen et al. (1995), who reported signicant accumulation of
Cu (176e790 mg kg
1) in ECMs. The highest value observed in our
study was only 162 mg kg
1 (in I. badia). Regrettably, no other data
were published that would allow further comparisons. With
respect to fungal species, both I. badia and A. muscaria are rather
weak Cu accumulators (Malinowska et al., 2004; Vetter, 2005) and
somewhat higher Cu contents have been reported for P. involutus
(Brzostowski et al., 2011a,b). High levels of Cu found in ECMs of
I. badia (mean 120 mg kg
1) and rather low in P. involutus (mean
67.3 mg kg
1) are therefore contrary to what was observed in the
fruit-bodies.
According to Kottke et al. (1998), ECMs of I. badia showed a
higher potential to store N, P, K, Mg, Fe, and Zn than other ECM
types. In our study, I. badia appeared to be the most efcient
accumulator of Ag, Cd, Cu, and Cl. On the other hand, concentra-
tions of Zn (mean 537 mg kg
1) were similar to those detected in
P. involutus (mean 529 mg kg
1) and T. terrestris (mean
492 mg kg
1).
We believe that ECMs from polluted sites represent interesting
subject for further studies. Especially, micro-PIXE analysis might
contribute to the knowledge of element distribution within ecto-
mycorrhizal tips (Turnau et al., 2001) and cultivable ECM species
with high metal(loid) accumulation capacities are suitable for
studies of element-fungus-plant interactions in pot experiments,
including the mechanisms underlying uptake, detoxication, and
immobilization of elements in fungal structures. The observation
that ECMs tend to accumulate higher concentrations of metals than
non-mycorrhizal ne roots indicates the sequestration of metals
within the hyphal structures which may protect associated plants
from metal overload.
Noteworthy, the mean Zn concentration calculated for I. badia
are in good agreement with 2600 mg Zn kg
1 reported in the fungal
mantle of Suillus luteus/Pinus sylvestris ECM from metal polluted
site (Turnau et al., 2001), whilst the levels of Cd and Cu in both the
I. badia and A. muscaria ECMs were more than an order of magni-
tude higher. It has been well documented that accumulation of
metals in fungi involves, besides the metabolism, independent
biosorption on the cell wall, cellular uptake and detoxication
(Bellion et al., 2006). It appears that high levels of Cd and Zn
tolerance in ECM fungi relies upon the safe compartmentalization
of those metals, mainly, but not exclusively, in the vacuoles (Bellion
et al., 2006; Ruytinx et al., 2013; Sa
ck et al., 2014).
Among the family Boletaceae, I. badia produces Cd-binding
cysteine (Cys)-rich phytochelatins ((PCs; [g-Glu-Cys]2Gly and [g
183
-Glu-Cys]3Gly (Collin-Hansen et al., 2007)), known as the major Cd-
detoxication peptides in plants and some yeasts in which the
cytosolic Cd-PC complex is transported into vacuoles for the
maximum detoxication (Clemens and Simm, 2003). Studies in
Amanita species revealed that ECM fungi can effectively eliminate
Ag toxicity through binding of free toxic metal ions with cytosolic,
cysteine-containing metallothioneins (MTs) (Osobova
et al., 2011;
Hlo zkova
et al., 2015). Several lines of evidence suggest that MTs
can be involved in detoxication of Cu (and Cd) in the mycelia of
P. involutus (Bellion et al., 2007), Hebeloma cylindrosporum (Ramesh
et al., 2009), Laccaria bicolor (Reddy et al., 2014), and Amanita
strobiliformis (Benes et al., 2016). Very recently, Reddy et al. (2016)
demonstrated that the mycorrhization of Eucalyptus tereticornis
with Pisolithus albus, in which Cd and Cu induced production of
MTs, reduced the uptake of Cd and Cu in host tissues 3-fold, indi-
cating metal barrier function of the mycobiont facilitated by MT
production.
4.3. Metalloids in ectomycorrhizae
When compared with soils, As concentrations in ECMs are
relatively low but they commonly reach tens of mg kg
1 in I. badia
and T. terrestris. Arsenic is known to be accumulated by macrofungi
(Stijve et al., 1990) and organic As compounds are usually present in
fruit-bodies (Nearing et al., 2014). However, the presence of the
corresponding biosythesis pathways in macrofungi has not yet
been demonstrated. Although Nearing et al. (2015) speculated that
the fruit-body, but not mycelium, itself may produce methylated As
compounds, the possibility that organic As species originate from
the associated soil microbiota could not be rejected.
There is a lack of information about how ectomycorrhizal fungi
adsorb and/or transform Sb at the soil-fungal-plant interface
(Pierart et al., 2015). The investigated site is heavily polluted by Sb,
with concentrations in Oe horizon commonly reaching higher
hundreds of mg kg
1. Despite the chemical similarity, Sb does not
correlate with As in macrofungi and only Suillus and Chalciporus
species are known to accumulate Sb in fruit-bodies (Borovi cka
et al., 2006). None of those genera, however, was covered by this
study and our results for ECMs indicate rather bioexclusion than
accumulation of Sb.
4.4. Chlorine in ectomycorrhizae
Available data on Cl concentrations in macrofungal fruit-bodies
indicate species-dependent accumulation of Cl; e.g., remarkably
high Cl concentrations were reported in Amanita spp. (Stijve, 1984;
Petrini et al., 2009) and low concentrations in P. involutus (Hedrich,

1988; Randa et al., 2005). High levels of Cl in ECMs of P. involutus,
similar to those of A. muscaria, thus represent an unexpected
outcome of this study.
The soil Cl concentrations at Lhota near Prbram correspond to
those found in Oe horizons at similar pristine habitats (Kabata-
Pendias, 2011; our unpublished data). Thus, the Cl levels in ECMs
reported in this study are likely not inuenced by smelter pollution.
The biological importance of Cl accumulation in fruit-bodies and
ECMs, however, remains unexplained and deserves further inves-
tigation. Chlorine (in chloride form) plays a role in fungal degra-
dation of lignine, humic substances, and phenolic organic matter
(Oberg et al., 1997) that constitute important components of the
soil environment. These compounds are often relatively resistant to
biological degradation and the chlorination step may accomplish
their mineralization (Matucha et al., 2007). Various components of
these potentially toxic materials may pass through plasma mem-
brane and damage the cell. This suggests that the accumulation of
Cl in fungal biomass may be important for the elimination of these
184
et al. / Environmental Pollution 218 (2016) 176e185
J. Cejpkova
potentially toxic compounds from the cytoplasm because the pro-
cess of chlorination-mediated degradation might be enhanced by
high chloride concentration (Lambeir and Dunford, 1983). Then, the
accumulation of Cl (chloride) in the fungal biomass may be hypo-
thetically interpreted as an adaptive mechanism enhancing the
detoxication of the fungal cell (Thuillier et al., 2014). Noteworthy,
macrofungi are known to produce various chlorinated compounds
such as chlormethane, chloro amino acids and others (Drehmel and
Chilton, 2002; Redeker et al., 2004). Some of those metabolites
were shown to be cytotoxic (Takahashi et al., 1992) and possible
(e.g, protective) biological functions of Cl-containing fungal me-
tabolites should be taken into consideration.
5. Conclusions
ECM fungi were found to highly accumulate trace elements in
their ECMs, particularly Ag, Cd, Cl, and Zn, which are also known to
be accumulated in fruit-bodies. Among the inspected fungal spe-
cies, Imleria badia was the most efcient accumulator, followed by
Thelephora terrestris. Amanita muscaria was the only species accu-
mulating V in ECMs. Concentrations of As in ECMs were signi-
cantly higher than those in roots and might possibly allow
inspecting chemical forms of As.
The proportions of I. badia and A. muscaria mycelia quantied in
wild-grown ECMs showed substantial variations, but the median
values were close to 5% (w/w) for both species. The assessment of
the element concentrations in ECM hyphae revealed in I. badia
mean Ag, Cd, Zn and Cl concentrations of 1680, 1510, 2670, and
37,100 mg kg
1 dry weight, respectively, which indicate high
element accumulation capacity of mycelia. Such accumulation of
metals in mycelia, followed by their detoxication within the fungal
cells, seemingly results in decrease of their bioavailability in soils.
We assume that the subtle proportion of Ag in easily extractable
fractions in forest soils reported in many environmental studies
could be at least in part caused by the highly effective Ag uptake by
fungi.
Acknowledgements
We thank four anonymous reviewers for valuable and
constructive comments which helped us to improve our manu-
script. This research was supported by the project GF16-34839L
(Czech Science Foundation); the results for Cl concentrations in
macrofungi were obtained within the frame of the project GAUK
200415 (The Charles University Grant Agency). INAA measure-
ments were carried out at the CANAM infrastructure of the NPI CAS
 
Re z supported through the project No. LM2011019 (Ministry of
Education, Youth and Sports of the Czech Republic). Institutional
support was provided by the Long-term Development Projects
RVO61389005, RVO67985831, and RVO61388971.
Appendix B. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2016.08.009.
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