f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 8 7 3 e8 8 0

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Growth responses to and accumulation of mercury by

ectomycorrhizal fungi

Sharron CRANEa,b,*, John DIGHTONb,c, Tamar BARKAYa,b

a
Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ 08901, USA
b
Graduate Program in Ecology and Evolution, Rutgers University, New Brunswick, NJ 08901, USA
c
Rutgers Pinelands Field Station, P.O. Box 206 New Lisbon, NJ 08064, USA

article info abstract

Article history: Heavy metals have been shown to negatively affect the growth of ectomycorrhizal fungi
Received 28 December 2009 (ECMF). In addition, ECMF have been shown to accumulate heavy metals and to protect
Received in revised form host trees from metal toxicity. However, specific literature on the interactions between
21 July 2010 ECMF and mercury (Hg) is scant. This paper describes the responses of ECMF to Hg in axe-
Accepted 12 August 2010 nic culture conditions. Six ECMF from an area with no known history of direct Hg contam-
Available online 20 August 2010 ination were tested to determine their sensitivity to Hg. ECMF were incubated on solid
Corresponding Editor: Anna Rosling medium amended with Hg (0e50 mM) as HgCl2 and the effect of Hg on radial growth was
determined. The effect of preexposure cultivation on Hg sensitivity, the effect of Hg on bio-
Keywords: mass production, and the ability to accumulate Hg were determined for four of the ECMF.
Heavy metal At micromolar concentrations, Hg significantly inhibited the radial growth rate of ECMF.
Hyperaccumulation This inhibitory effect was lessened in some ECMF when an established colony was exposed
Trajectory to Hg. Mercury lowered biomass production by some ECMF, and ECMF accumulate Hg from
a solid growth substrate in direct relation to the amount of Hg added to the media. Possible
implications for ECMF communities in Hg-impacted areas are discussed.
ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction atmospheric deposition (Fitzgerald et al. 1998). Mercury has

Ectomycorrhizal fungi (ECMF) are essential contributors to
mineral nutrient uptake by woody plants, and can play an im-
portant role in protecting host trees from stresses such as
drought, soil temperature and pH extremes, and toxic sub-
stances (Smith & Read 1997). In acidic forest soils, metals
such as mercury (Hg) are highly bioavailable because of in-
creased solubility and competition with Hþ ions for binding
sites on organic matter (Rieuwerts et al. 1998). Mercury is
a highly toxic metal with no known biological function. The
presence of Hg in soil can be due to geological processes or en-
vironmental contamination from either point sources or
been shown to change soil microbial community structure
and function at bioavailable concentrations of approximately
0.2 ppm (Rasmussen & Sorensen 2001). Although interactions
between ECMF and Hg may have implications for manage-
ment of impacted sites and understanding the biogeochemi-
cal cycling of Hg, to date very little is known.
Extensive research has been done regarding the effects of
heavy metals on filamentous fungi including ECMF. In general,
elevated concentrations of metals have adverse effects on
fungal growth (Gadd 1993; Meharg 2003). Physical symptoms
of metal toxicity include reduced hyphal extension, morpho-
logical changes in mycelia, reduced biomass and increased

* Corresponding author. Department of Biochemistry and Microbiology, 76 Lipman Dr., New Brunswick, NJ 08901, USA. Tel.: þ1 732 932
9763x332; fax: þ1 732 932 8965.
E-mail address: shahicks@rci.rutgers.edu
1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.funbio.2010.08.004
874
hyphal branching (Hartley et al. 1997; Fomina et al. 2003). A
considerable amount of intra- and interspecific variability
has been observed among the responses of ECMF to metals:
for example, a wide range of zinc tolerance indices was
reported among several strains of Suillus luteus, Suillus bovinus
and Rhizopogon luteolus using increase in fungal dry weight as
a measure of tolerance (Colpaert et al. 2005). Similar results
were found when several strains of ECMF representing five
species were screened using several metals: the toxicity of
cadmium and copper varied greatly among isolates of Paxillus
involutus, as did the toxicity of cadmium, copper and nickel
among isolates of S. luteus. Also, groups of strains of S. luteus
and Pisolithus tinctorius demonstrated greater overall tolerance
to copper, cadmium, and zinc than P. involutus, while several
strains of P. involutus were less affected by nickel than other
ECMF (Blaudez et al. 2000b).
Some studies reporting the Hg content of fungal sporo-
carps have included ectomycorrhizal species (Falandysz &
Chwir 1997; Svoboda et al. 2000; Vetter & Berta 2005). In an
area in Poland remote from any point source of pollution,
reported median Hg concentrations in tissue relative to that
of the underlying soil (bioconcentration factors) in Amanita
muscaria were 9 (cap) and 4.7 (stalk), and bioconcentration fac-
tors of Russula aeruginea (cap) were correlated to the Hg con-
tent of the soil (Falandysz & Chwir 1997). Additionally,
species of Russula and other ECMF were found to have concen-
trations of Hg several folds higher when collected near two
smelters (copper and mercury) than when collected at pristine
sites (Svoboda et al. 2000). Because the formation of sporo-
carps generally takes only a few days, Hg found in them is
expected to have come from the soil rather than from direct
deposition. Therefore, most of the Hg found in sporocarps is
likely to have been translocated from other parts of the myce-
lium (Svoboda et al. 2000). Translocation of solutes has been
observed in filamentous fungi as a result of both passive diffu-
sion and active translocation via rhizomorphs or other spe-
cialized mycelia (Cairney 1992), and ECMF have been shown
to translocate lead in axenic culture (Vodnik et al. 1998). Addi-
tionally, studies using the wood-rotting fungus Agrocybe aeger-
ita as a model system to investigate the translocation of Hg
and Cd by fungal mycelia indicated that both metals were
translocated away from the source to sporocarps and to outly-
ing substrate by actively growing mycelia (Brunnert & Zadrazil
1981, 1985).
Some ECMF are able to protect their hosts against metal
toxicity (Brown & Wilkins 1985; Denny & Wilkins 1987;
Colpaert & Van Assche 1992). This ability likely depends on
a number of factors, including the capacity of the fungus to
continue to proliferate through the soil substrate and to pro-
duce new biomass in the presence of high metal concentra-
tions (Colpaert & Van Assche 1992). It has been suggested
that ECMF biomass, both in the extraradical mycelia and in
the Hartig net, binds metal from the soil solution, effectively
reducing the metal concentration experienced by the host
roots (Denny & Wilkins 1987). Ectomycorrhizal trees are candi-
dates for use in the afforestation and reforestation of soils
contaminated with metals or radionuclides (Gray 1998). In ad-
dition to providing a barrier between toxic substances and the
tree root, ECMF may provide direct remediation to such soils
by providing additional binding sites for the contaminants
S. Crane et al.
(White et al. 1995). Although no clear connection has been
made between the growth response of ECMF to metal stress
and their ability to ameliorate the toxic effects of metals in
host trees (Jones & Hutchinson 1988; Aggangan et al. 1998),
the testing of ECMF in axenic culture has been considered as
a preliminary screening step in the selection of ECMF inocula
for reclamation projects (Blaudez et al. 2000b).
Both radial growth and mass gain are important measure-
ments of fungal growth. Radial growth of ECMF is important
because hyphae must be able to reach and colonize host
root tips if the ectomycorrhizal symbiosis is to develop. Addi-
tionally, resource acquisition is only possible if extraradical
hyphae are able to proliferate throughout the soil matrix. Bio-
mass of ECMF is also significant: dense hyphal growth is an in-
dication that the organism is in the exploitative phase of
growth and is actively utilizing soil resources. A possible role
for fungal biomass in Hg-impacted soil is as a sorbent for
Hg, especially at low pH (Ledin et al. 1996). Indeed, fungi
have been extensively investigated as sorbents for use in de-
toxifying contaminated water supplies (Tobin et al. 1994). If
ECMF are acting as Hg sorbents in the soil matrix, the exten-
sive presence of fungal mycelia may sequester Hg in less bio-
available forms, rendering the soil a more hospitable
environment for the growth of ECMF, their host trees, and
other soil organisms. We have therefore tested three hypoth-
eses: H1: the growth of ECMF is inhibited by Hg, as measurable
by both radial expansion and mass gain; H2: toxic effects of Hg
on ECMF are ameliorated in established colonies; H3: ECMF are
able to accumulate Hg from their environment. Here we de-
scribe the results from our investigation into the toxic effects
of Hg on five basidiomycetous and one ascomycetous ECMF.
Materials and methods
Culture of ECMF
Six ECMF were obtained from the culture collection at the
Rutgers University Pinelands Field Station in New Lisbon, NJ.
Amanita muscaria, Cenococcum geophilum, Laccaria laccata, Pilo-
derma bicolor, Pisolithus tinctorius, and Suillus decipiens were
originally isolated from locations in the New Jersey Pine Bar-
rens with no known history of Hg contamination. ECMF
were maintained on Modified MelineNorkrans (MMN) agar
(Marx 1969).
Experiment I: growth of ECMF in response to Hg
Several cylindrical agar plugs (4 mm diameter) were removed
from the edges of actively growing fungal colonies and incu-
bated on fresh media at room temperature for 3e7 d. Plugs
were then aseptically placed mycelium-side down in the cen-
ter of 60 or 90 mm Petri dishes containing fresh MMN agar
amended with concentrations of Hg ranging from 0 to
50 mmol l1 (mM). Treatment regimes chosen for each ECMF
were based on previous observations during a preliminary
study (data not shown). Mercury was added to molten auto-
claved media cooled to approximately 60  C using a stock so-
lution of 50 mmol l1 mercuric chloride in 0.1 N nitric acid.
Media used for Cenococcum geophilum, Pisolithus. tinctorius,
Responses of ectomycorrhizal fungi to mercury
Piloderma bicolor and Suillus decipiens was amended with strep-
tomycin (20 mg ml1) in order to prevent bacterial contamina-
tion of the cultures. Over time it became clear that bacterial
contamination would not become an issue; therefore, no
streptomycin was used during the screening of Amanita mus-
caria and Laccaria laccata. There has been no indication that
streptomycin affects the growth of C. geophilum, P. tinctorius,
P. bicolor, or S. decipiens. Fungal cultures were incubated in
the dark at room temperature (17e22  C). The cultures were
observed under a dissecting microscope and the outermost
edges were marked along four diameters (eight radii) every
few days. Because of their different growth rate trajectories,
the number of days (d) over which data were collected differed
among the ECMF, and are as follows: A. muscaria, 74 d; C. geo-
philum, 82 d; L. laccata, 40 d; P. bicolor, 84 d; P. tinctorius, 66 d; S.
decipiens, 40 d. Radial measurements for all time points were
recorded to the nearest 0.25 or 0.5 mm after the end of the in-
cubation period.
Experiment 2: the effect of colony size on toxicity of Hg,
the effect of Hg on biomass production, and the ability
of ECMF to accumulate Hg
Cylindrical plugs (0.8 cm diameter) of Amanita muscaria, Pisoli-
thus tinctorius and Suillus decipiens were preincubated on fresh
media at room temperature for 4e5 d to ensure even growth,
then transferred to MMN agar overlain with sterile cellophane.
Plugs of Laccaria laccata were not preincubated prior to transfer
because of the short lag phase of this organism. Cultures were
incubated at room temperature in the dark for several days to
develop established colonies. Cultures (including cellophane)
were transferred to plates amended with 0, 10, and 25 mM
Hg. Fungal colony radii were marked as described above on
the date of transfer. Cultures were incubated at room temper-
ature for an additional 11e24 d, after which final radial mea-
surements were taken (Table 1). The effect of Hg on the
radial growth of these established fungal colonies was com-
pared to observations made for selected time points in Exper-
iment 1 in order to evaluate the impact of colony age or size on
the ability of ECMF to buffer the influence of Hg on subsequent
colony growth.
At the end of the incubation period, all samples were
weighed and at least three samples of each organism were
dried at room temperature for several days to determine
fresh:dry weight ratios, which were used to estimate the dry
Table 1 e Incubation details of each species tested in
Experiment 2. In bold italics are the time points from
Experiment 1 chosen for comparison. Experiment 1
colonies were all considered to have a radius of 2 mm and
an age of 0 d upon exposure.
Age upon Hg Average colony Duration of
exposure (days) radius before Hg Hg exposure
exposure (mm) (days)
A. muscaria 14 17.5 24, 24
P. tinctorius 10 15.3 11, 16
L. laccata 9 8.66 11, 12
S. decipiens 16 14.7 21, 22
875
weight of all samples. The remaining samples were used for
total Hg analysis: these were stored at 4  C in concentrated
1:1 trace metal grade nitric:sulfuric acid until Hg analysis
was performed.
Samples in concentrated acid were heat digested (>70  C)
for at least 90 min. One or 0.5 ml of digested sample was then
removed to a clean test tube and a half-volume of 5 % potas-
sium permanganate was added to ensure complete oxidation
of all Hg in the sample. Immediately before analysis, 12 % hy-
droxylamine hydrochloride was added until the color of the so-
lution changed from purple to clear or clear-yellow: this color
change indicated complete reduction of the potassium per-
manganate. Additional dilutions were made as needed using
2 % hydrochloric acid, and Hg analysis was performed using
a Leeman Labs Hydra AA Hg analyzer (Hudson, NH).
Statistical analyses
Repeated measures analysis of variance (RMANOVA) was used
to determine whether Hg had an effect on the radial growth of
the six ECMF and whether the effect of Hg on these fungi
changed over time (Experiment 1). Comparisons of the impact
of Hg on radial growth measurements between young, smaller
cultures (Experiment 1) and established, larger cultures (Exper-
iment 2) were performed using the Student’s t-test. Multivariate
analysis of variance (MANOVA) was used to determine the effect
of Hg exposure on final radial measurement, biomass gain, and
Hg content (Experiment 2). Mean separation was performed us-
ing Tukey’s HSD post-hoc tests. All analyses described in this
paper were performed using Graphpad Prism version 4.00 (La
Jolla, California) or SAS version 9.1.3 (Cary, North Carolina).
Results
Colony establishment and growth in a range of Hg
concentrations
The six ECMF tested showed unique intrinsic growth rate pat-
terns when no Hg was added to the media (Fig 1). The radius of
Amanita muscaria expanded slowly for several days then grew
exponentially; Laccaria laccata, Pisolithus tinctorius, and Suillus
decipiens expanded quickly at the beginning of incubation,
then grew more slowly after several days; Cenococcum geophi-
lum and Piloderma bicolor displayed short lag phases followed
by steady, slow growth. RMANOVA indicated that Hg had a sig-
nificant negative effect ( p < 0.001) on the colony size of all six
ECMF tested, and that interaction between time and Hg was
significant at the 0.05 level for all ECMF with the exception
of C. geophilum, for which it was significant at the 0.1 level
(Table 1). This indicates that Hg concentration influenced
not only the final colony diameter, but also the trajectory of
the growth curve for all six isolates. Sensitivity of the six
ECMF to Hg varied. The final colony radii of A. muscaria and
L. laccata were no different when incubated with 5 mm Hg
than when no Hg was added, but the growth of both fungi
was negatively affected by this concentration at the beginning
of the incubation (Fig 1). The same is true for the effect of
10 mm Hg on P. bicolor, although the effect of this concentration
of Hg on the organism at the beginning of the incubation was
876 S. Crane et al.

A 45 D 30
P. bicolor
40
A. m uscaria
35
30 20
25
20
15 10
10
5
0 0
0 25 50 75 100 0 25 50 75 100
Average colony radius (mm)

B 20 E 40
P. tinctorius
C. geophilum
30

10 20

10

0 0
0 25 50 75 100 0 10 20 30 40 50 60 70

C 30 F 20
L. laccata S. decipiens

20

10

10

0 0
0 10 20 30 40 50 0 10 20 30 40 50

Days
Fig 1 e The effect of Hg on radial growth of (A) A. muscaria [6], (B) C. geophilum [3], (C) L. laccata [6], (D) P. bicolor [3],
(E) P. tinctorius [5], and (F) S. decipiens [5]. Numbers in brackets refer to the number of Hg concentrations to which that isolate
was exposed. Micromolar concentrations of Hg are signified by - (0), , (5), 10 : (10), O (20), A (25), and > (40). Means and
standard error of at least three replicates are presented, with the exception of C. geophilum grown at 25 mM Hg (two replicates).

minor. Pisolithus tinctorius was much more inhibited by high
(20 and 25 mM) than by low (5 and 10 mM) concentrations of
Hg, and an increase in the lag phase of this organism was
not observed until it was exposed to 20 mM Hg. The final colony
diameter of S. decipiens was nearly identical for all Hg concen-
trations tested (except 0 mM), but at earlier time points the ra-
dii were significantly different among concentrations. From
the results, it is clear that Hg, when added to growth media
at a high enough concentration, both increased lag phase
and decreased growth rate of ECMF (Fig 1, Table 2); however,
the concentration of Hg at which these effects were observed
varied among species.
were only inhibited by 50.6 and 77.9 %. Likewise, young cul-
tures of Amanita muscaria were almost completely inhibited
by both concentrations of Hg tested, but established colonies
were only inhibited by 30.7 and 74.8 %. Pisolithus tinctorius
Table 2 e Results of RMANOVA on the effect of Hg and the
interaction of time and Hg on their radial growth and
concentration of Hg (mM) at which an increased lag phase
or decreased post-lag growth rate was first seen.
Hg Time Increased Decreased
*Hg lag phase growth rate

p p
Effects of mercury on radial growth: comparison between new
and established fungal colonies A. muscaria <0.0001 <0.0001 5 20
C. geophilum <0.0001 0.097 10 25
Established colonies of all four isolates tested were less sensi- L. laccata <0.0001 <0.0001 10 5
P. bicolor <0.0001 0.0123 25 25
tive to the presence of Hg than newly developing colonies
P. tinctorius <0.0001 <0.0001 20 5
(Table 3). Young Laccaria laccata colonies were completely S. decipiens <0.0001 <0.0001 10 5
inhibited by both 10 and 25 mM Hg, yet established colonies
Responses of ectomycorrhizal fungi to mercury
Table 3 e Average percent inhibition of radial growth (SE) of both new and established ECMF colonies by 10 and 25 mM Hg.
Values were generated by dividing the growth measured at the time point reported in Table 1 (new colonies) or the growth
measured at the end of the experiment (established colonies) by the average growth of control cultures (no Hg added to the
media) and multiplying by 100. Statistical tests could not be performed on L. laccata because growth was completely
inhibited in all replicates at the time point chosen for Experiment 1, resulting in zero variance.
ECMF 10 mM Hg
New Established
A. muscaria 92.9 (1.93) 30.7 (3.64)
L. laccata 100 50.6 (6.02)
P. tinctorius 45.4 (1.98) 14.7 (1.97)
S. decipiens 26.2 (3.34) 2.92 (3.98)
showed similar results, although it was less inhibited by either
concentration of Hg than A. muscaria and L. laccata. Interest-
ingly, established colonies of Suillus decipiens were barely
inhibited by either concentration of Hg used.
Effects of mercury concentration on biomass of established
fungal colonies
Mercury reduced biomass of Laccaria laccata and Pisolithus tinc-
torius (Fig 2): this is consistent with the reduction in radial
growth seen in both new and established colonies of these
two organisms (Table 3). In contrast, there was no effect of
Hg concentration on the biomass of Amanita muscaria, al-
though radial growth of this organism was inhibited by Hg.
There was no change in biomass production of Suillus decipiens
at 10 mM Hg and only a slight increase at 25 mM Hg, which is
consistent with the finding that Hg did not affect the radial
growth of established colonies of S. decipiens.
Mercury accumulation by established colonies of ECMF
All four ECMF accumulated Hg in relation to the amount of Hg
added to the growth medium: dry weight concentrations of Hg
in fungal mycelia were lower when any of the organisms were
exposed to 10 mM Hg than when they were exposed to 25 mM
Hg (Fig 2). It is difficult to compare Hg accumulation among
the four species because of the various experimental condi-
tions; however, the two ECMF exposed to Hg for longer periods
(Amanita muscaria and Suillus decipiens) absorbed more Hg per
unit biomass at both 10 and 25 mM Hg than Pisolithus tinctorius
and Laccaria laccata (Fig 2).
Discussion
This study demonstrated for the first time a variety in growth
responses of ECMF to Hg dependent on the fungal species, col-
ony age and size, and media concentration of Hg. We have
also shown that ECMF accumulate Hg from their environment
in axenic culture. Studies on the effect of contaminants on ra-
dial growth of fungi rarely focus on trajectories of growth. In-
ferences from these studies may be less relevant to
ectomycorrhizal fungi where their rate of growth is important
for colonizing root tips that may be at different distances from
the initial site of fungal growth. When ECMF are exposed to
Hg, a dramatically increased lag phase such as was observed
in vitro with Laccaria laccata and Amanita muscaria (Fig 1) may
877
25 mM Hg
p New Established p
<0.0001 98.2 (1.84) 74.8 (4.15) 0.0008
NA 100 77.9 (2.43) NA
<0.0001 86.8 (4.44) 63.8 (6.44) 0.0007
0.0009 56.7 (4.94) 5.31 (5.82) <0.0001
affect the ability of an individual to quickly reach and colonize
a root tip. Although this suggestion would need to be con-
firmed using in vivo studies, differential effects of Hg on
ECMF growth and colonization of root tips could strongly in-
fluence community composition of ECMF on roots in areas
with high bioavailable Hg. A significant difference has been
found between the communities of sporocarps found at pris-
tine and Hg-impacted sites (Svoboda et al. 2000). Although
sporocarps are not typically indicative of belowground com-
munities, it is a reasonable hypothesis that an environmental
disturbance that impacts the aboveground fungal community
may also impact the belowground ECMF community. Other
studies have shown an effect of metals on mycorrhizal com-
munities. For example, community composition of Pinus syl-
vestris ECMF morphotypes shifted in response to nickel
deposition (Markkola et al. 2002). Additionally, reduced total
mycorrhizal colonization and ECMF diversity of Betula pendula
were observed in a bare heap uranium mining site compared
to a reference site (Staudenrausch et al. 2005), but no such dif-
ferences were observed between Quercus garryana ECMF com-
munities on (metalliferous) serpentine and nonserpentine
soils (Moser et al. 2009).
One of the only studies to look at the growth response of fil-
amentous fungi to Hg used a concentration that was so high as
to completely inhibit all isolates tested (Baldrian & Gabriel
1997). By using a range of concentrations we can see that
some ECMF may only be tolerant of or resistant to low levels
of Hg (e.g., Cenococcum geophilum, L. laccata, Piloderma bicolor),
and that there can also be threshold levels of the effect of
Hg on growth of ECMF, as there is a dramatic decrease in the
radial growth of all isolates in this study at Hg concentrations
higher than 10 mM (Fig 1). An increase in lag phase followed by
the resumption of a normal growth rate, for example in A.
muscaria at low Hg (Fig 1; Table 2), suggests the possible induc-
tion of an adaptive response to Hg (resistance), as is well docu-
mented for the bacterial response to Hg (Barkay et al. 2003).
Both inter- and intraspecific variations have been docu-
mented in ECMF radial growth responses to a number of
metals. For example, it was demonstrated that while radial
growth of a Suillus bovinus isolate was inhibited by 91 %
when exposed to 0.5 ppm, cadmium, while Paxillus involutus
and Rhizopogon subcaerulescens were only inhibited by approx-
imately 26 and 28 %, respectively (Kim et al. 2004). Also, four
isolates of C. geophilum demonstrated a range of sensitivities
to nickel (Goncalves et al. 2007). The C. geophilum isolates
from nonserpentine soil were more sensitive to nickel than
those from serpentine soil, providing some evidence that
878
Fig 2 e Biomass and Hg concentration in established cul-
tures of (A) A. muscaria, (B) L. laccata, (C) P. tinctorius and (D)
S. decipiens after several days’ exposure to Hg. Means and
standard error of at least three replicates are presented,
with the exception of L. laccata grown at 0 mM (two repli-
cates). Letters above bars (regular type for biomass, italics
for Hg concentration) indicate statistically significant dif-
ferences among groups.
S. Crane et al.
fungal provenance may influence metal tolerance. Colpaert
et al. (2004) have found similar results regarding zinc sensitiv-
ities among several closely related suilloid fungi and have sug-
gested that evolutionary adaptation to metal contamination
occurs among ECMF. In contrast, Dunabeitia et al. (2004)
showed that Rhizopogon luteolus, Rhizopogon roseolus and Sclero-
derma citrinum were all similarly inhibited by increasing con-
centrations of cadmium, copper and lead. To our knowledge,
our study is the first report of the effect of Hg on the growth
curve trajectories of ECMF.
The effect of Hg on the rate of growth of fungi depended
not only on the organism and Hg concentration used, but
also on the original size or developmental stage of the fungal
colony. Hyphal extension was less inhibited in established
colonies than in young colonies exposed to Hg (Table 3).
Thus, it may be suggested that successful colonization of roots
from an established mass of hyphae in soil or in association
with a root tip would be greater than that originating from
a germinating spore (single hypha) in the presence of Hg
contamination.
Reduced biomass production in response to metals with
a high degree of variability both between and within species
has been demonstrated for ECMF. For example, the cadmium
EC50 for Suillus granulatus was approximately 5.5 and 1575
times that of P. involutus and Suillus variegatus, respectively,
while its zinc EC50 was only 1.5 times that of P. involutus and
was not statistically different than that of S. variegatus
(Hartley et al. 1997). Biomass production of P. involutus, R. sub-
caerulescens, and S. bovinus was adversely affected by the pres-
ence of cadmium (Kim et al. 2003). In our study, established
cultures of ECMF exposed to 10 mM Hg exhibited between
0 and 50 % biomass production decrease (Fig 2). When exposed
to 25 mM Hg, these organisms exhibited between 0 and 75 %
biomass production decrease. Dunabeitia demonstrated that
R. luteolus, R. roseolus and S. citrinum had similar responses
when either radial growth or biomass was used as a metric
(Dunabeitia et al. 2004). Although all of the species tested dem-
onstrated both radial expansion and biomass gain in the pres-
ence or absence of Hg, there have been reports of continued
radial expansion of fungal colonies in the absence of signifi-
cant biomass increase in response to metal stress (Vodnik
et al. 1998; Fomina et al. 2005). In such cases, the growth was
in the form of fine, diffuse hyphae, which may be indicative
of weak exploratory growth. Investigation into the effect of
any disturbance on the growth of filamentous fungi must
take both radial growth and biomass gain into consideration.
Accumulation of Hg by fungal mycelia leading to detoxifi-
cation of the environment may partially explain the recovery
of radial growth seen in some ECMF cultures exposed to Hg, as
Hg bound to fungal biomass is likely to be less toxic than Hg in
solution. This may be an adaptation for Amanita, Pisolithus and
Suillus, where increased Hg loading was less inhibitory to
established colonies than to new colonies, did not have a large
effect on biomass production and where fungal biomass could
accumulate increasingly higher amounts of Hg (Fig 2; Table 3).
Some ECMF are capable of accumulating a variety of metals,
although hyperaccumulation of one metal does not imply
the ability to hyperaccumulate another: this may suggest
that there are several mechanisms that contribute to the accu-
mulation of metals by ECMF. In one study, P. involutus was
Responses of ectomycorrhizal fungi to mercury
shown to accumulate cadmium primarily in the vacuole
(Blaudez et al. 2000a). In symbiotic association with Picea abies,
Hebeloma crustuliniforme formed extracellular cadmium com-
plexes, but primarily sequestered zinc in the cytoplasm
when faced with an excess of these metals. These findings
are in agreement with the suggestion by Colpaert & Van
Assche (1987) that tolerance to one metal does not imply toler-
ance to others: in their experiment, Pisolithus tinctorius was
highly tolerant to copper but sensitive to zinc. It is possible
that the differences among metal tolerance mechanisms in
fungi are related in part to the fact that some metals (e.g.,
zinc) are essential micronutrients, while other such as cad-
mium and Hg have no known biological function in fungi
(Frey et al. 2000). Most metal-impacted sites have toxic con-
centrations of several metals, and it would be useful to under-
stand the effects of several different metals on resident ECMF
in metal contaminated areas and on any ECMF being consid-
ered for remediation efforts.
In conclusion, our preliminary findings on the effect of Hg on
ECMF are as follows: Hg inhibits both radial expansion and bio-
mass production of ECMF, although to different extents among
isolates. The inhibition of ECMF by Hg may be ameliorated in
established colonies, and ECMF are able to accumulate Hg rela-
tive to the amount present in the growth media. Although ECMF
do not always behave the same in symbiosis as they do in axenic
culture, carefully controlled growth and uptake studies must be
done if we are to begin to understand the complex interactions
between Hg and ECMF. Current work is underway to investigate
the effect of Hg on ectomycorrhizal communities and the ability
of ECMF to protect host plants from Hg stress.
Acknowledgements
We would like to thank two anonymous reviewers for their
helpful comments on the original version of this manuscript.
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