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Ectomycorrhizal communities associated with Populustremula growing on a heavy metal contaminated site

Doris KRPATAa,*, Ursula PEINTNERa, Ingrid LANGERb, Walter J. FITZb,Peter SCHWEIGERb

aInstitute of Microbiology, University Innsbruck, Technikerstrasse 25, 6020 Innsbruck, AustriabDepartment of Forest and Soil Sciences, University of Natural Resources and Applied Life Sciences,

Peter Jordan-Strasse 82, 1190 Vienna, Austria

a r t i c l e i n f o

Article history:

Received 30 October 2007

Received in revised form

30 January 2008

Accepted 14 February 2008

Corresponding Editor:

Derek T. Mitchell

Keywords:

Aspen

Ectomycorrhiza

ECM

Fungal community

Heavy metal

Populus tremula

Soil horizon

* Corresponding author.E-mail address: d.krpata@uibk.ac.at

0953-7562/$ – see front matter ª 2008 The Bdoi:10.1016/j.mycres.2008.02.004

a b s t r a c t

European aspen is one of the most widely distributed trees in Central Europe and is a typical

early colonizer of poor and disturbed soils. However, little is known about ectomycorrhizal

(ECM) fungi in these ecosystems. We examined the ECM community of European aspen

growing on a heavily contaminated site in southern Austria by analysing ECM roots, sorting

them into morphotypes, subjecting them to DNA extraction, PCR, and DNA sequencing.

ECM root symbionts were sampled two times in 2004. During this time, the below-ground

community structure was relatively stable; we found no evidence of taxa adapted to sum-

mer or autumn conditions and only two species varied widely in occurrence between soil

horizons. The ECM fungal community was diverse (54 species), rich in Basidiomycota (43

species), and dominated by Cenococcum geophilum and fungi with corticoid basidiomes

(e.g. Thelephoraceae).

ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction shifts in species composition (Smit et al. 1997; Turpeinen

Mining and smelting operations have resulted in a large

number of sites contaminated with heavy metals (some

100 K potentially contaminated sites in Western Europe)

(Quercia et al. 2006). Soil metal concentrations on these sites

frequently reach levels toxic to both plants (Cobbett 2003)

and microorganisms (Giller et al. 1998; Dai et al. 2004). As a con-

sequence, a reduced diversity of microorganisms (Moffett et al.

2003) has frequently been observed on such sites. Differences

in metal tolerance between organisms additionally result in

ritish Mycological Society

et al. 2004). Severe zinc (Zn) pollution can trigger the evolution

of an increased Zn tolerance in ECM fungi as was shown by

Colpaert et al. (2004, 2005) and Adriaensen et al. (2006) for Suil-

lus isolates. However, data concerning the effect of heavy

metal pollution on ectomycorrhizal (ECM) fungal communi-

ties are controversial: negative effects were reported by

Hartley et al. (1997), Hartley-Whitaker et al. (2000), and

Dunabeitia et al. (2004). Moreover, Ruhling & Soderstrom

(1990) reported a decrease of fruit body-producing species

with increased soil metal concentrations. This is in contrast

. Published by Elsevier Ltd. All rights reserved.

1070 D. Krpata et al.

to other data (Meharg 2003) reporting a high ECM fungal diver-

sity on metal contaminated sites.

Numerous studies have examined the above- as well as the

below-ground community structure of ECM fungi (Gardes &

Bruns 1996; Kernaghan & Harper 2001; Peter et al. 2001). Most

of these studies were conducted on coniferous trees with

only a few studies on broadleaf tree species. Of these, Populus

spp. have received some attention. Kaldorf et al. (2004) exam-

ined the ECM fungal community of transgenic hybrid aspen

(Populus tremula� P. tremuloides) clones planted on agricultural

land. They differentiated 23 ECM types by morphology, as well

as molecular methods, and found the community to be dom-

inated by a Tomentella sp. and Phialocephala fortinii. Other stud-

ies report slightly lower numbers of ECM types associated

with poplars (12–19) (Jakucs 2002; Kernaghan et al. 2003;

DeBellis et al. 2006). This compares with 43 putative mycorrhi-

zal fungi that produced fruit bodies in Populus tremuloides

dominated forests (Cripps & Miller 1993).

Poplars are of scientific interest because of the relatively

small size of their genome (Martin et al. 2004) and also because

of applied interest based on their use in short rotation forestry

(Dickmann 2006; French et al. 2006). They have further been

used for pollution monitoring (Madejon et al. 2004) and are

considered suitable for use in phytoremediation (Laureysens

et al. 2004). Screenings of Populus spp. for metal tolerance

and uptake revealed large differences between species and

clones (Djingova et al. 1999; Bittsanszky et al. 2005). Especially

a clone of European aspen (Populus tremula), collected from

a severely polluted site in southern Austria, was found to

accumulate and tolerate high concentrations of cadmium,

lead, and zinc in the leaves (Unterbrunner et al. 2006). Euro-

pean aspen is one of the most widely distributed trees in

Central Europe (Worrell 1995) and is a typical early colonizer

of poor and disturbed soils.

Few studies have investigated the ECM communities of

heavy metal disturbed sites: Mleczko (2004) investigated my-

corrhizal and saprobic macrofungi of two zinc wastes in

Southern Poland. He described 14 ECM types associated with

Pinus sylvestris and Betula pendula. Staudenrausch et al. (2005)

found 23 ECM types associated with Betula pendula at a ura-

nium mining heap.

ECM fungi are known to affect soil bioavailability and plant

uptake of heavy metals (Jentschke & Godbold 2000). Therefore,

they are predicted to play an important role in phytoremedia-

tion activities involving mycorrhizal hosts (Perotto & Martino

2001), such as, for example, European aspen. This conclusion

was reached based on the large amount of external hyphae

that stabilise soil structure and very effectively exploit soil

nutrients. Additionally, ECM fungi transfer elements very effi-

ciently directly to root cells. Tolerance to high soil metal con-

centrations and good functional compatibility with the plant

used in the phytoremediation activity are prerequisites for

effective ECM fungal symbionts (Khan et al. 2000). ECM fungi

associated with European aspen growing in metal-polluted

soil are expected to fit these characteristics.

In the wider context of a project aiming at testing the influ-

ence of mycorrhizal associations on metal uptake of poplars

suitable for phytoremediation purposes, the ECM fungal com-

munity on roots of European aspen growing on a heavily con-

taminated site in southern Austria was characterised by PCR

and ITS sequence analysis. The data were further used to eval-

uate the impact of soil factors (heavy metal content) on the

formation of the ECM community in organic and mineral

soil layers. The generated data are subsequently compared

with studies on diversity measures of ECM fungal communi-

ties from uncontaminated sites.

Materials and methods

Study site

The study site, which is next to the former lead smelter, is

characterised by a very rough relief formed by prehistoric

rockslide material from the mountain Dobratsch in 1348

(Krainer 1998). This area in Arnoldstein (Austria; N46 330 E13

420) has been affected by lead smelting from the end of the

15th century. In the 1950s smelting activities were extended

to the production of zinc and cadmium. Estimates of annual

emissions from 1989 to 1992, when the smelter was closed,

were 9–13 tonnes lead, 5 tonnes zinc and 10–100 kg cadmium

(Kasperowski 1993). Additionally, different sorts of wastes

such as slag materials were deposited on the site. All of these

activities have resulted in very high soil heavy metal concen-

trations (Kasperowski 1993). Smelting also resulted in severe

sulphur dioxide (SO2) emissions, which left the area mainly

deforested until 1979 when SO2 emissions were cut. From

then on, the barren land was partly re-vegetated by European

aspen (Populus tremula), goat willow (Salix caprea), and grami-

naceous species, such as Brachypodium pinnatum.

Three homogenous stands of aspen (A, B, C,) in the vicinity

of the former lead/zinc smelter were selected as sample plots

(approx. 250 m2 each). Aspen is clonal, and the stands could

well represent one individual with numerous ramets spring-

ing from a single root system. All ramets of aspen on the plots

were very similar in size and height. The estimated age of the

trees was about 20–25 y. In addition to aspen, few individuals

of other ECM species, such as Betula pendula, Salix caprea, and

Corylus avellana, and some saplings of Quercus robur and

Fraxinus excelsior grew on the plots. The soil of the sample plots

is a calcaric Cambisol [pH(H2O) 6.7–7.3] covered by a 5–8 cm

thick mor-like humus layer [pH(H2O) 5.8–6.2].

Soil sampling and physicochemical analysis

Each site was sampled following the same design. Samples

were taken from five randomly distributed spots using a soil

auger with an inner diameter of 7.5 cm. Auger cores were sep-

arated into the organic humus horizon (mor) and mineral soil

(10 cm) and stored in closed plastic bags. On site B a profile

was described and classified following the keys of the world

reference base for soil resources (FAO 1998). A representative

sample was taken from each horizon. Samples were trans-

ported in cooling boxes and stored thereafter in a cooling

chamber at 4 �C.

After homogenisation (mixing and, in the case of the

mineral horizons, sieving to <2 mm) soil subsamples were

air-dried for physicochemical characterisation following

standard procedures. Additionally, humus and mineral soils

were digested in acid mixtures of HClO4/HNO3 and HCl/HNO3,

Populus tremula growing on a heavy metal contaminated site 1071

respectively (Blum et al. 1996). NH4NO3 extracts of mineral

soils were carried out according to DIN 19730 (1995). Analysis

of lead, zinc, and cadmium was performed using inductively

coupled plasma mass spectrometry (ICPMS, Elan 9000 DRCe,

Perkin Elmer, Norwalk, USA).

Sampling

Fungal fruiting bodies were collected in 2004 and 2005. Identi-

fication of these was based on careful macro- and microscop-

ical analysis of morphological characters. Voucher material of

critical species is deposited in the herbarium IB (University of

Innsbruck).

In June 2004 (summer),a totalof15 soil blocks werecollected:

five blocks per plot (A, B, C), each block in close vicinity to one of

five randomly selected aspen trees. Soil blocks were 7.5 cm

square and 15–18 cm deep, including 10 cm deep mineral soil

and varying depths of the organic horizon. In September 2004

(autumn), soil blocks of the same size were taken adjacent to

the summer sampling patches. Each block was divided into

the mineral and the organic soil layer (Baier et al. 2006). These

soil samples were stored in plastic bags at 4 �C for 7 d at the

most until further processing. All roots of a soil sample were

washed out from the soil with tap water over a 2 mm sieve.

Roots of nontarget plants were excluded based on morphologi-

caldifferences. Roots collected in summer from thefive mineral

and organic samples of each plot were pooled, resulting in a to-

tal of three organic and three mineral root samples. Roots from

the autumn sampling were not pooled, resulting in a total of 15

organic and 15 mineral root samples.

For randomisation of selected root tips all roots of a sample

(pooled or not) were evenly distributed in a Petri dish of 14 cm

diam. The dish was subsequently placed on a 1� 1 cm grid

with randomly distributed 1� 1 cm squares marked in red.

All mycorrhizal systems positioned above these red-marked

squares, which represent 6.5 % of the total area of the dish,

were then selected for further analysis. Depending on the

quantity of mycorrhizal root tips in a sample, the procedure

of distributing the roots and selecting those on the red squares

was repeated up to three times to get a minimum number of

root tips (¼ 100 per sample). The number of selected root

tips was recorded for each soil sample. The selected mycorrhi-

zal systems were carefully cleaned under a binocular micro-

scope (SMZ-U, Nikon, Tokyo, Japan) by removing soil and

debris from the roots with tweezers. Individual root tips

were dissected and sorted according to their morphological

characteristics following Agerer (1987–2002): presence and

type of ramification, surface colour, texture, presence of

a mantle, emanating hyphae or rhizomorphs. Only mycorrhi-

zal root tips with the same morphology, from one sample (or

pooled sample) and from the same soil layer were joined,

resulting in a given morphotype sample. Within each mor-

photype sample several individual root tips were prepared

for DNA extraction, while others were subjected to compara-

tive morphological studies. In cases where DNA sequence

analysis gave several results for one presumed morphotype,

the total number of root tips of this morphotype was propor-

tioned according to the number of molecular results. Some

root samples were examined for arbuscular mycorrhizal root

colonisation at �50 magnification following standard clearing

and staining (Gazey et al. 1992). Thus, a total of 12 020 ECM root

tips of Populus tremula were randomly selected and sorted

from 30 soil cores (blocks) during summer and autumn 2004.

Molecular protocols

The rDNA ITS region, situated between the SSU (18S gene) and

the LSU (28S gene) of rRNA genes was amplified using the

primer pair ITS1� ITS4 for fruiting bodies and the primers

ITS1-F� ITS4 for mycorrhizal root tips (White et al. 1990; Gardes

& Bruns 1993). Unamplifiable samples, usually from brown

ECM root tips, were assumed to be dead. Also root tips with

multiple infections were excluded from subsequent analyses.

DNA extraction of fruiting bodies was carried out from

fresh material or dried herbarium specimen following stan-

dard protocols (Zolan & Pukkila 1986).

Of the 12 020 morphotyped ECM root tips, 209 were used for

molecular identification. Root tips of selected morphotypes

not exceeding 5 mm length (Gardes et al. 1991) were stored

in 1.5 ml Eppendorf tubes containing 50 ml CTAB buffer and

either processed immediately or stored at �20 �C. DNA was

extracted from individual root tips following Southworth (2000;

http://www.sou.edu/BIOLOGY/Faculty/Southworth/CTAB.htm).

Usually, only one tip was used for DNA extraction. Several tips

were extracted together only if they were part of one my-

corrhizal system, or if single mycorrhizal tips were very

small (less than 2 mm). The tips were ground in the

1.5 ml Eppendorf tubes containing 50 ml CTAB buffer using

a micropestle. After adding a further 550 ml CTAB buffer

(final concentration per sample: 12.5 mg hexadecyltrime-

thylammoniumbromide, 10 mM Tris–HCl (pH 8), 1.4 M

NaCl, 20 mM EDTA, 0.2 % b-mercaptoethanol) the samples

were incubated at 65 �C for 40–60 min. After centrifugation

for 7 min at 16 000 g the supernatant was precipitated us-

ing an equal volume of chloroform and centrifuged for

15 min at 16 000 g. The upper phase was transferred into

a new tube containing 750 ml cold (�20 �C) 98 % isopropyl

alcohol, which was put in the freezer for precipitation

(30 min to overnight). After centrifugation for 30 min at

16 000 g the pellet was washed with 200 ml cold (�20 �C)

70 % ethanol and centrifuged for 5 min at 7000 g. Superna-

tant was decanted and uncapped tubes invert until dry.

Dried DNA pellets were resuspended in 50 ml TE buffer

and directly used for PCR.

PCR was performed in a volume of 25 ml consisting of 5 ml

DNA and 20 ml PCR mix. The final concentrations were

0.6 mM Tris–HCL, 53.2 mM KCl, 1.5 mM MgCl2, 6.4 mM EDTA,

and 4 % glycerine, as well as 200 mM of each dNTP, 1.25 units

Taq polymerase (peqGOLD, all peqlab, Erlangen) and 0.4 mM

of each primer. PCR reactions were performed using a Techne

Unit Progene thermocycler (Techne, Cambridge) with the

following conditions: an initial step of 5 min at 94 �C was

followed by 40 cycles of denaturation at 94 �C for 1 min,

annealing at 50 �C for 55 s with annealing time increasing 3 s

each cycle, and extension at 72 �C for 45 s. Thermal cycling

was ended by a final extension at 72 �C for 6 min. Purified

PCR products (Amicon� Micron�-PCR Centrifugal filter de-

vices; Millipore, Billerica, MA) were sent to MWG-Biotech

(Ebersberg) for sequence analysis.

1072 D. Krpata et al.

DNA sequence analysis and taxonomic determinationof ECM fungi

After importing the sequences Populus ECM sequences and

closely related sequences obtained from public databases to

Sequencher�, the chromatograms were checked and all se-

quences were automatically aligned. Manual adjustment

was carried out with Se-Al alignment software (Rambaut

1996). Identification of mycorrhizas was based on our in-

house reference database (sequences created from voucher

material), BLAST searches (Altschul et al.1997), comparisons

with the most closely related sequences retrieved from the

public database UNITE (Koljalg et al. 2005), and the sequence

databases at the National Center for Biotechnology Informa-

tion (NCBI: http://www.ncbi.nlm.nih.gov/BLAST/).

ECM root sequences matching sequences from fruiting

bodies were designated by a genus and species epithet fol-

lowed by an IB voucher number. Sequences found on roots

but not as fruiting bodies were named based on data from

BLAST searches, sequence alignments, and advice from taxo-

nomic experts. We used a conservative approach to avoid

overestimation of species richness. Taxon categories were

assigned based on similarities to reference sequences as

follows: ECM fungal isolates were regarded as identified on

species level [1 operational taxonomic unit (OTU)], at se-

quence similarities �97 % with a reference sequence.

Diversity and cluster analyses

The ECM fungal communities associated with the roots were

described by the following five diversity measures: species

richness (S) is the total number of detected species. Simpson’s

Index (1-D) and Fisher’s alpha Index (Alpha Mean) were used

as measures of diversity, and Pielou’s J and the Shannon–

Wiener Index (H) were used as measures of evenness.

Abundance was defined as the total number of ECM root

tips of a species or group, and relative abundance was defined

as the total number of ECM root tips of a species or group

divided by the total number of all ECM root tips of all taxa.

We used relative abundances only and subsequent mentions

of abundance refer to these percentage values.

Frequency was defined as the number of soil cores (blocks)

in which a species or group was detected, and can, therefore,

be regarded as a measure of spatial heterogeneity. For fre-

quency calculations the five soil cores from each of the three

plots were pooled. Organic and mineral layers of pooled sam-

ples were treated separately. Sampling was carried out twice;

therefore, frequency calculations were based on a total of 12

pooled samples.

To estimate the number of undetected species, incidence-

based species richness estimators Jackknife1 (Burnham &

Overton 1979), ICE Mean, and Chao2 (Chao 1987) were calcu-

lated. These indices were performed using the program

EstimateS 8.0 (Colwell 2006).

Detrended correspondence analysis (DCA) was used to un-

ravel trends within the ECM fungal community. We tested

whether data grouped by each of the three categories (plot,

horizon, or season) were statistically different from random

associations. Multi-response permutation procedures (MRPP)

(Sørrenson) were applied to find separation between groups.

Species frequency was used with rare species down-weighted.

Cluster analyses and statistical tests were performed with PC-

ORD version 5.0 (McCune & Mefford 1999; McCune & Grace

2002).

The non-parametric Wilcoxon test for paired samples was

used to test for significant differences in the occurrence of se-

lected fungal taxa in organic and mineral soil layers. Only taxa

identified in five or more of the cores taken in the autumn

sampling were included in this analysis. Mann–Whitney U

tests were performed to test for homogeneity of seasons.

Only taxonomic groups represented by three or more species

were included in this analysis. P< 0.05 was considered as

statistically significant. These statistical tests were performed

using Statistica 6.0 software package (StatSoft Inc, Tulsa,

USA).

Results

Soils

Due to the long history of emissions, very high total heavy

metal concentrations were measured in the mineral soil

(2546–8068 mg kg�1 lead, 1957–3302 mg kg�1 zinc, and 23–

63 mg kg�1 cadmium) as well as in the organic horizon

(20 404–52 220 mg kg�1 lead, 8177–11 239 mg kg�1 zinc and 86–

111 mg kg�1 cadmium) of the three sample plots. Also

NH4NO3-extractable fractions in mineral soils were very high

(152–1335 mg kg�1 lead, 10 686–58 773 mg kg�1 zinc, 369–

2941 mg kg�1 cadmium).

Fungal community on ECM roots

In the summer, 5212 individual mycorrhizal root tips were

randomly selected. The mean number of selected ECM tips

per 100 ml soil amounted to 2529 (S.D.¼ 410; n¼ 3) for mineral

soils, and to 3459 (S.D.¼ 870; n¼ 3) for organic soils (range 542–

1202 root tips/sample; three pooled samples comprising five

cores each, organic and mineral layer separately). In autumn,

a total of 6808 individual mycorrhizal root tips were randomly

selected. The mean number of ECM tips per 100 ml soil

amounted to 1735 (S.D.¼ 318; n¼ 15) for mineral soils, and to

4263 (S.D.¼ 1895; n¼ 15) for organic soils (range 836–1952 root

tips/sample; organic and mineral layer separately).

The examination of Populus tremula root tips showed an

ECM mycorrhization degree of 95 %. Arbuscular mycorrhiza

was not observed. Due to the high abundance of brownish

black root tips a successful and reliable morphotyping was

limited. Sequences were obtained for an average of 53 % of

all processed root tip DNA extracts.

We recovered a total of 54 OTUs of ECM fungi on the roots

of P. tremula (Table 1). Basidiomycetes clearly dominated (43

OTUs) over ascomycetes (11 OTUs). Species richness of five

cores per plot and season (mineral and organic layer sepa-

rately) ranged from 7–15 OTUs in summer (mean 9 OTUs in

the organic horizon and 10 OTUs in the mineral horizon). In

autumn, we found 10–15 OTUs (mean 12 OTUs in the organic

horizon and 12 OTUs in the mineral horizon). The taxonomi-

cal groups with the highest below-ground species richness

Table 1 – Identification and abundance of ectomycorrhizal (ECM) fungal operational taxonomic units (OTUs) colonizingroots of Populus tremula in 2004

OTUs Accession number BLAST matchvoucher ID

Sim. (%) Abundance (%)

Total Organic Mineral

Cenococcum geophilum EF644122 DQ179119 97 17.6 8.4 9.2

Hebeloma mesophaeum* EF644129 IB2005309 100 9.3 8.4 0.9

Tricholoma scalpturatum* EF644164 IB2005700 100 5.2 1.2 4

Tomentella sp. 6* EF644157 IB2005315 100 3.9 3.9 0

Tom. subclavigera EF644163 AY010275 100 3.5 3.5 0

Heb. incarnatulum EF644128 AF430291 99 3.5 3.5 0

Scleroderma sp. 1 EF644144 UDB000044 91 3.3 1 2.3

Tomentella sp. 1 EF644152 UDB000951 93 3 0 3

Scleroderma areolatum EF644143 UDB001212 97 2.9 0 2.9

Cadophora finlandia EF644140 AF486119 98 2.9 2.7 0.2

Tomentella aff. stuposa EF644159 UDB000245 94 2.8 2.1 0.7

Tuber puberulum EF644165 UDB000122 99 2.5 1.6 0.9

Heb. populinum* EF644130 IB2004245 100 2.3 2 0.3

Inocybe sp. 1 EF644135 UDB000637 95 1.9 1.2 0.7

I. pseudoreducta* EF644136 IB2004185 100 1.9 1 0.9

Sebacina incrustans* EF644146 IB2004242 100 1.8 0 1.8

Tomentella punicae EF644151 UDB000959 99 1.7 0.2 1.5

Xerocomus rubellus* EF644171 IB2004272 100 1.7 0.4 1.3

Tomentella sp. 5 EF644156 UDB000250 94 1.7 0 1.7

Pseudotomentella sp. 1 EF644141 AJ889968 91 1.5 0.5 1.1

Cortinarius sp. 1 EF644123 UDB000167 98 1.5 1.5 0

Cortinarius sp. 3 EF644125 UDB000705 95 1.4 0.6 0.8

Unidentified Asco. sp. 1 EF644120 ASU68331 96 1.4 0 1.4

Tomentella sp. 4 EF644155 UDB000267 95 1.3 0 1.3

Inocybe sp. 2 EF644134 UDB000765 96 1.2 0 1.2

Tom. atramentaria* EF644148 IB2004029 100 1.1 0.9 0.3

Tomentella sp. 7 EF644158 AF272941 96 1.1 0.5 0.7

Peziza badia* EF644139 IB2004271 100 1.1 0.9 0.2

Unidentified Asco. sp. 2 EF644127 AY213652 95 1.1 1.1 0

Sebacina sp. 1 EF644145 AF490393 92 1 0.2 0.8

Tom. atramentaria* EF644147 IB2004189 100 1 1 0

Inocybe sp. 4 EF644133 UDB000639 95 1 0 1

Hymenoscyphus ericae EF644131 AJ430151 97 0.9 0.8 0.1

Cortinarius hemitrichus* EU003181 IB2004246 100 0.8 0.8 0

Wilcoxina sp. 1 EF644170 DQ069052 98 0.8 0.5 0.2

Inocybe sp. 3 EF644154 UDB000636 72 0.8 0 0.8

Tom. galzinii EF644150 UDB000264 98 0.7 0.7 0

Tomentella sp. 2 EF644153 UDB000951 93 0.7 0.3 0.4

Laccaria laccata* EU003182 IB2004243 100 0.6 0.6 0

Tuber sp. 2 EF644166 AF106890 95 0.6 0.6 0

Tom. aff. subclavigera EF644162 AF272939 93 0.5 0.5 0

Tuber sp. 1 EF644167 DQ011848 98 0.5 0.1 0.4

Pseudotomentella sp. 2 EF644142 AJ889968 93 0.5 0.5 0

Boletus luridus* EF644121 IB2004270 100 0.5 0 0.5

Cortinarius sp. 2 EF644124 UDB000067 95 0.5 0 0.5

Tom. stuposa* EF644160 IB2005314 100 0.4 0 0.4

Cortinarius sp. 4 EF644126 DQ097870 94 0.4 0.4 0

Tomentella sp. 3 EF644132 UDB000954 92 0.4 0 0.4

Tom. aff. stuposa EF644161 UDB000244 93 0.4 0.4 0

I. flocculosa* EF644137 IB2004200 100 0.3 0 0.3

Leccinum populinum* EF644138 IB2004174 100 0.3 0 0.3

Unidentified Helotiales EF644169 DQ182427 99 0.2 0 0.2

Unidentified ECM EF644168 AB218075 91 0.2 0.2 0

Tom. ferruginea EF644149 UDB000256 100 0.2 0.2 0

Accession number, the best Blast match, voucher ID, similarity (Sim. in percentage) are given. Asterisks indicate 100 % match with sequences

obtained from fruiting bodies. Abundances are given in total and for organic and mineral soil separately. Affinities of OTUs with similarities

<97 % to closely related taxa are shown with ‘aff.’ (¼affinis) preceding the species epithet. Tom. refers to Tomentella, Heb. to Hebeloma, Asco to

ascomycete.

Populus tremula growing on a heavy metal contaminated site 1073

0

0,5

1

1,5

2

2,5

3

3,5

0 10 20 30 40 50 60OTUs

Fig 1 – Logarithmic distribution of 54 OTUs found in total (A)

in 2004, 37 OTUs in summer (>) and 41 OTUs in autumn ( ),

ranked according to the number of root tips of each OTU.

The rank of each OTU from 1 (the most abundant OTU) to n

(the rarest OTU) is plotted on the x-axis. The y-axis repre-

sents the logarithm of the relative abundance of each OTU.

The most dominant OTUs in summer were Cenococcum

geophilum (17.0 %), Tomentella sp. 6 (8.3 %) and Tricholoma

scalpturatum (5.3 %); whereas in autumn C. geophilum

(18.4 %), Hebeloma mesophaeum (15 %) and Tomentella sub-

clavigera (5.6 %) were most abundant. In total C. geophilum

(17.6 %), H. mesophaeum (9.3 %) and Tricholoma scalpturatum

(5.2 %) were the most dominant OTUs.

1074 D. Krpata et al.

were Tomentella (17 OTUs), Inocybe (6 OTUs), Cortinarius

(5 OTUs), Hebeloma, and Tuber (each 3 OTUs).

Unambiguous identification of mycobionts (at least at ge-

nus level) was possible for 51 OTUs. Sequences of 16 mycorrhi-

zas were found to match at 100 % similarity with sequences

obtained from reference material collected in Arnoldstein

(Table 2). ECM root tips of two OTUs were identified as Tomen-

tella subclavigera, and Tomentella ferruginea, respectively, based

on their high (>99 %) ITS sequence similarity with sequences

from public databases. Sequences of 11 further OTUs had an

ITS sequence similarity between 99 and 97 %, and were

regarded as being identified on species level. Twenty OTUs

showed a high similarity (>91 %) with known fungi. These

OTUs were identified on species complex, genus, or lower

level (Table 1). One sequence had a similarity of 72 % with

a locked sequence from UNITE (Ellen Larsson, pers. comm.).

Indentification of four OTUs to species complex level was

not possible due to lack of reference data, but none of them

were dominant on root tips.

The morphotypes of Cenococcum geophilum were initially

separated into different groups to prevent it being mistaken

for other black, shining ECM with emanating hyphae e.g. Phia-

locephala fortinii. Eleven sequences for these groups indicated

the presence of two OTU subtypes of C. geophilum (similarity

97 %) which did not correspond to the morphological groups.

C. geophilum was consequently identified by morphotyping,

these two subtypes were not further discriminated.

This study revealed a few abundant and a large number of

rare ECM fungal species on Populus tremula roots (Table 1). Spe-

cies accumulation curves show a characteristic exponential

rate of increase of OTUs (Fig 1) similar to other ECM ecosys-

tems. Twenty-one representative species of three genera

formed more than 50 % of all mycorrhizal root tips (C. geophi-

lum 17 %, Tomentella 24 %, and Hebeloma 15 %). As typical for

environmental data, the distribution of fungal taxa was un-

even (Fig 3). Results of five cores of each of three plots were

pooled in summer, as well as in autumn, mineral and organic

Table 2 – Fungal fruit body collections used in this studyfor identification mycobionts (100 % match withectomycorrhizal root tips of Populus tremula) withcollection number and GenBank accession numbers

Voucher name IB number Accession number

Boletus luridus IB2004270 EF644104

Cortinarius hemitrichus IB2004246 EF644105

Hebeloma mesophaeum IB2005309 EF644106

Heb. populinum IB2004245 EF644107

Inocybe flocculosa IB2004200 EF644108

I. pseudoreducta IB2004185 EF644109

Laccaria laccata IB2004243 EF644110

Leccinum populinum IB2004174 EF644111

Peziza badia IB2004271 EF644112

Sebacina incrustans IB2004242 EF644113

Tomentella atramentaria IB2004029 EF644114

Tom. atramentaria IB2004189 EF644115

Tomentella sp. 6 IB2005315 EF644116

Tom. stuposa IB2005314 EF644117

Tricholoma scalpturatum IB2005700 EF644118

Xerocomus rubellus IB2004272 EF644119

layers were treated separately, resulting in a total of 12 sam-

ples. The most widely distributed taxa were C. geophilum,

found ten times, and Hebeloma mesophaeum, Pseudotomentella

sp. 1, and Tricholoma scalpturatum which were found seven

times each. Xerocomus rubellus occurred six times. Cadophora

finlandia, Inocybe sp. 1, and Inocybe pseudoreducta were found

four times. Eight OTUs were triplicates (occurring three times),

and 21 OTUs were duplicates (occurred two times). Seventeen

OTUs occurred once.

Commonly used diversity indices Shannon–Wiener diver-

sity index (H)¼ 2.00, evenness (J)¼ 0.84, Simpson’s diversity

index (1–D)¼ 0.19, and Fisher’s alpha diversity index¼ 8.06

suggest that overall diversity of the underground ECM com-

munity (organic and mineral horizons separately from sum-

mer and autumn) was relatively high.

Species richness calculations based on all samples (five

cores pooled of each plot, both seasons, organic and mineral

layer separately) estimated a total richness of 70 OTUs (Jack-

knife1), and ICE mean estimated total richness to 71 OTUs

(Fig 2). Considering the samples from autumn only (cores

not pooled, organic and mineral layer separately) Jackknife1

amounted to 62 OTUs and ICE Mean estimated richness to

74 OTUs. Based on these estimates our sampling detected

a minimum of 73 % of the estimated richness.

Rank–abundance curve (Table 1) shows that C. geophilum

was the most abundant and common species, occupying

17.6 % of all root tips, followed by Heb. mesophaeum colonizing

9.3 % of all ECM root tips, and by Tricholoma scalpturatum

0

10

20

30

40

50

60

70

80

0 1000 2000 3000 4000 5000 6000 7000Root tips

Fig 2 – Species-Area-Curves for 41 OTUs observed in au-

tumn (dashed black line) and for 54 OTUs observed in total

(dashed grey line). First-order Jacknife richness estimator

amounted to 62 OTUs in autumn (grey line) and to 70 OTUs

in total (black line).

Populus tremula growing on a heavy metal contaminated site 1075

(5.2 %). Most of the ECM species (40 OTUs), such as Laccaria lac-

cata and I. flocculosa had low abundances (<2 %). These 40 ECM

mycobionts contributed together to 37.2 % of all ECM root tips.

However, several of these low-abundance ECM fungi were

evenly distributed: Pseudotomentella sp. 1 (abundance: 1.5 %)

was found in 58 % of all samples, and X. rubellus (abundance:

1.7 %) occurred in 50 % of all samples (Table 1, Fig 3). Rank–

abundance plots (Fig 1) compare total 2004 data with data

from summer and autumn 2004. In summer C. geophilum

was the most abundant and common species, found on

17.0 % the root tips, followed by Tomentella sp. 6 (8.3 %) and

Tricholoma scalpturatum (5.3 %); in autumn C. geophilum

(18.4 %), Heb. mesophaeum (15 %) and Tomentella subclavigera

(5.6 %) were most abundant. In total, C. geophilum (17.6 %),

Cenococcum

geophilum

Hebelo

ma m

esophaeum

Pseudotom

entella sp

. 1

Tric

holo

ma scalp

turatum

Xerocom

us rubellus

Cadophora fin

landia

Inocybe sp

. 1Inocybe pseudoreducta

8 O

TUs

21 O

TUs

17 O

TUs

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10Frequency

OT

Us

Fig 3 – Frequency of Populus mycobionts: Number of OTUs

detected as ‘‘uniques’’ (occurring once) duplicates (detected

twice), triplicates, quadruplicate and more than four times

(shown as numbers). In total 54 OTUs were found (n [ 12

pooled samples: three plots, two seasons, mineral and or-

ganic soil layers separately. A pooled sample consists of five

pooled soil cores from one soil layer of each of three plots.)

Heb. mesophaeum (9.3 %), Tricholoma scalpturatum (5.2 %)

were the three dominating species.

Spatiotemporal patterns in the ECM community

Multi-Response Permutation Procedures (MRPP) showed no

significant differences between individual plots, seasons,

and horizons (P> 0.05). However, when MRPP was constrained

by horizons (Euclidean P¼ 0.098, Sørrenson P¼ 0.119), Euclid-

ean distance measure indicates a weak effect of horizons.

Similarly, detrended correspondence analysis (DCA) gave no

good separation for plots, seasons, and horizons. When con-

sidering single OTUs non-parametric Wilcoxon tests for

paired samples showed Tricholoma scalpturatum to be signifi-

cantly more common in mineral soil (P¼ 0.043) and Cadophora

finlandia to be significantly more common in organic layers

(P¼ 0.043). No trend was observed for the other two tested

species (Cenococcum geophilum, Hebeloma mesophaeum). Com-

paring the occurrence of taxa between seasons Mann–Whitney

U tests indicated a tendency for Hebeloma spp. to be more

frequent in autumn. No trend was observed for the other three

taxonomic groups examined (Cortinarius spp., Inocybe spp.,

Tomentella spp.).

Discussion

Our study is among the first to extensively describe the

below-ground ECM fungal community of a heavily metal

contaminated site. Other broadleaf tree studies were mainly

conducted on sites not affected by anthropogenic pollution.

Moreover, this study is among the first dealing with fungal

communities of European aspen.

ECM fungi protect themselves and their hosts from heavy

metal pollution by, for example, binding them into cell-wall

components or by storing high amounts of heavy metals in their

cytosol (Bellion et al. 2006). Heavy metals often co-precipitate

with DNA and interfere in all the subsequent molecular analy-

ses (Desai & Madamwar 2006; Fortin et al. 2004; Hinoue et al.

2004; Wilson 1997). Therefore, heavy metals could also have

affected the comparatively low PCR success rate (53 %) of our

Populus root tip DNA extracts.

Recently, the effect of heavy metal-contaminated soils on

the performance of P. tremula was demonstrated by Hermle

et al. (2006) (e.g. reduction of biomass, decrease of photosynthe-

sis and transpiration). However, a potential influence of the

ECM community associated with these poplars was not

included in this study. ECM formation usually decreases, and

alterations in species composition occurs as a result of

increasing concentrations of, for example, soil-applied lead

(Chappelka et al. 1991). However, our monitoring showed ecto-

mycorrhiza to be the major mycorrhizal type of P. tremula, as

arbuscular mycorrhiza was not observed and non-mycorrhized

root tips were very rare. Ectomycorrhiza symbiosis leads to an

increase of potential nutrient-absorbing surface and thus also

to an increased potential to interchange water and also pollut-

ants (Rousseau et al. 1994).

The overall picture of the fungal community detected on

this heavy metal contaminated site is quite surprising: We

expected to find few, highly dominating fungal taxa that

1076 D. Krpata et al.

were well adapted to this stressful environment. This initial

hypothesis was also supported by other studies of ECM com-

munities of zinc and uranium polluted soils: one morphotype

(an unidentified Pinirhiza arenosa) formed 70 % of all analysed

mycorrhizas followed by less abundant Tricholoma scalpturatum,

Hebeloma mesophaeum, and members of Thelephoraceae on roots

of Pinus sylvestris and Betula pendula growing in a zinc waste

(Mleczko 2004); three mycobiont species (Phialophora sp., Lactar-

ius decipiens, and Amanita muscaria) dominated the ECM commu-

nity of B. pendula growing at a uranium mining heap

(Staudenrausch et al. 2005). The high diversity and evenness of

the detected fungal communities in our study is contrary to

these findings. Our initial hypothesis of a low ECM diversity

could not be corroborated as 54 fungal taxa were detected as

mycobionts in this heavily contaminated site. A comparison

with other data indicates that diversity values of our contami-

nated Populus sites (Shannon-Wiener index H¼ 2; Simpson’s

diversity index 1-D¼ 0.189) lay within the range observed in

other studies on broadleaved trees, although data of similar

studies are not directly comparable due to different definitions

of OTUs: Smith et al. (2007) reported H¼ 1.077 on oak, DeBellis

et al. (2006) reported H¼ 3 on aspen, and H¼ 4.2 on birch.

Richard et al. (2005) found 1-D¼ 0.244 on saplings of Quercus,

while Smith et al. (2007) reported 0.525 on mature oaks. Also

studies of conifer-dominated forests reported similar values

[Ishida et al. (2007) reported H¼ 2.7–3.5 in conifer–broadleaf

mixed forests; Fransson et al. (2000) reported H¼ 1.96–2.44 in

a Norway spruce forest; Cline et al. (2005) reported 1.19 on

mature Douglas fir].

Basidiomycetes (43 OTUs forming 71 % of ECM root tips)

were the most numerous fungi associated with roots of

P. tremula. The species composition of our P. tremula stand

was comparable with those of mature forests (e.g. Horton

et al. 1999; Stendell et al. 1999; Jakucs 2002). However, many

of the taxa detected in Arnoldstein were typically observed

in early successional or disturbed habitats (Cenococcum, Hebe-

loma, Inocybe, Scleroderma, Tomentella, Tuber, Wilcoxina) (Nara

et al. 2003; Rudawska et al. 2006; Muhlmann et al. 2008) reflect-

ing the short and disturbed history of our Populus sites.

We detected a few species characteristic for aspen stands

(e.g. Hebeloma populinum, Leccinum populinum). As is typical

for communities of any taxonomic group (Putman 1994;

Horton & Bruns 2001), a few abundant and a large number of

rare ECM fungal species were found on P. tremula roots.

Dahlberg et al. (2001) compiled a review of 49 ECM fungal com-

munity studies of coniferous and deciduous forests. They

found Cenococcum geophilum, corticoid basidiomycetes and

Thelephoraceae to be the three most abundant or frequent

ECM fungal taxa, as recorded from mycorrhizas. It is now

widely known that species belonging to the Thelephoraceae

are among the most frequent and abundant ECM species in

Europe and Northern America (Horton & Bruns, 2001; Koljalg

et al. 2000), they are generalists and important mycorrhizal

partners of many deciduous trees and conifers. This is in

accordance with our study and a study on ECM of transgenic

aspen (Kaldorf et al. 2004). Conversely, the results obtained

in the latter study differ considerably regarding species com-

position and diversity. Different environmental conditions,

as well as different plant ages, may cause these discrepancies;

the estimated age of the naturally grown aspen trees in

Arnoldstein was about 20–25 y. In contrast, the aspen clones

investigated by Kaldorf et al. (2004) were planted in an exper-

imental field and did not exceed the age of six years. There-

fore, typical early-stage ECM fungi, such as Laccaria laccata,

were among the dominating mycobionts of young transgenic

plants, whereas this fungus was found with low abundance

(0.6 %) in our study.

Thirty-three fungal ECM taxa, which do not form conspic-

uous fruiting bodies (mainly Tomentella spp.), were detected on

the roots of P. tremula. These species colonized 59 % of all root

tips. Tomentella spp. form thin, resupinate fruiting bodies on

bare ground or on wooden substrates and are easily over-

looked. Thus, only sequences from fruiting bodies of four

Tomentella species (out of 17 on the root tips) were found to

match at 100 % similarity with sequences obtained from root

tips.

Cenococcum geophilum does not form fruit bodies but sclero-

tia of C. geophilum were found in the soil. However, it was not

possible to obtain cultures from C. geophilum. This anamorphic

ascomycete with worldwide distribution appeared to be the

best adapted mycobiont to the conditions in our plots, forming

17.6 % of all P. tremula mycorrhizal root tips. This agrees with

data from experiments with soil-applied lead, which was

found to lead to an increase in occurrence of C. geophilum

ECM on Pinus taeda up to 300 % compared with uncontami-

nated control soil (Chappelka et al. 1991). Similarly, Cripps

(2003) reported a prolific occurrence of C. geophilum on aspen

in areas of high pollution.

Obviously, all fungal lineages of this specific site are toler-

ant to the high heavy metal concentrations of the environ-

ment. The high degree of mycorrhization suggests that ECM

fungi play an important role in heavy metal uptake or avoid-

ance of the plants. Fungal immobilisation of heavy metals

could be another potential mechanism by which mycorrhizal

fungi alleviate metal toxicity to their hosts (Joner & Leyval

1997). However, interactions are very complex as they vary

in a fungal species- and metal-specific manner: e.g. cadmium

was almost exclusively localized in fungal cell walls of the

Hartig net, whereas zinc was accumulated in both cell walls

and the fungal cytoplasm (Frey et al. 2000); suilloid mycorrhi-

zas significantly enriched lead and zinc in their mantle,

although no such biofiltering effect was observed for

mycorrhizas formed by other taxa (Turnau et al. 2002). During

this study, cultures of several fungal species found in

Arnoldstein were obtained from fruit bodies and from

mycorrhizal root tips. Further studies on the heavy metal

concentrations of abundant mycobionts and of synthetic my-

corrhizae are planned to study a potential selective enrich-

ment of heavy metals at high concentrations within

mycorrhizal fungal structures (ECM mantle and Hartig net

hyphae).

Sequences of 16 fungal species detected as mycorrhizal

partners were found to match perfectly (>99 %) with se-

quences obtained from fruit bodies collected at the site. Thirty

percent of the ECM species found below-ground were also

found as fruiting bodies. This is higher than data reported in

other studies (e.g. Jonsson et al. 1999: 17 % in Scotch pine

stands; Peter et al. 2001: 22 % in a Norway spruce forest; Ishida

et al. 2007: 13 % in mixed conifer–broadleaf forests; Smith et al.

2007: 20 % when considering only epigeous taxa), but lower

Populus tremula growing on a heavy metal contaminated site 1077

than the 45 % detected in an extensively (8 years) sampled

xeric Quercus forest, where sampling included also hypogeous

taxa (Smith et al. 2007). Pritsch et al. (1997) observed fruiting

bodies from eight species out of a total of 16 ECM species asso-

ciated with black alder (Alnus glutinosa). In their study epige-

ous fruiting bodies were collected monthly over one growing

season. Similarly, Cripps (2004) found half of the mycorrhizal

fungi associated with P. tremuloides as fruiting bodies above

ground. The varying correspondence between detected fungal

fruiting bodies and ECM taxa often depends on the sampling

effort and the available taxonomical expertise.

The number of mycorrhizal Populus root tips per 100 ml soil

(1735 to 4263) was similar to or slightly higher than the num-

ber of Picea abies root tips per 100 ml soil (1117 to 2370)

reported from an Austrian site (Marth 2007).

Our results revealed trends of some species of the ECM

community to preferentially occur in different soil layers

(Genney et al. 2006), e.g. Tricholoma scalpturatum in the mineral,

and Cadophora finlandia in the organic horizon. This is in agree-

ment with Tedersoo et al. (2003), who studied a mixed, mostly

deciduous forest and found Cadophora finlandia mostly in the

A-horizon. In contrast, studies of boreal forests (Rosling et al.

2003) showed C. finlandia to preferentially colonise roots in

the mineral soil. Interestingly, in our study no preferences

for a certain soil layer could be observed of C. geophilum.

This contrasts with the studies of Goodman & Trofymow

(1998) and Fransson et al. (2000) who found that C. geophilum

was more common in the organic layer than in the mineral

soil. We speculate that the even distribution of C. geophilum

could be related to the high heavy metal concentrations which

generally lead to a considerable increase of C. geophilum abun-

dances (Chappelka et al. 1991).

Acknowledgements

We sincerely thank Friederike Gobl and Regina Kuhnert-

Finkernagel for assistance and helpful comments. This study

is part of the FWF Project P170120-B08 ‘Mycorrhizal associa-

tions of zinc/cadmium-accumulating poplars: perspectives

for phytoremediation?’. And last but not least the authors

wish to thank the two anonymous reviewers for their useful

comments.

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