Fungal community structure under goat willows (Salixcaprea L.) growing at metal polluted site: the potentialof screening in a model phytostabilisation study

Marjana Regvar • Matevz Likar •Andrej Piltaver . Nives Kugonic - Jane E. Smith

Abstract Goat willow (Salix caprea L.) was selectedin a previous vegetation screening study as a potentialcandidate for the later-stage phytostabilisation effortsat a heavily metal polluted site in Slovenia. The aimsof this study were to identify the fungi colonisingroots of S. caprea along the gradient of vegetationsuccession and to estimate their colonisation levels inrelation to metal pollution in order to reveal itsmycorrhizal status at the site. Additionally the metalaccumulation capacity of S. caprea and photosynthet-

ic pigments were analysed as indications of its fitnessat four differentially polluted plots. Despite highconcentrations of leaf accumulated Cd, no significantdifferences in photosynthetic pigment concentrationswere observed. The roots were colonised by arbus-cular mycorrhizal (AM) fungi, ectomycorrhizal (EM)fungi, and dark septate endophytes (DSE), with EMas the dorninant type on all the plots. Molecularcharacterisation showed poor correlation of the rootEM community with the above-ground sporocarpdiversity. Members of Sordariaceae were the mostfrequent colonisers with an average colonisation of21 % of all root tips, followed by Thelephoraceae with10%. DSE colonisation increased with increasing Pbconcentrations and decreasing organic matter (OM).

Keywords Fungal endophytes . Heavy metalpollution ITS rRNA . Mycorrhiza PhotosynthesisSalix

Introduction

Representatives of Salix (willow) species are wellknown pioneer plants with high tolerance to excessheavy metal concentrations (van der Heijden 2001;Punshon and Dickinson 1997; Unterbrunner et al.2007; Regvar et al. 2006). Once established, they helpstabilize the soil surface, reduce soil wind and watererosion, while the demand of their perennial root

1997; Leyval et al. 1997; Markkola et al. 2002).Because of the selection pressure of metal toxicityEM genotypes develop on metal enriched soils(Hartley et al. 1997; Leyval et al. 1997; Markkolaet al. 2002; Colpaert et al. 2004; Adriaensen et al.2005) with the ability to alleviate the effects ofheavy metal toxicity for themselves and their hosttrees (Adriaensen et al. 2005, 2006; Krznaric et al.2009), by providing a more balanced access tomineral nutrition (Marschner and Dell 1994). Fur-thermore, literature data (Kayama et al. 2006)suggest the adaptation to heavy metal enriched soilsmay depend upon the interaction of specialisedgenotypes of both host trees and EM fungi.

Willows are also known to be colonised by darkseptate endophytes (DSE) (Kovacs and Szigetvari2002; Trowbridge and Jumpponen 2004) that areubiquitous root-associated and possibly symbioticfungi (Jumpponen 2001). Their prevalence in extremehabitats and their alleged role in protecting plantsunder abiotic stress (Haselwandter and Read 1982;Mullen et al. 1998; Ruotsalainen 2003) make thestudies on DSE even more compelling.

The objectives of the present study were (1) toevaluate metal accumulation capacity and photosyn-thetic pigments, as indicators of stress, in Salix caprea(goat willow) from the plots with differing metallevels, in order to confirm its physiological suitabilityfor phytostabilisation, (2) to identify fungal symbiontscolonising S. caprea along the gradient of vegetationsuccession, and (3) to establish the extent of coloni-sation with different groups of fungal symbionts inrelation to metal levels, as data from such screeningstudies are an important step in identification ofpotential fungal symbionts for their application inphytoremediation efforts, For this, S. caprea shrubs atfour plots with differing soil metal levels weredeliberately selected at the increasing distances fromthe lead smelter as the main pollution source (aerialpollution) and examined for metal accumulationcapacity and colonisation with various fungal partners.

Materials and methods

Site description

The study site is located in Zerjav (northern Slovenia;46°8' N, 14°51' E), between 500 m and 700 m above

system for water decreases the risk of contaminantleaching (Sander and Ericsson 1998). Willows havebeen successfully used for the re-vegetation of heavilycontaminated land (Dickinson 2000) and proved to bea useful part of a decontamination scheme of soilswith increased Cd and Zn concentrations (Hammer etal. 2003; Unterbrunner et al. 2007). As such, willowsare considered to be significant for future reforesta-tion in phytoremediation efforts.

Toxicity of excess metals (Cd, Pb, Ni, Cu) affectsplant metabolism, including synthesis of photosyn-thetic pigments due to metal binding to proteinsulphydryl groups (Van Assche and Clijsters 1990),or by direct destruction of photosynthetic pigmentsthrough generation of highly active oxygen radicals(pinto 2003). Resulting changes of photosyntheticproperties can lead to severe physiological stress andchanges in carbon allocation, with possible impact onbelow-ground fungal communities and their associa-tions with plant roots (Smith and Read 2008).

Several studies have focused on benefits of mycor-rhizal symbioses for the plant hosts growing on heavymetal enriched sites (Leyval et a1. 1997; Markkola etal. 2002; Colpaert et a1. 2004; Adriaensen et al. 2005),with the aim of possible biotechnological applicationssuch as phytoremediation (Krupa and Kozdroj 2007).Willows are known to form mycorrhizal associationswith both ectomycorrhizal (EM), and arbuscularmycorrhizal (AM) fungi (Kovacs and Szigetvari2002; Trowbridge and Jumpponen 2004; Cazares etal. 2005, Obase et al. 2007). Hosting differentmycorrhizal types might be of functional importancefor plant nutrition (Read and Perez-Moreno 2003)and has been shown to contribute to metal toleranceof host plants (Hartley et a1. 1997; Vogel-Mikus eta1. 2006), as fungi can reduce the metal uptake bythe plant by sequestration, extracellular precipitationand biosorption to the cell walls (Gadd 1993; Bellionet a1. 2006).

In addition, several studies have focused onbenefits of mycorrhizal symbioses for the plant hostsgrowing on heavy metal emiched sites (Leyval et al.1997; Markkola et al. 2002; Colpaert et a1. 2004;Adriaensen et al. 2005), with the aim of possiblebiotechnological applications such as phytoremedia-tion (Krupa and Kozdroj 2007). Extremely pollutedsites are reported to have lower rates of mycorrhizalcolonisation, fewer fungal propagules and lowerfungal species diversity (Gadd 1993; Hartley et al.

reference material (NIST SRM 2711, Montana Soil)was used for analytical quality assurance. Soilorganic matter content was determined by theWalkey-Black method of wet combustion (Nelsonand Sommers 1982). Soil pH was measured in a soil:KCI OM) (1 :5) suspension.

Root fungal colonisation

Root samples were collected under each plant (5 ploCI)

app. 20-30 em from the stem ofthe shrub by followingthe root from the base of the stem to the root withunbranching last root branch, to assure their prove-nance. Approximately 109 of terminal roots werecollected at the depth 5-20 em below the soil surface.

Root samples of S. cap rea were cleaned of soildebris and examined with stereomicroscopy (Leica,MZ8, Wetzlar, Germany). Ectomycorrhizal root tipswere assigned to individual morphotypes (A-G),distinguished by differences in colour, size, and thetype of ramification (Agerer 1996-2006). The colo-nisation levels were estimated on a 6 x 6-cm grid (36squares planC], five plants ploCI

) and expressed foreach individual EM morphotype as a percentage of allexamined root tips.

For the assessment of AM and DSE colonisationthe roots were cleared by autoclaving in 10% KOHfor 15 min (0.1 MPa, 121°C) and stained in 0.05%trypan blue for 40 min (Phillips and Hayman 1970).The frequencies of DSE and AM colonisation (F%)were estimated on the same l-cm fragments (20fragments plant-1, five plants plot-1) (Zeiss, KF2,Oberkochen, Germany) according to Trouvelot et al.(1986). Non-septate hyphae with vesicles and onlyoccasional arbuscules were counted as AM, whereas

sea level, close to a lead smelter facility. A profounddisturbance with severe erosion on the most heavilyaffected sites is the result of lead mining activitiesinitiated in the 15th cent. Four plots (P1-P4) of ca.300 m2 (Table 1) were deliberately selected along thegradient of vegetation succession (Regvar et al.2006). Earlier stages of the succession are dominatedby herbaceous perennials: Minuartia gerardii (Willd.)Hayek, Sesleria caerulea (L.) Ard., Calamagrostisepigeios (L.) Roth, Calamagrostis varia (Schrad.),Thlaspi praecox Wulfen and Thymus serpyllum agg.In the later succession stages broadleaf species suchas Salix spp., Acer spp. and Betula pendula Roth. arefound more abundantly.

The underlying bedrock on the plots is composedmainly of Triassic limestone and metalliferous dolo-mite (calamine). The soils are stony of a rendzina type,polluted with lead (Pb), cadmium (Cd) and zinc (Zn).

Soil analyses

Soil and plant material for the analyses were collectedin spring and fall 2000. Soil samples were collectedaccording to a standard procedure (DIN ISO 10381-1)at the section of a concentric circle around the node ofcoordinates, on six points. The samples were homog-enized, dried at 36°C, ground in a ceramic mill, andpassed through a 2-mm plastic sieve. Samples wereground further prior to metal analyses in an agatemill (10 min) and passed through a l50-um sieve.Micro-wave assisted wet digestion of the sampleswas performed in aqua regia. After filtration, thecontents of Cd and Pb were determined usingsimultaneous multi-element atomic absorption spec-trometry (perkin-Elmer SIMAA 6000). A standard

nucleotide, 500 nM concentrations of each primer,0.75 U of Taq polymerase (Promega, Madison, WI,USA), and 12.5 ul of 100-fold diluted template.

ITS-RFLP patterns were produced using restrictionenzymes HinfI and MboI (New England BioLabs,Beverly, MA, USA). If two root tips of the samemorphotype yielded two different RFLP patterns,additional root tips (up to eight) were analysed inorder to achieve reliable estimates of RFLP patterndiversity. After the analysis, colonisation levels wereassigned to every RFLP pattern. Because the majorityof fungal species from roots remained unknown, theITS region was sequenced for phylogenetic analysis.

Sequencing and sequence analysis

Prior to sequencing, the amplicons were cleaned andligated into pGEMT-Easy vector (Promega, Madison,WI, USA). The recombinant vector was used fortransforming cells of E. coli JMl09. The trans-formants were plated on LB agar plates containing50 ug/ml of ampicilin and X-Gal/IPTG. Screening forrecombinant cells was carried out with blue/whiteselection. Three clones of each amplicon were usedfor sequencing. Prior to cycle-sequencing reactionswith T7/SP6 primers, the presence of inserts in thevectors was confirmed with colony PCR using T7 andSP6 primers. Sequences are available at the GenBank(accession numbers AY916066-AY916071 andAY916073-AY916077). The sequences were sub-jected to GenBank searches, using the default optionof gapped-BLAST (Altschul et al. 1997) and alignedwith the closest matches and additional representa-tives of the groups from the GenBank. Neighbor-joining analysis was performed using MEGA4(Kumar et al. 2008). The robustness of the internalbranches was assayed by bootstrap analysis (1,000runs). After the analysis, colonisation levels wereassigned to the corresponding fungal sequences.

Statistical analysis

Data were analysed with Statistica® (Statsoft, Inc.).One-way ANOVA with Duncan's post hoc test wasused for evaluation of differences in measured soiland plant parameters as well as in colonisation levelsof AM, DSE and EM among different plots.

Translocation factors (TF=Cshoot/Croot) were calcu-lated to quantify root to shoot translocation and

inter/intracellular melanised hyphae with microscler-otia were recorded as DSE.

Plant analyses

For determination of plant accumulated metals andphotosynthetic pigments, fully developed leaves offive plants per each plot were collected randomly(five samples plane) around the whole tree canopy).Subsamples were taken from each sample for both,photosynthetic pigments and element analysis. Deter-mination of metals accumulated in S. caprea rootswas done on the root samples used for estimation ofEM colonisation.

For metal analysis, plant samples were dissolved inconcentrated HNO3 in a microwave oven (CEM MSP1000, Matthews, NC, USA), filtrated, and determinedaccording to the same protocol as soil samples. Fordetermination of photosynthetic pigments, leaveswere immediately frozen in liquid nitrogen andextracted in 100% cold (4°C) acetone according tothe method of Lichtenthaler (1987).

PCR amplification and RFLP genotyping

Sporocarps were collected on all plots for identificationand DNA analysis. The sporocarps were freeze-dried(Christ, Alpha 2-4, Osterode am Harz, Germany) anddeep-frozen (-70°C). A small piece of sporocarp tissueat the junction of the stem (stipe) and cap (pileus) wasused for DNA extraction.

Two root tips of each EM morphotype detected ona root system of each individual plant (102 samples intotal) and two samples from each species found assporocarps were selected for DNA extraction. TheITS region of the nrRNA gene was amplified (with70% success rate) for molecular characterisation usingrestriction fragment length polymorphism (RFLP) andsequencing. DNA extraction and RFLP protocolsfollowed those of Gardes and Bruns (1993).

PCR conditions for ITS1F-ITS4 (White et al. 1990;Gardes and Bruns 1993) primer pairs were 1 min at94°C, followed by 35 cycles of 35 s denaturation at94°C, followed by 53 s annealing at 55°C, and 30 s ofelongation at 72°C. Time of elongation step wasincreased for 5 s per cycle. A final extension wasperformed at 72oC for 10 min. The PCR reactionmixture (25 ul)contained: 2.5 ul 10x PCR buffer,2.5 mM MgC12, 200 uM concentrations of each

individual plots was 6.8±0.2, while organic matterincreased with closing of vegetation cover from 3.2%at P1 to 25.2% at P4.

The highest concentrations of plant accumulatedmetals were measured at P2 (Table 2), with maximalconcentrations of up to 101 and 147 mg kg-1 of Cdand 695 and 1,460 mg kg-1 of Pb, in the leaves androots respectively. A relatively large portion (42-61%) of Pb and even larger portion (63-83%) of Cdwas accumulated in the shoots. Photosynthetic pig-ments showed no statistically significant differencesamong the plots (Table 2).

Root fungal colonisation of Salix caprea

Microscopic examination of stained root segments ofSalix caprea revealed presence of hyphae and distinctmicrosclerotia of dark septate endophytes (DSE), aswell as vesicles and non-septate hyphae of AM fungi.AM colonisation levels (F%) ranged from 0 on P1 to3.0%± l.6% on P2 representing an average of 1.0%±0.4% within the total colonisation level (Table 2). Thehighest level of DSE colonisation was observed on P1(20.4%±7.0%) whereas on plots P2-4 it did not varysignificantly with less than 5% of all root segmentscolonised. EM colonisation levels with 44.5%± 1.8%of colonised root tips on average represented the

bioaccumulation factors (BAF=Cshoot/Csoil) were cal-culated to quantify the accumulation of the individualmetal relatively to the soil concentrations.

We used Mantel test and ordination to evaluate therelationships among colonisation levels, RFLP patternand sporocarp data with plant EM community compo-sition and available environmental factors (Cd, Pb,organic matter, pH). Mantel test was performed usingVegan v1.15library inR v2.7.2 andBray-Curtis distancematrices. Tests were run with 10,000 permutations,

To evaluate colonisation levels and RFLP data sets,we used an indirect gradient analysis. Community datawere first ordinated using nonmetric multidimensionalscaling (MDS) (Kruskal I964a, b) in R v2.7.2(metaMDS, 20 iterations) and then the relationship ofdata with environmental gradients was estimated usingevfit function of Vegan library (1,000 permutations).

Results

Soil properties, metal accumulation and physiologicalproperties of S. caprea

Higest soil Cd concentrations were found on P2,whereas Pb was most enriched on P 1 and decreasingtoward P4 (Table 1). Average pH of the soil at

sequence restriction fragments of sporocarps and EMroot tips resulted in two matches. Morphotype Bshowed similarity with the RFLP pattern of Hebelomamesophaeum (Tricholomataceae), while one of theRFLP pattems of morphotype D was similar to thatof Cortinarius decipiens (Cortinariaceae).

All fungal RFLP pattems exceeding total coloni-sation of roots above 1.5%, as well as the representa-tives of EM morphotypes G and C (less than 1.5% ofall root tips) were sequenced. Sequencing yielded 11fungal sequences (Table 4). Assuming that they sharethe same trophic strategy as their closest GenBankrelatives, two of the ascomycete sequences withsimilarities to Leotiomycetes could be plant endo-phytes, whereas the other 9 sequences most likelyrepresent EM fungi. The three most abundant fungalspecies (Table 4, AY916066-AY916068) colonizingS. caprea belonged to the ascomycete family Sordar-iaceae (Fig. la) and grouped within morphotype E.Other ascomycete taxa were represented by sequencesfrom Sarcosomataceae (morphotype F) and ascomy-ceteous endophytes from Leotiomycetes (morpho-types A and G). Basidiomycete sequences belongedto Thelephoraceae (morphotype A and D) andCortinariaceae (morphotype D) (Table 4, Fig. 1b).Unfortunately, no sequences from the morphotypes Band C were obtained.

Ascomycetes dominated the EM fungal communi-ty at the most degraded and exposed plots respective-ly (P1 and P3; Fig. 2a), while basidiomycetes formedthe bulk of EM fungal species at the least pollutedplot (P4; Fig. 2a). In general Sordariaceae colonisedon average 40% of EM root tips (21% of all root tips),followed by Thelephoraceae with 22% (10%), Sarco-somataceae 5% (2.5%), genus Hebeloma/Tricholoma-taceae 4% (2%), and Cortinariaceae 4% (2%).

highest portion of colonisation among the three fungaltypes.

Sporocarp EM community

Sporocarps of seven mycorrhizal species belonging tofive different genera were recorded over a 2-yearperiod (Table 3). Hebeloma species, e.g. H. meso-phaeum (Pers) Quelet and H. collariatum Bruchetwere the most abundant species, producing 113 (41%of the total) and 80 (29% of the total) sporocarps,respectively. Also, H. mesophaeum was the onlyspecies producing sporocarps on all four plots.Inocybe was the second most abundant sporocarp-producing genus (26% of the total), although itsrepresentatives were only found on P3. Otherrecorded species were: Lactarius subdulcis (pers. exFr.) S.F. Gray, Suillus bovinus (Fr.) O. Kuntze,Cortinarius decipiens (Pers.: Fr.) Zaw that formed5% of total sporocarps. On average 3-4 sporocarpforming species were found on plots 2-4 with thehighest number of species on P3, whereas H.mesophaeum was the only species found on1.

Analysis of relationship of sporocarp data withenvironmental factor or EM plant community com-position, using Mantel test showed no statisticallysignificant correlation.

Description of EM community on roots of S. caprea

Morphotyping resulted in seven distinct EM morpho-types (labelled A-G), which yielded 26 differentRFLP patterns. Morphotypes B, C and G were onlyrepresented by 1-2 RFLP patterns, while othermorphotypes showed greater genetic heterogeneity(4-7 RFLP patterns). A comparison of the ITS

Unidentified RLFPs that represented 47% of all RLFPpatterns colonised 17% of all EM root tips (8% of allroot tips) (Fig. 2b).

Relations between environmental factorsand colonisation

Mantel test showed significant positive correlationbetween colonisation levels and environmental varia-bles (R=0.47, p<0.0001). The nonmetrical multidi-mensional scaling (MDS) returned two-dimensionalordinations for colonisation data (Stress = 6.07, non-metric fit R2 = 0.99; Fig. 3). Joint plots of thecolonisation data with fitted environmental gradientsshowed positive correlation of DSE colonisation levelswith increasing Pb concentrations and decreasing OM.EM and AM colonisation levels showed no correlationwith environmental gradients. The indirect ordinationanalysis of RFLP data showed no statistically signif-icant correlations with environmental gradients.

Discussion

Salix caprea was shown to accumulate high levels ofPb and Cd. The majority of Pb was immobilised inthe roots of S. caprea, which is also common for otherSalix species and other plant genera (Dahmani-Mulleret al. 2000; Pulford and Watson 2003), indicating theless efficient internal transport of Pb from roots toshoots (Huang and Cunningham 1996). Cd showedhigher mobility and reached leaves/roots ratio of 0.8at the most polluted plot. Several studies have shownthat willows can accumulate high levels of Cd(Landberg and Greger 1996; Sander and Ericsson1998; Vandecasteele et al. 2002; Vandecasteele et al.2005), although they are considered as good accumu-lators rather than hyperaccumulators of Cd (Reevesand Baker 2000). Still, their high biomass productioncould make willows useful in the removal of Cd fromthe soil. In our case of high soil metal concentrations,metal removal makes little sense; nevertheless the

concentrations indicate the tolerance of Salix to theexisting levels of pollution.

Despite the high levels of plant accumulated metalsand several reports on the impact of heavy metals on

photosynthetic pigments (Van Assche and Clijsters1990) we observed no differences in chlorophyll orcarotenoid content among the willows growing atdifferent levels of pollution, further confirming its

enhance their competitiveness and establishment inpolluted environments, although the understanding ofthe ecological role of fungal melanin in DSE fungi isstill lacking. Even though the nutritional role of DSEcolonisation on roots is often unclear (Smith and Read2008), enhanced host mineral nutrition was shown forPhialocephala fortinii on Salix glauca (Fernando andCurrah 1996) indicating their presumable functionalrole at highly polluted sites. However, as no con-elationbetween colonisation levels of S. caprea with differentfungal groups and plant accumulated metal concen-trations, bioaccumulation factors or translocation fac-tors could be observed, the benefit of fungalcolonisation in this extreme environment may be aresult of functional importance of individual fungalspecies or an effect of improved nutrition and toleranceto stress factors other than heavy metal pollution. Inaddition, a more precise examination of the signifi-cance of fungal isolates on the growth of S. caprea,and their persistence on polluted sites over a longerperiod of time is needed for evaluation of theimportance of different fungal colonisers on S. capreafitness and survival at a polluted site.

In order to reveal the most frequent ectomycor-rhizal colonisers, the EM root tips were sorted tomorphotypes that were characterized with RFLPtyping. Sequencing of the most frequent RFLPpatterns from ectomycorrhizal root tips revealedasco- and basidiomycete taxa previously reported toform ectomycorrhizal associations with Salix species(Jumpponen et al. 2002; Trowbridge and Jumpponen2004; Obase et al. 2007; Miihlmann and Peintner2008). In all, 15 EM taxa were revealed on the rootsof S. caprea studied at the polluted site, by combiningthe EM phenotyping and sporocarp identification. Themembers of Sordariaceae were the most frequentcolonisers with an average colonisation of 21 % of allroot tips, followed by Thelephoraceae with 10%. Astudy from a primary succession site at the forefrontof a receding glacier also reported Sordariaceae asdominant EM fungi of willows (Trowbridge andJumpponen 2004). The presence of "sordariales"EM could be extremely important data as we havelittle knowledge on the EM of those ascomyceteouslineages. No statistically significant correlations ofenvironmental gradients with RFLP data ordinationcould be found, which could be a result of the absenceof DSE fungi among the sequenced fungal groups, asonly EM root tips were used. Still, ascomycetes

physiological suitability for phytostabilisation of thesite.

Distinct AM, DSE, and EM structures were found inassociation with roots of S. caprea. Examination of S.caprea root showed that on average 44% of root tipswere colonised by EM fungi. DSE colonised 6% ofexamined root segments, while AM fungi wereobserved only on 1 % of the examined root segments.Similar colonisation levels were reported also for otherSalix species (van der Heijden and Vosatka 1999;Trowbridge and Jumpponen 2004: Cazares et al. 2005;Obase et al. 2007). AM colonisation may be favouredin natural environments where EM propagules aresparse or absent or at early establishment (Smith et al.1998). Indeed, highest AM colonisation levels coin-cided with lowest EM colonisation levels. In our study,DSE colonisation increased with higher soil Pb contentand lower organic matter content. Presence of melaninin the cell wall of DSE fungi could contribute to theirheavy metal tolerance (Gadd 1993) and consequently

USDA-SLO grant MSZS 3411-99-71-0026 Symbiotic interac-tions of plants and fungi on polluted sites in Zerjav; MSZS L1-5146-0481-03 Tolerance of organisms in stressed ecosystemsand the potential for phytoremediation with sponsors MPI,Mezica Mine and Mobitel d.d.; MSZS PO-0522-0481-03Ecology and environmental protection; COST 8.38 ManagingArbuscular Mycorrhizal Fungi for Improving Soil Quality andPlant Health in Agriculture.

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An additional group consisting of representativesfrom Helotiales, which includes root biotrophs andmycorrhizal species (Vralstad 2004) was observed.These ascomycetes could be intercellular endophytesor rhizoplane colonisers and their role for S. capreadeserves further attention. Besides DSE, ascomyceteEM fungal taxa seem to be most numerous at theplots with high metal and low levels of organic matterand more sparse vegetation cover (e.g, early succes-sional stages).

Conclusions

• Despite the high levels of plant accumulatedmetals we observed no differences in chlorophyllor carotenoid content among the willows growingat different levels of pollution, suggesting hightolerance of S. caprea to increased heavy metalpollution, thus making it suitable for phytoreme-diation efforts.

• Root colonisation of willows with three clearlydistinct functional fungal groups may add to theadaptability of willows on the polluted sites.

• Members of Sordariaceae were the most frequentcolonisers with an average colonisation of 21 % ofall root tips, followed by Thelephoraceae with10%. Still, only DSE colonisation increased withhigher levels of pollution and low levels oforganic matter suggesting their potential function-al role for the S. caprea growing at metal enrichedsoils and stressing the need for their furthercharacterisation in screens of fungal symbiontswith the potential application of phytoremedialstudies.

Acknowledgements The authors wish to thank Dr. ThomasHorton and Ms. Donaraye McKay for their help with molecularwork and valuable suggestions. The work was supported by

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