Environmental Pollution 133 (2005) 233–242

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Zn, Cd and Pb accumulation and arbuscular mycorrhizalcolonisation of pennycress Thlaspi praecox Wulf. (Brassicaceae)

from the vicinity of a lead mine and smelter in Slovenia

Katarina Vogel-Mikus*, Damjana Drobne, Marjana Regvar

Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, SI-1000 Ljubljana, Slovenia

Received 23 September 2003; accepted 15 June 2004

Thlaspi praecox Wulf. (Brassicaceae) is a newly discovered Cd, Zn and Pb hyperaccumulatorable to form symbiosis with arbuscular mycorrhizal fungi.

Abstract

Significant hyperaccumulation of Zn, Cd and Pb in field samples of Thlaspi praecox Wulf. collected from a heavy metal pollutedarea in Slovenia was found, with maximal shoot concentrations of 14 590 mg kg�1 Zn, 5960 mg kg�1 Cd and 3500 mg kg�1 Pb.

Shoot/root ratios of 9.6 for Zn and 5.6 for Cd show that the metals were preferentially transported to the shoots. Shoot bio-accumulation factors exceeded total soil Cd levels 75-fold and total soil Zn levels 20-fold, further supporting the hyperaccumulationof Cd and Zn. Eighty percent of Pb was retained in roots, thus indicating exclusion as a tolerance strategy for Pb. Low level

colonisation with arbuscular mycorrhizal fungi (AMF) of a Paris type was observed at the polluted site, whereas at the non-pollutedsite Arum type colonisation was more common. To our knowledge this is the first report of Cd hyperaccumulation and AMFcolonisation in metal hyperaccumulating T. praecox.� 2004 Elsevier Ltd. All rights reserved.

Keywords: Thlaspi praecox; Arbuscular mycorrhiza; Hyperaccumulation; Phytoremediation; Heavy metals

1. Introduction

There has been growing interest in plants that hyper-accumulate heavy metals due to their unique physiologyand potential use in phytoremediation for decontami-nating polluted soil (McGrath, 1998; Zhao et al., 2003).Several heavy metal hyperaccumulating species belong-ing to the genus Thlaspi were reported, among themThlaspi caerulescens hyperaccumulating Zn/Cd (Bakeret al., 1994), Thlaspi rotundifolium ssp. cepaeifoliumhyperaccumulating Pb (Brooks, 1998; Wenzel and

* Corresponding author. Tel.: C386 1 4233388; fax: C386 1

2573390.

E-mail address: katarina.vogel@uni-lj.si (K. Vogel-Mikus).

0269-7491/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2004.06.021

Jockwer, 1999), and T. praecox Wulf. hyperaccumulat-ing Zn (Reeves and Brooks, 1983a). Thresholds forplant hyperaccumulation (shoot dry weight) were set at10 000 mg kg�1 (1%) Zn, 100 mg kg�1 (0.01%) Cd and1000 mg kg�1 (0.1%) Pb (Reeves and Baker, 2000).Levels of accumulated metals vary depending on metalconcentrations in the substrate and plant concentrationsin laboratory experiments usually far exceed the valuesof field specimens from the same populations (Bakeret al., 1994; Lombi et al., 2000). T. caerulescens wasreported to accumulate up to 21 000 mg kg�1 Zn,660 mg kg�1 Pb and 164 mg kg�1 Cd in shoots underfield conditions (Baker et al., 1994) whereas in hydro-ponic growth the dry foliar concentration reached up to14 187 mg kg�1of Cd without showing any toxicitysymptoms (Lombi et al., 2000). T. rotundifolium ssp.

234 K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

cepaeifolium from the Gailitz river bed, Austria, andCave del Predil, Northern Italy, was reported to accu-mulate up to 1934 and 8200 mg kg�1 of Pb, respectively(Reeves and Brooks, 1983b; Wenzel and Jockwer, 1999),and T. praecox was reported to accumulate up to 2.1%of Zn (Brooks, 1998), whereas its uptake of Cd and Pbto our knowledge was not reported.

Hyperaccumulating plants represent an excellentmodel for studying mechanisms of metal uptake(Wenzel and Jockwer, 1999), whereas their interactionswith rhizosphere organisms, including arbuscular my-corrhizal fungi (AMF), have been generally neglected(Salt and Kramer, 2000). Previous reports on arbuscularmycorrhizal (AM) colonisation from polluted sitesmainly referred to non-accumulating species such asViola calaminaria (Tonin et al., 2001), Biscutella leavi-gata and Plantago lanceolata (Orlowska et al., 2002).Hyperaccumulating plants, on the other hand, havebeen widely assumed to be non-mycorrhizal (Leyvalet al., 1997; Pawlowska et al., 2000; Coles et al., 2001).Only recently AM colonisation of Berkheya coddii andother Ni-hyperaccumulating members of the Asteraceaefrom ultramafic soils from South Africa was reported(Turnau and Mesjasz-Przybylowicz, 2003), as well assome meadow Thlaspi sp. (Brassicaceae) (Regvar et al.,2003). The present work is a continuation of this study,and was conducted to (i) reveal possible Zn, Cd and Pbhyperaccumulating capacity of T. praecox from a heavymetal polluted area in the Southern limestone Alps,Slovenia; (ii) asses the metal tolerance strategies adoptedby the plant with respect to Zn, Cd and Pb; (iii) comparethe morphology of AM colonisation developed atpolluted and non-polluted sites; and (iv) compare AMcolonisation levels of T. praecox from a polluted anda non-polluted site.

2. Materials and methods

2.1. Site description

Seven sampling plots (1–7) were selected within anarea heavily affected by lead mining and smeltingactivities in Zerjav, in the southern limestone Alps inNorthern Slovenia (ca. 600–700 m above sea level)according to their increasing distance from the mainsource of pollution (Table 1). The first three plots (1–3)were selected in the valley of the lead smelter chimneys,where vegetation was previously severely damaged bySO2, forest fires and elevated heavy metal concentra-tions. The other four (4–7) plots were selected on the hillrising above the valley where the vegetation was affectedmainly by high heavy metal concentrations. The non-polluted reference plot (indicated by C) was selectednear Vrhnika in central Slovenia, ca. 600 m above sealevel. The base of the polluted site was composed mainly

of Triassic limestone and metalliferous dolomite (cala-mine) with dominating grassland vegetation, while thebase of the non-polluted reference site was mainlycomposed of Triassic limestone harbouring a grasslandvegetation. Geographical locations were determined bythe GPS system. Plant species were identified as in thestandard Slovenian flora (Martincic et al., 1999).

2.2. Chemical analyses

Plant material and soil samples for chemical analyseswere collected on 14 April 2002 at the non-polluted siteand on 8 May 2002 at the polluted site during theflowering period ofT. praecox. The number ofT. praecoxspecimens collected per site was adjusted according to thesize of the natural population from 1 to 9 specimens inorder to avoid drastic depletion of plant populations.Together 31 plants from the polluted site and 7 from thenon-polluted reference site were analysed.

Soil samples were taken from the rhizosphere of a par-ticular plant. The plants were carefully dug from thesubstrate and the majority of bulk soil was manuallyremoved from the roots. Only the substrate closelyattached to the root system was analysed. After dryingat 30 �C for one week soil samples were sieved (!2 mm),homogenized, and divided into subsamples. For theanalysis of total metals 0.1 g of the soil was mineralisedbywet digestion in 3.5 ml of aqua regia (ultrapuremixtureof concentratedHNO3/HCl (Merck)Z 1/3 (v/v)) onanAlblock and then diluted with 5 ml of 0.2% HNO3 andanalysed by flame atomic absorption spectrometry(Perkin Elmer AAnalyst 100). For the analysis of theexchangeable metal fraction the protocol of Baker et al.(1994) was followed. Ammonium acetate (NH4Ac) waschosen as extractant because of its well-proven use as ameasure of the plant available fraction of soil (Ernst,1996). In short, 1 g of soil sample was added to 20 ml of1 M NH4Ac solution pHZ 7.0 and shaken for 2 h at23 �C. The extract was then filtered through a 0.4 mmMilipore membrane filter and analysed by AAS (PerkinElmerAAnalyst 100). Soil pHwasdetermined in thewaterfraction by diluting 1 g of dried soil in 12.5 ml of MiliQwater (}Ohlinger, 1995) andorganicmatterwasdeterminedby wet combustion according to Kandeler (1995).

Prior to the analysis of the plant material, leaves androots of T. praecox were separated and carefully washedwith tap and distilled water in order to remove any sur-face soil or dust deposits, and then (with the exception ofthe fine roots used for the determination of AMFcolonisation) frozen in liquid N2 and freeze-dried for oneweek. For metal analyses, a 30 mg aliquot of freeze-driedand milled aboveground or underground plant materialwas mineralised by wet digestion in 3.5 ml of ultrapuremixture of concentrated HNO3/HClO4 (Merck)Z 7/1(v/v) on an Al block after 24 h incubation period in thesame mixture at room temperature. The samples were

235K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

Table 1

Detailed description of sampling locations of T. praecox in Slovenia

Site Geographic

parameters

Location Substrate pH water Organic

matter (%)

Dominating plant species or plant

association and relative plant cover

1 46 �28#43.2$N,

14 �51#57.7$EOpposite the lead

smelter chimneys on

a 30 � slope.

Stony, very eroded, the

bedrock covered with a

thin (1 cm) layer of partly

decomposed organic

matter and humus.

7.2G 0.0 2.5 G 0 Minuartia gerardii (Willd.)

Hayek (Cariophyllaceae); 70%

2 46 �28#49.9$N,

14 �51#59.4$E250 m from the lead

smelter chimneys on a

40 � slope.

Bedrock covered with

20 cm of partly

decomposed organic

matter and humus.

7.4G 0.2 6.3 G 0.8 Seseleria caeruela (L.)

Ard. (Poaceae); 70%

3 46 �28#49.9$N,

14 �58#58.5$EStreambed under the

2nd site.

Stony, the bedrock

covered with a thin (1 cm)

layer of partly decomposed

organic matter and humus.

7.2G 0.0 7.2 G 0 Salix. sp. (Salicaceae); 50%

4 46 �28#50.0$N,

14 �58#57.9$E20 � slope opposite the

2nd site.

Bedrock covered with

10 cm of partly decomposed

organic matter and humus.

7.4G 0.1 4.5 G 1.2 Thlaspi praecox Wulf. (Brassicaceae),

Salix sp. (Salicaceae); 70%

5 46 �28#26.5$N,

14 �51#56.0$ETop of the hill above

the lead smelter

chimneys.

Bedrock covered with

developing rendzina partly

decomposed organic matter

and humus.

6.1G 0.1 34.6 G 2.5 Thlaspi praecox Wulf., Erysimum

sylvestre Scop. (Brassicaceae),

Thymus serphyllum agg. (Lamiaceae)

and Seseleria caeruela (L.) Ard.

(Poaceae); 90%

6 46 �28#20.4$N,

14 �51#51.7$ETop of the hill 200 m

farther from the

5th site.

Bedrock covered with

developing rendzina, partly

decomposed organic matter

and humus.

6.0G 0.2 42.3 G 3.4 Calamagrostis varia (Schrad.)

Host (Poaceae), Erica carnea L.

(Ericaceae) and Acer pseudoplatanus

L. (Aceraceae); 100%

7 46 �28#18.0$N,

14 �51#48.0$ETop of the hill 400 m

farther from the

5th site.

Bedrock covered with

developing rendzina and

humus.

7.3G 0.1 44.3 G 4.6 Calamagrostis varia (Schrad.)

Host (Poaceae), Erica carnea L.

(Ericaceae) and Acer pseudoplatanus

L. (Aceraceae); 100%

C 45 �58#25.0$N,

14 �14#25.0$EChalk meadow

near the road.

Bedrock covered with

rendzina.

7.7G 0.1 8.6 G 0.8 Gentiano-Koelerietum plant

association; 100%

1–7 – polluted site, C – reference unpolluted site.

then diluted with 5 ml of 0.2% HNO3 and analysed byAAS (Perkin Elmer AAnalyst 100). Shoot bioaccumula-tion factors, one of the hyperaccumulation criteria, werecalculated as quotients of shoot metal concentrationsand total or NH4Ac extractable soil metal concentra-tions (Baker et al., 1994; Robinson et al., 1998).

2.3. AMF colonisation

Plant material for AMF colonisation analysis fromthe polluted sites was collected at the same dates as forchemical analyses. In total, 38 plants were examined. Thecolonisation of each plant was determined on thirty 1 cmlong fine root segments. After washing and clearing with10% KOH, the roots were stained in 0.05% trypan blueaccording to the modified method of Philips andHaymann (1970) and examined under a Karl Zeiss (KF10) light microscope. Mycorrhizal frequency (F%),mycorrhizal intensity (M%), mycorrhizal intensity ofcolonised root fragments (m%), arbuscular richness

(A%)andarbuscular richness of colonised root fragments(a%) were assessed according to Trouvelot et al. (1986).

2.4. Statistical analysis

The relations between soil and plant metal con-centrations were analysed using multiple regression.Pearson’s correlation coefficients (r) were used whencalculating correlations between soil pH, organic matterand plant metal contents. Statistica Statsoft� (version5.0) software was applied.

3. Results

3.1. Accumulation of Zn, Cd and Pb

The soil from the polluted site was highly enriched inZn, Cd and Pb with maximal concentrations of up to5490 mg kg�1 Zn, 390 mg kg�1 Cd and 67 940 mg kg�1

236 K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

Pb. The reference site contained up to 280, 5 and164 mg kg�1 of Zn, Cd and Pb, respectively. Significanthyperaccumulation of metals was found in shoot tissues(dw) of T. praecox with maximal concentrations of14 590 mg kg�1 (1.5%) Zn, 5960 mg kg�1 (0.6%) Cd and3500 mg kg�1 (0.4%) Pb (Fig. 1). A considerable samplevariation between soil and plant metal concentrationsboth within and between the sites was observed,pointing to the heterogeneous nature of the pollutedarea and to the high variability both within and betweenplant populations.

NH4Ac extractable fractions ranged from 8.5 to27.6% for Zn, 16.5 to 44.5% for Cd and 9.6 to 40.0%for Pb. A significant linear correlation between total and

y = 269,94x0,40

R2 = 0,60

y = 841,99x0,11

R2 = 0,10

100

1000

10000

100000

100 1000 10000

Soil total Zn (mg/kg)

Plan

t Z

n (m

g/kg

)

shoots

roots

a)

y = 17,32x0,89

R2 = 0,86

y = 12,66x1,00

R2 = 0,80

1

10

100

1000

10000

1 10 100 1000

b)

y = 43,67e0,00x

R2 = 0,54

y = 109,21e0,00x

R2 = 0,52

0

3000

6000

9000

12000

15000

0 20000 40000 60000 80000

c)

shoots

roots

shoots

roots

Plan

t C

d (m

g/kg

)P

lan

t P

b (m

g/kg

)

Soil total Cd (mg/kg)

Soil total Pb (mg/kg)

Fig. 1. Relationships between (a) Zn, (b) Cd and (c) Pb concentrations

in shoots and roots of T. praecox and total metal soil concentrations.

NH4Ac extractable concentrations was found for Zn(rZ 0.94: P! 0.001) and Cd (rZ 0.94: P! 0.001), butfor Pb an exponential relationship between total andNH4Ac extractable Pb ( yZ 0.13x1.03; R2Z 0.97) wasfound indicating saturation of the NH4Ac fraction atvery high total soil Pb concentrations. The NH4Acextractable fraction of Cd and Zn represented only 3%and 7% of total uptake by the shoots, whereas 2–36%(average 10%) of NH4Ac extractable Pb was incorpo-rated into the shoots and 10–92% (average 41%) intothe roots.

Shoot (dw) Zn concentrations significantly linearlycorrelated with total soil Zn concentrations on a loga-rithmic scale (R2Z 0.60) (Fig. 1a), thus confirming thehyperaccumulation of Zn by Thlaspi praecox. However,no correlation was found between root and total soil Znconcentrations. In addition, the same regression modelcould be applied for the description of the relationshipbetween shoot and root Cd concentrations with totalsoil Cd concentrations (R2Z 0.80) (Fig. 1b), thusconfirming the hyperaccumulation of Cd as well.Although shoot Pb concentrations exceeded the thresh-old levels set for hyperaccumulation, the exponentiallyincreasing regression of shoot Pb with total soil Pbconcentrations (R2Z 0.54) indicates exclusion of Pb asa tolerance strategy (Fig. 1c).

Analysis of the effects of pH and organic mattercontent on plant metal concentrations revealed that Znand Cd plant and Pb root concentrations werenegatively affected by soil pH (Table 2), while on theother hand Zn and Cd shoot concentrations positivelycorrelated with organic matter content (Table 2).Additionally, shoot Pb concentrations were negativelycorrelated with soil organic matter content (Table 2).

Shoot/root ratios ranged from 0.8 to 9.6 (average 3.2)for Zn, 0.4 to 5.6 (average 1.4) for Cd and 0.02 to1.5 (average 0.3) for Pb. The mean shoot/root ratios O1for Zn and Cd revealed very efficient transport ofaccumulated Zn and Cd to the shoots, whereas about

Table 2

Effects of pH and organic matter on metal shoot and root concen-

trations

Plant metal

concentrations

pH Organic

matter (%)

Zn shoot �0.41** ns

Zn root �0.42*** 0.52***

Cd shoot �0.54*** ns

Cd root �0.64*** 0.62***

Pb shoot ns ns

Pb root �0.43*** �0.41**

Pearsons’s correlation coefficients between soil properties and tissue

metal concentrations in T. praecox and their significance are given.

Significance levels: *P! 0.05, **P ! 0.01, ***P ! 0.001, ns – non-

significant.

237K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

80% of accumulated Pb was immobilised in roots. Thesignificant linear regression of Zn shoot/root ratio on alogarithmic scale (R2Z 0.31) indicated increased trans-port of Zn to shoots at higher total soil Zn concen-trations (not shown). On the other hand, a horizontallinear regression of Cd shoot/root ratios with total metalconcentrations was observed, indicating that Cd shoot/root ratios may not be plant traits dependent on soil Cdcontents. No regression model could be fitted to therelationship between Pb shoot/root ratios and total Pbconcentrations.

Bioaccumulation factors based on metal concentra-tions in shoots and total or NH4Ac extractable metalconcentrations of the rhizosphere soils revealed thediffering capacity of T. praecox for the accumulation ofthe three metals in shoots (Fig. 2). A significantly higherability to accumulate Cd was observed in the T. praecoxpopulation from the polluted site (3–75; average 16.6)compared to the population from the non-polluted site(4–29; average 11.0), whereas no differences in Zn and Pbaccumulation were found between the two populations(Fig 2b). The bioaccumulation factors presented on alogarithmic scale linearly decreased with increasing metalcontents in the soil (Fig. 2). Higher determination coeffi-cients of the bioaccumulation factors based on NH4Acextractable rather than total metal concentrations (withthe exception of Cd in the population from the non-polluted site), were observed indicating better exploita-tion of the NH4Ac extractable as total metal pool.

3.2. Morphology of AMF colonisation

Colonisation of T. praecox with AMF from thepolluted site was characterized by a cell to cell spread ofrunning hyphae, forming distinct constrictions at pas-sages through the cell walls. Numerous coils with in-frequent intercalary arbuscules of Paris type mycorrhizawere formed in the inner cortical cell layers as the mainfeature of the colonisation pattern at the polluted sites(Fig. 3). Infrequent spores were formed on terminalhyphae. In contrast, straight intraradical hyphae weremore characteristic at the non-polluted site. Arbuscules,when present, almost exclusively formed on straightintraradical hyphae (Fig. 4), thus exhibiting a moreArum type mycorrhizal colonisation. Coils alwaysformed in outer cortical cells and never containedarbuscules. When present, they were even more frequentthan arbuscules. Intraradical spores were occasionallyobserved.

3.3. Colonisation levels

Low to moderate colonisation levels of T. praecoxroots collected at the non-polluted site were observed,with maximal mycorrhizal frequencies (F%) of up to70%; however, levels of 20–30% were more common

(Fig. 5). No colonisation was observed on the three mostpolluted plots in Zerjav, but on less polluted plotsmycorrhizal frequencies of up to 30% were occasionallyobserved, with levels of 10% the most common.Arbuscules were formed only when total soil concen-trations did not exceed 4100, 85 and 33 000 mg kg�1 ofZn, Cd and Pb, respectively (Fig. 5). When calculated onthe basis of NH4Ac extractable Zn, Cd and Pb, thelimits of arbuscular formation were at 900, 26 and5200 mg kg�1, respectively.

1

1

10

100

1000

10 100 1000 10000

Zn

b

io

accu

mu

latio

n facto

rs

a)

0,001

0,01

0,1

1

10

100

1000

0,1 10 100 1000C

d b

iaccu

mu

latio

n facto

rs

b)

0,001

0,01

0,1

1

10

1 10 100 1000 10000 100000

Pb

b

io

accu

mu

latio

n facto

rs

c)

Soil total Zn (mg/kg)

Soil total Cd (mg/kg)

Soil total Pb (mg/kg)

BF-total

BF-NH4Ac

y = 15510,82x-0,87

R2 = 0,86

y = 269,94x-0,60

R2 = 0,78

y = 2252,74x-0,79

R2 = 0,54

y = 510,69x-0,75

R2 = 0,51

y = 248,94x-1,39

R2 = 0,88

y =388,97x-2,80

R2 =0,95

BF-total-P

BF-NH4Ac-P

BF-total-N

BF-NH4Ac-N

y = 0,84x-0,40

R2 = 0,47

y = 5,45x-0,42

R2 = 0,61

BF-total

BF-NH4Ac

Fig. 2. Relationship between shoot bioaccumulation factors (BF)

based on total and ammonium acetate extractable (NH4Ac) metal

fractions and total soil metal concentrations. (a) Zn, (b) Cd, (c) Pb. P –

the population from the polluted site (–), N – the population from the

non-polluted site (----).

238 K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

Fig. 3. Typical formation of Paris type mycorrhiza in T. praecox from polluted site. Dense coils of fungal hyphae with hyphal constrictions (Co) are

observed; barZ 20 mm.

Mycorrhizal intensity (M%) of up to 35%, mycor-rhizal intensity in colonised root fragments (m%) of upto 60% and arbuscular richness in colonised rootfragments (a%) of up to 95% were most frequent atthe non-polluted site. At the polluted sites M% was!20%, and m% and a% were !10%. However, ina few cases m% of up to 70% and a% of up to 100%were observed.

4. Discussion

Among those plant species recognized to haveimportant metal remediation capabilities only 25 showthe capacity to accumulate three or more metals(McIntyre, 2003). T. praecox from the vicinity of a lead

Fig. 4. Typical formation of Arum type mycorrhiza in T. praecox from

non-polluted site. Arbuscules formed on straight intraradical hyphae

are observed; barZ 20 mm.

mine and smelter in Slovenia is able to accumulate 1.5%Zn, 0.6% Cd and 0.4% Pb. According to Reeves andBaker (2000) and Reeves and Brooks (1983b) 4.4% Znand 0.2% Cd in T. caerulescens and 0.8% Pb in T.rotundifolium subsp. cepaeifolium were the highest valuesof metal accumulation reported within the genusThlaspi. These results indicate remarkable hyperaccu-mulation of the three metals in T. praecox, which showsa promising potential for further phytoremediationstudies. Hyperaccumulation of Cd is of particularimportance because so far T. caerulescens and Arabi-dopsis halleri are the only species identified as hyper-accumulators of Cd in the natural environment (Brooks,1998; McGrath et al., 2001).

Accumulation and exclusion are two basic strategiesby which plants respond to elevated concentrations ofheavy metals. In metal accumulator species shoot/rootratios greater than 1 are common, whereas in metalexcluder species the factors are typically lower than 1(Baker, 1981). Accumulation of Zn and Cd is suggestedas a tolerance strategy by T. praecox on the basis ofregression analysis, which is also typical of T. caer-ulescens (Zhao et al., 2003). In addition, shoot/root Znand Cd ratios higher than 1 indicate a very efficientability to transport metals from roots to shoots, mostlikely due to efficient Zn and/or Cd transportersystem(s), presumably similar to that reported for T.caerulescens (Lasat et al., 2000; Lombi et al., 2000; Zhaoet al., 2002), and probable sequestration of Zn and Cd inleaf vacuoles and apoplast (Vazquez et al., 1992; Kupperet al., 1999; Lasat et al., 2000).

Shoot Pb concentrations exceeded the hyperaccumu-lation criterion with a maximal concentration of3500 mg kg�1, which may therefore classify T. praecoxas a Pb hyperaccumulator as well. However, accordingto Baker (1981), exclusion of Pb was suggested asa tolerance strategy on the basis of the relationship

239K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

between shoot and total soil Pb concentrations. Shoot/root ratios of 0.3 were common for Pb, which is typicalof excluders (Baker, 1981; Dahmani-Muller et al., 2000).Furthermore, about 80% of Pb was immobilised in theroots of T. praecox, which is also common for otherThlaspi species and other plant genera (Punz andSchinninger, 1995; Dahmani-Muller et al., 2000), in-dicating the lack of internal transport of Pb from rootsto shoots (Huang and Cunningham, 1996).

Plant metal concentrations greatly depend on metalconcentrations in the soil (Baker et al., 1994; Lombiet al., 2000; Peralta-Videa et al., 2002); however, metalsconsidered readily available for plant uptake often

0

10

20

30

40

50

60

70

80

0 1000 2000 3000 4000

Soil total Zn (mg/kg)

F%

A%a)

0

10

20

30

40

50

60

70

80

0 20 40 60 80

Soil total Cd (mg/kg)

F%

100

A%b)

0

10

20

30

40

50

60

70

80

0 5000 10000 15000 20000 25000 30000 35000

Soil total Pb (mg/kg)

F%

A%c)

Fig. 5. Mycorrhizal frequency (F%) of T. praecox as a function of total

metal concentrations (a) Zn, (b) Cd and (c) Pb. Bubble size reflects the

size of arbuscular richness (A%).

present only a small portion of the total metal contentof the soil (Blaylock and Huang, 2000). McGrath et al.(1997) demonstrated that more than 90% of total Znuptake in T. caerulescens comes from the non-extract-able soil metal fraction. The NH4Ac extractable fractionrepresents only 3% and 7% of total Cd/Zn uptake in theshoots of T. praecox, indicating that the plant can tapinto non-extractable metal pools as well. Besides metalconcentrations, metal availability is also influenced byother characteristics of the soil system such as pH,organic matter content and cationic exchange capacity,which control the solubility and therefore the availabil-ity of metals (Marschner, 1995; Moreno et al., 1996).Enhanced uptake of Zn, Cd and Pb in T. praecox wasobserved at lower pH as previously reported forT. caerulescens (Robinson et al., 1998). When growingon Cd/Zn polluted soil, hyperaccumulators contain highconcentrations of metals, which may return to the soilafter plant death. It was reported that metals (Cd, Zn)associated with leaves of the hyperaccumulatorT. caerulescens became very mobile after incorporationinto the soil (Perronet et al., 2000), which could explainthe positive correlation with organic matter content andCd and Zn root concentrations in T. praecox. Comparedto Zn and Cd, Pb is generally considered insoluble andunavailable for plant uptake (Blaylock and Huang,2000). Furthermore, a higher affinity of Pb for organicmatter complex formation than for Zn and Cd wasreported (Martinez and Motto, 2000; Sauve et al., 2000;Tipping et al., 2003), which could be one of the reasonswhy organic matter contents negatively correlated withPb shoot concentrations of T. praecox.

The bioaccumulation factors (shoot/soil concentra-tion ratio) are more important than shoot concentrationswhen considering the potential of phytoremediation ofa given species (Zhao et al., 2003). In T. praecox, Znbioaccumulation factors based on total Zn soil concen-trations ranged up to 20, whereas accumulation factors ofup to 40 were reported for T. caerulescens (Zhao et al.,2003). In addition, Cd bioaccumulation factors of theT. praecox population from a polluted site of up to 75were the same as in T. caerulescens of the SouthernFrance Ganges ecotype (Zhao et al., 2003), indicatinga very high capacity of T. praecox for Cd accumulation.Pb bioaccumulation factors �1 observed in T. praecoxwere in the same range as for British populations ofT. caerulescens, indicating very low soil Pb exploitation(Baker et al., 1994). Bioaccumulation factors in generalare reported to decrease with increasing soil metalconcentrations (Robinson et al., 1998; Zhao et al.,2003). The decrease in accumulation ratio may be dueto the saturation of metal uptake and/or root to shoottransport when internal metal concentrations are high(Zhao et al., 2003). The activation of a low Zn/Cd affinitytransport systems at higher soil Zn/Cd concentrationswas reported for T. caerulescens (Lasat et al., 1996;

240 K. Vogel-Mikus et al. / Environmental Pollution 133 (2005) 233–242

Lombi et al., 2001), which may also be true for T.praecox.

Metal uptake in shoots and roots also depends onaccompanying rhizosphere microorganisms, which bytheir activities alter the rhizosphere conditions andconsequently the metal availability (Leyval and Joner,2001). Arbuscular mycorrhizal fungi (AMF) are knownto inhibit or enhance metal uptake in plant tissues(Heggo et al., 1990; Ietswaart et al., 1992; Joner andLeyval, 1997; Joner et al., 2000). However, only a fewreports on AMF colonisation of hyperaccumulatingplant species are known. Among these, the Ni hyper-accumulator Berkheya codii (Asteraceae) and other Nihyperaccumulating plants from ultramafic soils in SouthAfrica were reported to be consistently colonised byAMF (Turnau and Mesjasz-Przybylowicz, 2003). In thepresent paper we report AMF colonisation in T. praecoxfrom polluted sites in Slovenia. The colonisation patternof T. praecox was characterized by the formation ofParis type mycorrhiza, which could be an adaptation ofplant–fungal symbiosis to adverse soil conditions at thepolluted site, since a more Arum type mycorrhiza wascommon at the non-polluted site.

AMF colonisation levels were significantly lower orabsent on the most Zn, Cd and Pb polluted plots. Thereason could be the loss of AMF propagules on pollutedsites, disturbance of vegetation and erosion of the mostpolluted locations (Pawlowska et al., 1996; Pawlowskaet al., 2000), or the establishment of an early succes-sional plant community consisting of non-mycorrhizalruderals such as Minuartia gerardii (Caryophyllaceae),which do not support AMF colonisation. The non-polluted site, on the other hand, hosted a stable activelygrowing meadow community with different plant species(Table 1), which harbours higher abundance anddiversity of AMF (Peat and Fitter, 1993; Regvar et al.,2001). Although tolerant strains of AMF were isolatedfrom polluted soils (Weissenhorn et al., 1993), mycor-rhiza formation and development could also be altereddue to metal toxicity (Leyval et al., 1997; Leyval andJoner, 2001).

Sequencing of the 28S- and 18S-rDNA PCR-prod-ucts of T. praecox from non-polluted sites revealedcolonisation by Glomus intraradices (Regvar et al.,2003), so further experimental work has to be done inorder to identify AMF colonising T. praecox on heavymetal polluted areas in Slovenia and to highlight the roleof mycorrhiza and its possible influence on metal uptakein this newly discovered Cd hyperaccumulating plant.

5. Conclusions

� T. praecox (Brassicaceae) from the vicinity of a leadmine and smelter in Zerjav, limestone SouthernAlps, is able to accumulate 1.5% of Zn, 0.6% of Cd

and 0.4% of Pb. Hyperaccumulation of Cd and Znand exclusion of Pb were observed as plant tolerancestrategies in response to high soil metal levels.

� T. praecox formed AM symbiosis on both heavymetal polluted and non-polluted sites. Plants fromthe polluted site formed Paris type mycorrhiza,whereas at the non-polluted site a more Arum typemycorrhiza was observed.

� No AMF colonisation was observed on the most Zn,Pb and Cd polluted plots. Low to moderate levelsof mycorrhizal frequency (FZ 20–30%) were, how-ever, observed on less and non-polluted plots.

Acknowledgements

The authors are indebted to Dr. Corinne Leyval forher valuable comments on the manuscript and Dr.Katarina Kosmelj and Dr. Peter Kump for their helpwith statistical analyses. The work was supported by thefollowing projects: Tolerance of Organisms in Stressedecosystems and the Potential for PhytoremediationMSZS L1-5146-0481, Ecology and Environmental Pro-tection MSZS PO-0522-0481, Joint effects of binarymixtures of cadmium, zinc and mercury on selectedanimals and plants MSZS J1-3186, COST 8.38 entitledManaging Arbuscular Mycorrhizal Fungi for ImprovingSoil Quality and Plant Health in Agriculture and MPI,Rudnik Mezica, Crna na Koroskem.

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