Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution

Environmental Pollution 157 (2009) 280–286

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Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution Doris Krpata a, Walter Fitz b, Ursula Peintner a, Ingrid Langer b, Peter Schweiger b, * a b

Institute of Microbiology, Innsbruck University, Technikerstraße 25, A-6020 Innsbruck, Austria Institute of Soil Science, University of Natural Resources and Applied Life Sciences, Peter Jordan-Straße 82, A-1190 Vienna, Austria

Populus tremula and associated ectomycorrhizal fungi accumulate zinc and cadmium to similar concentrations.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 April 2008 Received in revised form 17 June 2008 Accepted 22 June 2008

Concentrations of Zn and Cd were measured in fruitbodies of ectomycorrhizal (ECM) fungi and leaves of co-occurring accumulator aspen. Samples were taken on three metal-polluted sites and one control site. Fungal bioconcentration factors (BCF ¼ fruitbody concentration: soil concentration) were calculated on the basis of total metal concentrations in surface soil horizons (BCFtot) and NH4NO3-extractable metal concentrations in mineral soil (BCFlab). When plotted on log–log scale, values of BCF decreased linearly with increasing soil metal concentrations. BCFlab for both Zn and Cd described the data more closely than BCFtot. Fungal genera differed in ZnBCF but not in CdBCF. The information on differences between fungi with respect to their predominant occurrence in different soil horizons did not improve relations of BCF with soil metal concentrations. Aspen trees accumulated Zn and Cd to similar concentrations as the ECM fungi. Apparently, the fungi did not act as an effective barrier against aspen metal uptake by retaining the metals. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Metals Accumulation Ectomycorrhizas Fruitbodies Populus tremula

1. Introduction Many fungi accumulate substantial amounts of metals such as zinc (Zn) and cadmium (Cd) in their fruitbodies (Tyler, 1980; Alonso et al., 2003). Especially Cd concentrations are generally much higher in fruitbodies than in shoots of plants growing at the same site (Krupa and Kozdroj, 2004; Rudawska and Leski, 2005a). Fruitbody metal concentrations generally increase with increasing soil metal concentrations (Kalacˇ and Svoboda, 2000). On polluted sites they may reach levels that constitute a serious health hazard to humans if the fruitbodies were consumed (Zimmermanova´ et al., 2001; Cocchi et al., 2006). Other soil characteristics that affect fruitbody metal concentrations are organic matter content and soil pH (Gast et al., 1988). Not all fungi accumulate metals to the same extent (Alonso et al., 2003). Differences between fungi in fruitbody metal concentrations have been suggested to reflect the capacity of the mycelium to absorb metals from the substrate (Tyler, 1980). The physiological basis of metal accumulation into fungal fruitbodies is, however, still poorly understood (Gadd, 1993). Fungal trophic status has been found to affect metal concentrations. Ectomycorrhizal (ECM) fungi usually have lower metal concentrations than for example

* Corresponding author. E-mail address: [email protected] (P. Schweiger). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.06.038

saprotrophs (Ru¨hling and So¨derstro¨m, 1990). This has been attributed to a relative to saprobic fungi much lower production of extracellular catabolic enzymes by ECM fungi (Rudawska and Leski, 2005a), which receive most to all of their carbon directly from associated host plants (Hampp and Schaeffer, 1998). Some ECM fungal species, however, have generally high metal concentrations such as those in the genus Amanita (Lepsˇova´ and Mejstrˇik, 1988; Rudawska and Leski, 2005a). Differences in metal concentrations between different parts of the fruitbody have also been observed (Peintner and Moser, 1996; Rudawska and Leski, 2005b). Metal accumulation in fungi has frequently been expressed in terms of bioconcentration factors (BCF; Gast et al., 1988). These describe the ratio between fruitbody metal concentrations and those in the substrate. Similar expressions have been used to describe metal accumulation by higher plants (Unterbrunner et al., 2007). In both fungi and plants double-logarithmic plots of Zn accumulation factors versus substrate Zn concentrations have been found to decrease linearly (Gast et al., 1988; Unterbrunner et al., 2007). A direct comparison between co-occurring fungi and plants in their decreasing metal accumulation with increasing substrate concentrations is, however, lacking. Expressions of BCF have generally been based on total substrate metal concentrations measured in the uppermost 5–10 cm of the soil below the litter layer (Gast et al., 1988; Alonso et al., 2003). In plants, metal concentrations are frequently more closely correlated with labile than with total soil metal concentrations (Robinson

D. Krpata et al. / Environmental Pollution 157 (2009) 280–286

et al., 2000; Nolan et al., 2005). In a study with Populus alba, this correlation for Cd was even further improved when concentrations in soil 25–40 cm below the surface were used (Madejo´n et al., 2004). Although not specifically discussed by the authors, this may reflect the distribution of actively absorbing roots of this tree in the soil profile. Similar to roots, the abundance of ECM fungi differs between horizons in the soil profile (Dickie et al., 2002). Additionally, some fungi colonise roots throughout the soil profile, while others are restricted to either organic or mineral soil horizons (Rosling et al., 2003). The objectives of this work were to (1) compare the use of total versus labile soil metal concentrations for correlations between ECM fungal BCF and substrate metal concentrations; (2) assess differences in BCF between ECM fungal taxa; (3) relate differences in BCF between ECM fungi with their predominant occurrence in either organic or mineral soil; (4) compare ECM fungal BCF with accumulation factors of co-occurring plant species. 2. Material and methods An area in the vicinity of a former lead (Pb)/Zn-smelter in southern Austria (near Arnoldstein/Carinthia) was selected for this study. The extent of metal pollution of the area has previously been summarized (Friesl et al., 2006). Fruitbodies of ectomycorrhizal fungi were collected during 2004 on three heavily polluted sites of approximately 250 m2 each. A less-polluted site approximately 3.5 km upwind from the smelter was included as a control site. European aspen (Populus tremula) was the dominant tree species on all four sites. The understory of the polluted sites was mainly made up of Tor grass (Brachypodium pinnatum) and to a lesser extent Lily-ofthe-valley (Convallaria majalis). The understory of the control site was more diverse and typical for temperate deciduous woodlands. The soils on the three heavily polluted sites were calcaric Cambisols covered by a 5–10 cm thick mor-type humus horizon. In contrast, the control site had no such organic horizon. For soil characterisation, soil samples were taken on each site from five randomly distributed spots using a soil auger with an inner diameter of 7.5 cm. In the field, auger cores were separated into the organic humus horizon and the upper 10 cm of mineral soil. In the laboratory, soil samples were stored at 4  C until further processing. Aspen roots were removed from the soil prior to any analyses for characterisation of the root-associated ectomycorrhizal community (Krpata et al., 2008). Air-dry mineral soil samples were sieved to <2 mm and their physicochemical properties characterised following standard procedures (Blum et al., 1996). The soil ¨ sterreichisches was digested in aqua regia for analysis of total metal content (O Normungsinstitut, 1999) and extracted with NH4NO3 for analysis of labile metal fractions (Pru¨eß, 1997, DIN 19730). The pH (H2O) of air-dry humus horizons was analysed after removal of coarse organic material (1:12.5 w/v). For analysis of total metal content, oven-dried humus horizons were ground with mortar and pestle and digested in HClO4/HNO3 (1:4). Trace element concentrations in diluted digests were measured by ICP-MS (Elan 9000 DRCe, PerkinElmer). A total of 114 fruitbodies from 22 ectomycorrhizal fungal species were collected for analysis of their metal concentrations. For comparison, fruitbodies of a saprobic fungus (Psathyrella candolleana) were also collected. Any adhering substrate particles were carefully removed, and fruitbodies were additionally washed in distilled water. This was done to ensure no contamination from the substrate (Stijve et al., 2004). Oven-dry samples of entire fruitbodies were ground and subsamples digested in a mixture of HClO4/HNO3. Digests were brought to a volume of 50 ml with Milli-Q water. Elemental concentrations in these solutions were measured by ICPMS. Washed leaf samples of aspen, Tor grass and the Zn and Cd hyperaccumulator Arabidopsis halleri (only growing on one site) were also analysed. For quality assurance, reference materials were processed and analysed identically. The accuracy of soil metal analyses was assessed by including EUROSOIL7 as an internal standard (Weissteiner et al., 1999). Analyses of fungal, plant and humus material were checked against certified plant reference material (oriental tobacco leaves, CTA-OTL-1, Institute of Nuclear Chemistry and Technology, Warzawa, Poland; purchased from LGC Promochem, Germany). Measured metal concentrations of the reference materials were generally above the certified values. Overestimation of total Cd and Zn concentrations were within 5 and 10%, respectively. A number of different bioconcentration factors (BCF) with respect to Zn (ZnBCF) and Cd (CdBCF) were calculated for the fungi. Subscripts were used to differentiate between values for BCF calculated for different soil horizons. CdBCFtot refers to the accumulation of Cd relative to its total concentration in the uppermost 5 cm of the soil. CdBCFlab indicates the accumulation relative to the labile Cd concentration in the mineral soil. All regressions were computed in StatGraphics. Slope coefficients were considered significant at p < 0.05. For the comparison of BCF relations of various ECM fungal taxa and between fungi and plants, fitting a common curve was compared to fitting separate curves. The significance of improvement in description for separate

281

curves was tested using the principle of the extra sum of squares (Mead and Pike, 1975).

3. Results Physicochemical properties of the soils are given in Table 1. Mineral soils were quite similar, except that the soil on the control site had a much lower carbonate concentration. Total and labile soil concentrations of Zn, Cd and Pb on the polluted sites were considerably above the normal concentration range (Table 2a, b). Concentrations were generally highest in the organic horizon. In mineral soils, total metal concentration ranges were 13–425, 1800– 10 820 and 1090–4056 mg kg1 for Cd, Pb and Zn, respectively. Even on the control site mean soil metal concentrations still exceeded normal concentration ranges (Table 2a, b). An analysis of metal concentrations down the soil profile showed heavy enrichment in Zn, Cd and Pb down to a depth of approximately 20 cm of the mineral soil with normal concentrations further down (data not shown). All fungal species collected and analysed, including the saprotroph P. candolleana, are listed in Table 3 with their fruitbody Zn and Cd concentrations. The data are quite variable, as seen when plotted against total Zn and Cd concentrations in the uppermost 5 cm of the soil (Fig. 1a and b; only ECM fungi). Mean Zn concentrations ranged from 250 to 500 mg g1. Mean Cd concentrations were in the range 10–20 mg g1, with more than 75% of the samples above 5 mg g1. For Zn, fruitbody concentrations did not increase with increasing soil concentrations (p ¼ 0.61). A slight and significant increase was observed for Cd (p ¼ 0.03). Log10-transformed Zn bioconcentration factors calculated for all fruitbodies were linearly correlated with log10-transformed total Zn concentrations in the upper 5 cm of the soil (ZnBCFtot; p < 0.01; R2 ¼ 0.57; Fig. 2a). On the polluted sites this corresponded to the Zn concentration in the organic horizon. The relationship was much weaker when calculated on the basis of total Zn concentrations in the mineral soil (R2 ¼ 0.33). The closest fit (R2 ¼ 0.77) was obtained, when log10 ZnBCFlab values were correlated with labile Zn concentrations in the mineral soil (Fig. 2b). Very similar results were obtained for Cd (Fig. 3a and b), where the closeness of the fit for the correlations increased from R2 ¼ 0.51 for log10 CdBCFtot to R2 ¼ 0.70 for log10 CdBCFlab (Fig. 3a and b). The effect of fruitbody Zn concentrations on Cd accumulation was tested in a multiple regression and was found to have a slight and positive effect (p ¼ 0.012). Values for ZnBCFlab differed between different fungal taxa (Fig. 4). Log–log relations of the decreasing ZnBCFlab values with increasing soil Zn concentrations were calculated for Amanita spp., Hebeloma spp., Inocybe spp. and Tricholoma scalpturatum. Parallel lines were obtained for Amanita spp., Hebeloma spp. and T. scalpturatum. On the contaminated sites, the highest ZnBCFlab values Table 1 General physicochemical characteristics of the soils Parameter

Site 1 Mean

Organic horizon pH (H2O)

5.8

Site 2 SD 0.17

Mean 6.20

Site 3 SD

Mean

0.10

6.19

Mineral soil Texture Loamy Sand Loamy Sand 32 0.6 38.6 2.0 Sand (g kg1) 54 1.1 53.7 2.5 Silt (g kg1) 14 1.0 7.6 1.5 Clay (g kg1) 6.7 0.16 7.10 0.20 pH (H2O) CaCO3 (g kg1) 69 20 93 32 1 50.0 8.3 37.0 18.0 Corg (g kg ) 159 22 109 23 EC (mS cm1) 41 261 16 CEC (mmolc kg1) 246

Loamy 35.0 53.6 11.4 7.28 163 40.8 147 300

Control SD

Mean

0.12 –

SD –

Sand Loamy Sand 5.3 42.9 2.0 4.3 48.3 2.7 2.6 8.7 2.0 0.14 6.55 0.35 18 10 11 14.3 59.7 20.7 26 125 17 40 290 73

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D. Krpata et al. / Environmental Pollution 157 (2009) 280–286

Table 2 Mean (þ/-SD) soil heavy metal concentrations from all experimental sites Site 1 Mean

Site 2 SD

Mean

Site 3 SD

(a) Total metal concentrations (mg kg1) Organic horizon Cd 105 13 86 23 Pb 52 220 10 100 20 404 7830 Zn 8655 1960 8177 2581 Mineral soil Cd 63 Pb 8068 Zn 3302

12 2615 716

24 3564 1957

10 1665 890

Control

Table 3 List of fungal species and their fruitbody Zn and Cd concentrations Normal

Mean

SD

Mean

SD

Range

111 20 706 11 239

11 4750 1358

– – –

– – –

– – –

23 2546 2165

11 878 879

5.7 459 571

3 131 281

0.1–0.6 2–80 10–80

411 303 10 686

277 246 9602

27 24 514

9 18 280

<5 <20 <250

1

(b) Labile metal concentrations (mg kg ) Mineral soil Cd 2941 1062 369 640 Pb 1335 610 152 81 Zn 58 773 29 658 18 435 4995

The concentration range normally observed on uncontaminated sites is also given.

were noted for T. scalpturatum. This fungus had ZnBCFlab values similar to those of Hebeloma spp. individuals collected on soil with labile Zn concentrations lower by a factor of 0.41. For Amanita spp. this factor was 0.54. For Inocybe spp., the log–log plot of ZnBCFlab versus labile soil Zn had a steeper slope with relatively lower values at the highest labile soil Zn concentration (Fig. 4). Fungal taxa did not differ in CdBCFlab (data not shown). On the control site, mean concentrations of Zn and Cd in aspen leaves were 340 and 3.8 mg g1, respectively (Table 4). They increased to on average 750 mg g1 Zn and 12.6 mg g1 Cd on the contaminated sites. Lower concentrations were measured in Tor grass, 115 mg g1 Zn and 0.35 mg g1 Cd (Table 4). A. halleri accumulated Zn to just above 20 000 mg g1 and Cd to 67 mg g1. Negative log–log relationships were obtained for plant Zn (Fig. 5a) and Cd (Fig. 5b) accumulation factors when plotted against the respective labile soil metal concentrations. Especially in the case of Cd accumulation factors were much higher in aspen than in Tor grass. For comparison, the one sample of the hyperaccumulator A. halleri is included in the figures. Correlations of ECM fungal BCF and aspen accumulation factors for both Zn and Cd with the labile soil concentrations of the respective metal (Fig. 6a and b) were not significantly different. 4. Discussion Concentrations of Zn are within the range of values generally reported for ECM fungal fruitbodies (Tyler, 1980; Vetter et al., 1997; ˇ anda, 2007). In the case of Cd, Alonso et al., 2003; Borovicˇka and R greatly elevated concentrations were observed in this study. More than 75% of all fruitbodies contained more than 5 mg Cd g1. In a screening of metal concentrations in fruitbodies from mainly uncontaminated sites (Tyler, 1980) 80% had concentrations below 5 mg Cd g1. Cd concentrations above 10 mg g1, as frequently measured in this study, have previously been reported from other heavily contaminated sites (Gast et al., 1988; Svoboda et al., 2006). As frequently observed (Gadd, 2007), fruitbody Cd concentrations were not correlated with soil Cd concentrations. Large variations in especially Zn concentrations were observed within the same and closely related taxa. Two species in the genus Inocybe collected on the control site, Inocybe maculata and Inocybe rimosa, had considerably elevated Zn concentrations (>500 mg g1). Individuals of I. maculata collected on the heavily contaminated sites tended to have lower Zn concentrations (p < 0.01). Another species in that genus, Inocybe pseudoreducta, had the lowest Zn concentrations on one of the contaminated sites. A similarly large

Fungal species

Control site

Site 1

Site 2

Site 3

Cd Zn Cd Zn Cd Zn Cd Zn (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1)

Ectomycorrhizal Amanita phalloides Mean 91.7 13.0 SD 12.5 0.025 (n) (2) (2) Amanita vaginata Mean 571 10.1 403 SD (n) (1) (1) (1) Cortinarius hemitrichus Mean 243 (n) (1) Cortinarius sertipes Mean 206 5.85 (n) (1) (1) Cortinarius sp. subg. Telamonia Mean 85.5 5.95 (n) (1) (1) Hebeloma mesophaeum Mean SD (n) Hebeloma velutipes Mean 191.05 3.56 178 SD 94.47 1.35 81 (n) (3) (2) (5) Inocybe fuscidula Mean SD (n) Inocybe pseudoreducta Mean 51.2 SD 58.7 (n) (4) Inocybe maculata Mean 581 17.4 SD 85 6.1 (n) (7) (7) Inocybe rimosa Mean 593 4.20 SD 49 2.76 (n) (5) (5) Inocybe sp. Mean SD (n) Laccaria laccata Mean 135 12.3 165 SD 65 5.30 50 (n) (4) (4) (4) Lactarius glyciosmus Mean 167 SD 48 (n) (2) Leccinum populinum Mean 296 (n) (1) Paxillus involutus Mean 274 SD 85 (n) (2) Peziza badia Mean 614 SD 113 (n) (3) Russula lutea Mean 186 5.8 (n) (1) (1) Scleroderma verrucosum Mean 598 SD 29 (n) (3)

48.5 (1)

560 154 (4)

36.0 13.6 (4)

488 123 (4)

72 53 (4)

453 225.89 (5)

25.7 15.14 (5)

334 157 (6)

7.81 6.14 (4)

431 125 (5)

12.2 6.1 (5)

16.6 (1)

6.6 4.5 (3)

285 39 (4)

35.9 11.2 (4)

249 102 (5)

6.29 2.91 (5)

291 60 (5)

4.7 1.29 (4)

13.1 12.4 (7)

93.3 72.1 (4) 15.6 3.4 (2) 19 (1) 5.3 0.08 (2) 22.9 3.0 (3)

7.9 0.52 (3)

777 174 (5)

7.0 2.5 (5)

D. Krpata et al. / Environmental Pollution 157 (2009) 280–286 Table 3 (continued)

a

Control site

Site 1

Site 2

Site 3

Cd Zn Cd Zn Cd Zn Cd Zn (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1)

Thelephora caryophyllea Mean 442 19.1 (n) (1) (1) Tricholoma scalpturatum Mean SD (n) Xerocomus rubellus Mean 573 36.2 (n) (1) (1) Saprobic Psathyrella candolleana Mean SD (n)

762 265 (5)

27.9 3.59 (4)

203 26 (5)

695 102 (5)

34.7 3.9 (5)

699 236 (5)

43.0 19.8 (5)

600

Fruitbody Cd concentration (µg g-1)

0,01 0,001 0,0001

control site site 1 site 2 site 3 500

1000

3000

5000

10000

10000 1000 100 10 1 0,1 0,01

control site site 1 site 2 site 3 1

10

100

300

Labile soil Zn concentration (µg g-1)

400

Fig. 2. Log–log plots of bioconcentration factors (BCF) of Zn calculated for all ECM fungi on the basis of (a) total and (b) NH4NO3-extractable soil Zn concentrations.

200

0

2000

4000

6000

8000

120

10000

149

control site site 1 site 2 site 3

100

12000

193

80 60 40 20 0

0,1

0,001 0,1

Soil Zn concentration (µg g-1)

b

b Fruitbody ZnBCF labile (unitless)

Fruitbody Zn concentration (µg g-1)

control site site 1 site 2 site 3

800

0

1

Total soil Zn concentration (µg g-1)

1200 1000

10

0,00001 300

127 26 (5)

Mean and standard deviation are given as well as the number of measurements within the detection range.

a

Fruitbody ZnBCF total (unitless)

Fungal species

283

0

20

40

60

80

100

120

Soil Cd concentration (µg g-1) Fig. 1. Concentrations of (a) Zn and (b) Cd in all fruitbodies of ectomycorrhizal fungi collected at the experimental sites. Data are plotted against total soil metal concentrations in the uppermost 5 cm soil. The line within the box shows the median and the boundaries of the boxes indicate the 25th and 75th percentiles. The whiskers indicate the 10th and 90th percentiles. All outliers are also plotted.

variation in Zn accumulation was previously found within another genus (Russula; Vetter et al., 1997). It was attributed to potential differences in Zn accumulation capacities between different sections within that genus. The general pattern of fungal metal accumulation agrees well with previous observations. T. scalpturatum accumulates high Zn concentrations (Vetter et al., 1997), and for Amanita spp. many reports on increased Cd concentrations are available (Ru¨hling and So¨derstro¨m, 1990; Rudawska and Leski, 2005a). Saprobic fungi, such as P. candolleana in this study, accumulate Cd to even much higher levels (Svoboda et al., 2006). The weak correlation between Cd concentrations in the fruitbodies and the soil supports previous conclusions that fungal fruitbodies are of only limited value as bioindicators of Cd contamination (Gast et al., 1988; Kalacˇ and Svoboda, 2000). Bioconcentration factors for Zn in this study are well below values reported elsewhere (Alonso et al., 2003; Malinowska et al., 2004). This is most likely due to the much lower substrate Zn concentrations measured in those studies (<100 mg Zn g1 soil). A comparison of the obtained ZnBCFtot values with data from a study covering a very wide range of substrate concentrations (Gast et al., 1988) shows, however, good agreement. Also the CdBCFtot values are relatively low even when compared to values obtained for contaminated sites (Gast et al., 1988; Svoboda et al., 2006). However, soils at most of those sites contained less than 1 mg g1 Cd which is lower than Cd concentrations at the control site in this study.

284

D. Krpata et al. / Environmental Pollution 157 (2009) 280–286 Table 4 Zn and Cd concentrations in leaves of European aspen and Tor grass

Fruitbody CdBCF total (unitless)

100

Plant Control site Site 1 Site 2 Site 3 species Zn Cd Zn Cd Zn Cd Zn Cd (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1) (mg g1)

10

Populus tremula Mean 340.0a SD 117.3 (n) (4)

1

control site 1 site 2 site 3

0,01

0,001

2

10

100

Fruitbody CdBCF labile (unitless)

957.3b 116.1 (2)

15.6z 3.4 (2)

974.0b 375.3 (3)

16.6z 5.0 (3)

108 38.1 (2)

132 36.6 (3)

0.42 0.14 (3)

106.7 31.6 (3)

0.28 0.06 (3)

0.37 0.19 (2)

200

assume that this affected the results only marginally, since fungal mycelia that produce the fruitbodies will have extended well beyond the soil below the fruitbodies (Cairney, 2005). In one study, even no mycelium could be detected directly below fruitbodies of the very same species (Peintner et al., 2007). By their foraging

10000

1000

100

a

10

1

control site 1 site 2 site 3

0,1 0,01

0,1

1

10

Labile soil Cd concentration (µg g-1) Fig. 3. Log–log plots of bioconcentration factors (BCF) of Cd calculated for all ECM fungi on the basis of (a) total and (b) NH4NO3-extractable soil Cd concentrations.

In most previous studies, fungal BCF were calculated based on soil metal concentrations directly below the fruitbodies (Alonso et al., 2003). Contrary to this, averaged metal concentrations from five randomly chosen spots of each site were used in this study. We

100 10 1 0,1

Amanita spp. Tricholoma scalpturatum Hebeloma spp. Inocybe spp.

0,01 0,001

0,1

1

10

100

300

Labile soil Zn concentration (µg g-1) Fig. 4. Log–log plots of Zn bioconcentration factors (BCF) on the basis of NH4NO3extractable soil Zn concentrations calculated individually for various ECM fungal taxa: Tricholoma scalpturatum (dotted line), Amanita spp. (solid line), Hebeloma spp. (dashed line), and Inocybe spp. (dash-dotted line).

1000

100

10

1

Populus tremula Brachypodium pinnatum Arabidopsis halleri 1

10

100

300

Labile soil Zn concentrations (µg g-1)

Plant accumulation factor (unitless)

1000

10000

0,1 0,1

b

10000

BCF (unitless)

486.0ab 8.1xy 78.4 1.8 (3) (3)

Mean and standard deviation are given as well as the number of measurements within the detection range. Superscripts indicate significant differences in plant metal concentrations between the sites.

Total soil Cd concentration (µg g-1)

b

3.8x 1.6 (4)

Brachypodium pinnatum Mean SD (n)

0,1

Plant accumulation factor (unitless)

a

1000

100

10

1

0,1

0,01 0,01

Populus tremula Brachypodium pinnatum Arabidopsis halleri 0,1

1

10

Labile soil Cd concentrations (µg g-1) Fig. 5. Log–log plots of accumulation factors of (a) Zn and (b) Cd calculated for European aspen (Populus tremula; solid line) and Tor grass (Brachypodium pinnatum; dotted line) on the basis of NH4NO3-extractable soil metal concentrations. The single measurement for the Zn/Cd hyperaccumulator Arabidopsis halleri is also given.

D. Krpata et al. / Environmental Pollution 157 (2009) 280–286

a Accumulation factor (unitless)

10000

1000

100

10

1

0,1 0,1

Hebeloma spp. Populus tremula 1

10

100

300

Labile soil Zn concentration (µg g-1)

Accumulation factor (unitless)

b

10000

1000

100

10

1

0,1 0,01

Inocybe spp. Populus tremula 0,1

1

10

Labile soil Cd concentration (µg g-1) Fig. 6. Log–log plots of (a) Zn and (b) Cd accumulation into European aspen (Populus tremula; dotted line) and selected ECM fungal taxa (solid lines) calculated on the basis of NH4NO3-extractable soil metal concentrations.

behaviour (Agerer, 2001), mycelia thus integrate the heterogeneity of soil chemical characteristics. Fungal BCF values were in previous studies generally related to total metal concentrations in the upper 5–10 cm substrate below the litter layer (Gast et al., 1988; Alonso et al., 2003; Rudawska and Leski, 2005a). A double-log plot of these data resulted in correlations with regression coefficients of R2 ¼ 0.51 for Cd and R2 ¼ 0.57 for Zn. Much closer correlations were obtained when BCF were expressed on the basis of labile soil metal concentrations in the mineral soil (for Zn: R2 ¼ 0.77; for Cd: R2 ¼ 0.70). Metal uptake by plants is generally more closely correlated with labile than with total soil metal concentrations (Robinson et al., 2000; Nolan et al., 2005). The labile soil Cd pool, e.g., which comprises the Cd in solution and the Cd sorbed to exchange surfaces (Adriano, 2002), is the main source of phyto-available Cd (Young et al., 2000). We are unaware of any information on soil Cd pools available to ECM fungi. In vitro experiments (Blaudez et al., 2000) suggest ECM fungal uptake from the same pool. This is supported by the close correlation of log10 CdBCFlab with log10-transformed labile soil Cd concentrations. The previously observed weak correlation between fungal CdBCFs and soil Cd (Gast et al., 1988) is thus most likely due to the use of total soil Cd concentrations and also the inclusion of saprobic fungi in their analysis. Ectomycorrhizal fungi differ in their occurrence and relative distribution between different horizons in the soil profile (Dickie

285

et al., 2002). The analysis of ECM root tips (Rosling et al., 2003) and also data on ECM fungal mycelia in soil (Genney et al., 2006) have shown preferential occurrence of some fungi in either mineral or organic soil horizons. Others occur throughout the whole soil profile. For the metal polluted sites in this study, species in the genus Hebeloma tended to more frequently colonise aspen root tips in the organic horizon (Krpata et al., 2008). Species in the genus Inocybe and T. scalpturatum were more frequent in the mineral soil, where also Amanita spp. tend to more commonly occur (Dickie et al., 2002). In an attempt to include this information in the analysis, CdBCF for all ECM fungi were related to total Cd concentrations of the substrate in which they were known or assumed to more frequently occur. This resulted in only a very slight improvement compared to a relation based on total Cd concentrations in just the mineral soil (from R2 ¼ 0.31 to R2 ¼ 0.35). A correlation between the CdBCF of only the two Hebeloma spp. and total Cd concentrations in the organic horizons of the three metal-polluted sites gave a much poorer fit (R2 ¼ 0.17) than a correlation of CdBCFlab (R2 ¼ 0.86). Nonetheless, we think that in future studies a consideration of ‘available’ Cd in the organic horizons (Sauve´ et al., 2003), in combination with data on the horizon-specific distribution of the fungi may contribute to improved correlations of BCF. Aspen Zn and Cd concentrations were intermediate between those measured in the hyperaccumulator A. halleri and the excluder Tor grass. They were in the range of concentrations previously reported for accumulators such as aspen and various Salix spp. grown on metal contaminated soil (Hermle et al., 2007; Unterbrunner et al., 2007). They were also similar to Zn and Cd concentrations measured in the fruitbodies of various ECM fungi collected at the same sites. Normally, ECM fungal fruitbodies contain higher metal concentrations than leaves of co-occurring plants (Krupa and Kozdroj, 2004; Rudawska and Leski, 2005a). This has been considered a consequence of the plant-protective role of these root-associated fungi. ECM fungi are known to act as a barrier against excessive soil metal concentrations (Frey et al., 2000; Jentschke and Godbold, 2000). Plants mycorrhizal with ECM fungi tolerant to high soil concentrations of various metals remained physiologically healthier than non-mycorrhizal plants or plants associated with metal-sensitive ECM fungi (Adriaensen et al., 2006). This included higher chlorophyll concentrations in their foliage, lower shoot metal concentrations and higher phosphate and ammonium uptake rates when exposed to high metal concentrations (Adriaensen et al., 2005). By their barrier-function, ECM fungi contribute to generally higher metal concentrations in tree roots compared to leaves (Dickinson and Pulford, 2005). These data were obtained mainly from trees that are not considered accumulators. Recent studies on accumulator trees, on the other hand, found similar root and leaf Cd concentrations (Sell et al., 2005; Unterbrunner et al., 2007). The latter study included aspen samples from the very same sites analysed here, and roots of these aspen were heavily mycorrhizal (95% of all root tips; Krpata et al., 2008). Apparently, the ECM fungi at the examined sites did not act as a barrier by retaining the metals. Such a function would be expected to also have resulted in elevated fruitbody metal concentrations (Thomet et al., 1999). Any effect of the fungi on aspen metal uptake thereby remains unclear with potentially large differences between fungal taxa. Besides having no effect some fungi may still have protected their host and simultaneously avoided metal accumulation by downregulation of transporters or increased metal efflux (Bellion et al., 2006). Alternatively, ECM fungi may have transported and further transferred the metals to the roots and thereby increased aspen metal uptake. Such a potential difference in ECM function with respect to metal uptake between accumulators and excluders is supported by results from recent studies (Sell et al., 2005; Baum et al., 2006).

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